Live attenuated influenza vaccine (LAIV) for pigs Hyun Mi, Pyo April, 2014

1. Influenza A virus 2. Influenza vaccine 3. Live Attenuated Influenza Vaccine 4. LAIV SIV/606 Contents

Influenza A virus Emerging Infectious Disease (2012) Kessell A. et. al. Orthomyxoviridae

Influenza A virus CDC, Public Health Image Library, ID# , Spanish Flu 2009 Pandemic H1N1, Yoshihiro Kawaoka Courtesy of Yoshihiro Kawaoka, University of Wisconsin-Madison 100nm

Influenza A virus ViralZone: Swiss Institute of Bioinformatics

Influenza A virus from healthmediconline.com H3N8

Influenza vaccine

Live attenuated influenza vaccine (LAIV) Currently available LAIV - Human: FluMist (intranasal live influeza vaccine, MedImmune) - Equine: attenuated, cold adapted live equine 2 influenza virus live canaripox vector expressing HA of H3N8 Advantages compare to killed/split influenza vaccine - induce strong and long lived immunity - feasible administration route - provide cross protective immunity Disadvantages - Possibility of reversion to virulence or reassortant - Restricted usage in healthy individuals - Maintenance or storage

1. Generation of reassortant LAIV from Attenuated donor Clin Exp Vaccin Res 2012;1:35-49 Classical attenuation method Cold-adaptation

2. Genetically modified recombinant viruses Modification of HA cleavage site - Stech et al., Nat Med 2005;11: Masic et al., J Virol 2009; 83: Replacement of Arg to Val allows HA cleavage by Elastase NS segment truncation mutant - Hai et al., J Virol 2008; 82: Fail to IFN antagonistic function and attenuated in IFN competent systems Mutant virus carrying bivalent HAs - Introduce heterologous HA into one of the segment - Gao et al., J Virol 2010;84 H1N1 carrying 9 segment, H1/H3 - Pena et al., J Virol 2013; 87 H9N2 with H5 in NS segment - Uraki et al., J Virol 2013; 87 H1N1 with H5 in PB2 segment - Masic et al., J Virol 2013; 87 H1N1 with H3 in NA segment

Selective packaging model Ozawa and Kawaoka (2011) Virus Research, 162

Objectives Produce LAIV candidate Evaluate the attenuation in pigs Efficacy test in pigs - Intratracheal vaccination - Intranasal vaccination

A strategy to generate recombinant LAIV candidate HA (Tx98 H3N2) 183nt 157nt19nt 28nt SK02 NA 3’ 5’ TGA NA PA (SIV/SK02) H1-HA (SIV/SK02) PB1 (SIV/SK02) PB2 (SIV/SK02) NP (SIV/SK02) M (SIV/SK02) NS (SIV/SK02) H3-HA (SIV/TX98) A) B) Masic et al., JVIMasic et al., JVI (2013) 87(18):

α-H1 HA α-H3 HA α-M1 C) B) M SIV/TX98 SIV/SK02 SIV-606 D) SIV/TX98SIV/SK02 SIV-606 Mock SK02 TX98 SIV-606 H1 H3 RT-PCR A) α-H1 HA α-H3 HA α-M1 Characterization of H1-H3 rSIV

WT SIV/SK02SIV-606 A) B) H1-H3 rSIV growth properties Trypsin+++ NA-+-

Pathogenicity study GroupInoculum Concentration (PFU ml -1 ) 1 (n=3)MEM- 2 (n=5)SIV/SK02 (H1N1) (n=5)SIV/SK02 (H1N1) (n=5)SIV/TX98 (H3N2) (n=5)SIV/TX98 (H3N2) (n=5)SIV-606 (H1/H3) (n=5)SIV-606 (H1/H3)10 6 Day 0 Day 5 Virus infection, IT Necropsy Rectal temperature and clinical sign

A) B) C) Rectal temperature

Gross lesion score in lung

Histopathology MEM SIV/SK02 Low dose SIV/SK02 High dose SIV/Tx98 High dose SIV/Tx98 Low dose SIV-606 High dose SIV-606 Low dose B C D E F G

Viral titer in lung tissue 5.9E46.5E4 1.8E4 3.7E4 2.4E1

Group Vaccination 1 st (day 0) 2 nd (day 21) Challenge (day31) 1 (n=5)MEM SIV/SK02 (H1N1) 2 (n=5)MEM SIV/TX98 (H3N2) 3 (n=5)H1H3 rSIV SIV/SK02 (H1N1) 4 (n=5)H1H3 rSIV SIV/TX98 (H3N2) 5 (n=3)--- Immunoprotection study after IT vaccination Trial 1 Day 0 Day 21 Day 31 Day 36 1 st Vaccination 2 nd Vaccination Necropsy Virus challenge

Antigen specific serum IgG titer Vaccination: MEM SIV-606 MEM SIV-606 MEM SIV-606 Day 0 Day 21 Day 31 A) ** *** B) *** Vaccination: MEM SIV-606 MEM SIV-606 MEM SIV-606 Day 0 Day 21 Day 31

** Vaccination: MEM SIV-606 MEM SIV-606 MEM SIV-606 Day 0 Day 21 Day 31 A) *** Vaccination: MEM SIV-606 MEM SIV-606 MEM SIV-606 Day 0 Day 21 Day 31 B) Antigen specific nasal IgA titer

A) B) ** * Rectal temperature after the viral challenge

Gross lesion score

A) B) C) D) E) Unvaccinated and no viral challenged pig MEM vaccinated andSIV/SK02 challenged pig MEM vaccinated and SIV/Tx98 challenged pig SIV-606 vaccinated and SIV/SK02 challenged pig SIV-606 vaccinated and SIV/Tx98 challenged pig Histopathology

Viral titer in lung tissue

Group Vaccination 1 st (day 0) 2 nd (day 21) Challenge (day31) 1 (n=5)MEM SIV/SK02 (H1N1) 2 (n=5)H1H3 rSIV SIV/SK02 (H1N1) 3 (n=5)MEM SIV/Tx98 (H3N2) 4 (n=5)H1H3 rSIV SIV/TX98 (H3N2) 5 (n=3)--- Immunoprotection study of Intranasal vaccination Trial 2 Day 0 Day 21 Day 31 Day 36 1 st Vaccination 2 nd Vaccination Necropsy Virus challenge

Antigen specific serum IgG titer Vaccination: MEM SIV-606 MEM SIV-606 MEM SIV-606 Day 0 Day 21 Day 31 Vaccination: MEM SIV-606 MEM SIV-606 MEM SIV-606 Day 0 Day 21 Day 31 A) B)

Virus neutralizing antibody titer Day 31Day 36 Day 31Day 36 A) B)

Evaluation of protective efficacy A) B) C)

A) B) Antigen specific IgG on Day 36

Antigen specific IgA on Day 36 A) B)

Cell mediate immune response on Day 36 A) B) C) D)

Summary 1.A novel LAIV candidate harboring H1 and H3 HAs was generated by rewiring NA segment. 2.LAIV candidate maintained the characteristics of parental virus while having H1 and H3 HAs. 3.Experimental infection of LAIV candidate did not caused any signs of typical SIV infection which indicates attenuation of the virus. 4.Vaccination of LAIV candidate protected the pigs from both parental H1N1 and H3N2 viral challenge. 5.Range of cross protection and vaccine regimen will be further studied.

Future study 1.Maternal antibody interference 2.Challenge study in Ferret model

Acknowledgement Dr. Yan Zhou Dr. Alexander Masic Animal care staff at VIDO All A121 lab members

-Non profit research centre own by U of Saskatchewan - Annual budget $12 million employees from 20 countries (26 Scientists, 18 PDFs, 34 Technical staff, 44 Grad students, 17 Admin) - 7 Research Programs and two supporting programs : Emerging Disease and Microbial Virulence (BSE, Bacterial Biology & Antimicrobials, influenza, Johne’s disease) Immune modulation Neonatal Immunization Bacterial Vaccine Development (Food Safety, Bovine Mastitis, Mycoplasma) Viral Pathogenesis and Vaccine Development (DNA immunization, HepC,PRRSv) Vectored Vaccines (Bovine and Porcine Adenovirus vectors) Pathogenomics (Pathogenomics of innate immunity, Stress, Disease and Delivery)

Chemistry, GMP & Genomics Services Clinical Research & Epidemiology (3 Veterinarians) - Certified Intervac CL3 facility ($140 million) - Attained ISO9001 Certification in 2013

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