1. EXPLORING INFLUENZA VACCINE PRODUCTION TECHNOLOGIES- PRESENT AND FUTURE Gaurav Gupta, Zydus Cadila, Ahmedabad

2. CONTENTS  INFLUENZA AND ANTIGENIC VARIABILITY  CURRENT INFLUENZA VACCINES & TECHNOLOGIES  LIMITATIONS AND FUTURE SCOPE FOR IMPROVEMENT  FUTURE APPROACHES FOR INNOVATIVE INFLUENZA VACCINES

3. INFLUENZA AND ANTIGENIC VARIABILITY

4. 4 Types of influenza viruses Three distinct types of influenza virus A, B, and C, Most cases of the flu, especially those that occur in epidemics or pandemics, are caused by the influenza A virus, which can infect a variety of animal species too. The B virus, which normally is only found in humans, is responsible for many localized outbreaks. The C virus is morphologically and genetically different from the other two viruses and is generally nonsymptomatic, so it has a little medical concern.

5. Horimoto T, Kawaoka Y: Influenza: lessons from past pandemics, warnings from current incidents. Nat Rev Microbiol 2005, 3:591-600.  Family Orthomyxoviridae  Eight negative-stranded RNA segment  Six code for internal proteins: RNA polymerase proteins (PB2, PB1 and PA) nucleoprotein (NP) matrix protein (M) non structural protein (NS)  Two code for the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) Targets of protective immune response INFLUENZA VIRUS

6. Influenza Clinical Features Incubation period 2 days (range 1-4 days) Abrupt onset of fever, myalgia, sore throat, nonproductive cough, headache Severity of illness depends on prior experience with related variants

7. Influenza Complications Pneumonia – secondary bacterial – primary influenza viral Myocarditis Death 0.5-1 per 1,000 cases

8. Influenza Antigenic Changes Antigenic Shift – major change, new subtype – caused by exchange of gene segments – may result in pandemic Example of antigenic shift – H2N2 virus circulated in 1957-1967 – H3N2 virus appeared in 1968 and completely replaced H2N2 virus – In April 2009, the virus appeared to be a new strain of H1N1(Swine Flu) which resulted when a previous triple reassortment of bird, swine and human flu viruses further combined with a Eurasian pig flu virus.

9. Influenza Antigenic Changes Antigenic Drift – minor change, same subtype – caused by point mutations in gene – may result in epidemic Example of antigenic drift – in 2002-2003, A/Panama/2007/99 (H3N2) virus was dominant – A/Fujian/411/2002 (H3N2) appeared in late 2003 and caused widespread illness in 2003-2004

10. CURRENT INFLUENZA VACCINES

11. 11 Types of influenza vaccines Live-attenuated influenza vaccines Inactivated trivalent influenza vaccines Cold-adapted influenza vaccines trivalent - Whole virus vaccine - Split vaccine - Subunit vaccine - Virosome vaccine

12. Influenza Vaccines immunity Inactivated subunit (TIV) – intramuscular – trivalent – split virus and subunit types – duration of immunity 1 year or less Live attenuated vaccine (LAIV) – intranasal – trivalent – duration of immunity at least 1 year

13. Timing of Influenza Vaccine Programs Influenza activity can occur as early as October In more than 80% of seasons since 1976, peak influenza activity has not occurred until January or later In more than 60% of seasons the peak was in February or later

14. Inactivated Influenza Vaccine Efficacy 70%-90% effective among healthy persons younger than 65 years of age 30%-40% effective among frail elderly persons 50%-60% effective in preventing hospitalization 80% effective in preventing death

15. Inactivated Influenza Vaccine Adverse Reactions Local reactions15%-20% Fever, malaisenot common Allergic reactionsrare Neurologicalvery rare reactions

16. LAIV Efficacy in Healthy Children 87% effective against culture-confirmed influenza in children 5-7 years old 27% reduction in febrile otitis media (OM) 28% reduction in OM with accompanying antibiotic use Decreased fever and OM in vaccine recipients who developed influenza

17. LAIV Efficacy in Healthy Adults 20% fewer severe febrile illness episodes 24% fewer febrile upper respiratory illness episodes 27% fewer lost work days due to febrile upper respiratory illness 18%-37% fewer days of healthcare provider visits due to febrile illness 41%-45% fewer days of antibiotic use

18. LIMITATIONS AND FUTURE SCOPE FOR VALUE ADDITION

19. Vaccines need to be changed due to high antigenic variation every season. Pandemic situations requires large quantities of vaccines to be produced. Classical technologies does not permit high yields of certain influenza strains- type B. Immunity provided in not strong enough for protecting all age groups with one type of vaccines- live/Inactivated

20. LIMITATIONS AND FUTURE SCOPE FOR VALUE ADDITION Live vaccines have risk to virulence reversion High doses of antigen required in inactivated vaccines for immunization. Clinical trials is not feasible to be evaluate safety & efficacy every year for new strains.

21. FUTURE APPROACHES FOR INNOVATIVE VACCINES

22. Universal Influenza vaccine protocol Selection of antigens: based on HA and NAGeneration of VLP’s-monovalent and multivalentProcess and analytical developmentMouse immunogenecity studiesChallenge studies in animal models- ferrets, MonkeysClinical studies

23. Universal Influenza vaccine covering broad antigenic diversity using efficient production technologies could be the solution.

24. Zydus Approach

25. Mouse Immunogenicity studies Task ETNA BIOTECH Year 2014/15Year 2015 apr-14 mag-14 giu-14lug-14 ago-14 set-14 ott-14 nov-14 dic-14 gen-15 feb-15 mar-15 apr-15 mag-15 giu-15lug-15 ago-15 set-15 ott-15 nov-15 dic-15 gen-16 feb-16 mar-16 MICE IMMUNOGENICITY STUDIES PRELIMINARY MOUSE STUDIES 3.1 Generation of reference mouse sera 3.2 Pilot mouse studies MOUSE VACCINATION STUDIES 3.3 Seasonal Influenza Vaccine 3.4 Hexavalent Group 2HA vaccine Go/no-go criteria 3.5 VN, qELISA, cELISA, HI DATA ANALYSIS 3.6Identification of Multivalent Lead Vaccine Candidate ADJUVANT EXPLORATION 3.7 Screening of readily available adjuvant

26. Lead vaccine candidate finally selected will go further for preclincal studies in Ferrets/Monkeys.

27. Summary & Conclusions Vaccine based on high yielding substrates will enhance coverage of influenza vaccination in developing countries. Technologies based on cells will be the key to produce influenza vaccines in low cost at higher level of production. Designing and exploring universal Influenza vaccines could lead to effective prevention of Influenza globally.

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