RECOMBINANT VACCINES Live or Killed

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1 Antigens and Vaccines Molecular biology drives Innovation Antigen Gene of Interest Recombination RECOMBINANT VACCINES Live or Killed

2 Antigens and Vaccines Molecular biology drives Innovation Antigen Gene of Interest Replacement Deletion Insertion Foreign Gene Updated V. Gene deleted V. Vector V.

3 THE VECTOR VACCINE CONCEPT Entering a new world...

4 The Concept of Vector Vaccine F NDV = Donor NDV NDV genome (RNA) rhvt- F F (Fusion) gene HVT HVT Insertion site HVT = Vector Inserted F gene

5 The Concept of Vector Vaccine Vector vaccine is injected The HVT vector replicates, Fusion gene is expressed, and immunity is induced.

6 The Concept of Vector Vaccine WHAT IS A VECTOR VACCINE? a Vector a Donor an inserted gene an insertion site a promoter a terminal sequence etc. etc.

7 The Concept of Vector Vaccine WHAT IS A VECTOR VACCINE? a Vector a Donor an inserted gene an insertion site a promoter a terminal sequence etc. etc. Patents + Patents + Patents + Know How A VECTOR IS NOT A VECTOR!

8 WHY HVT AS VECTOR?

9 The Concept of Vector Vaccine MATERNALLY DERIVED ANTIBODIES INTERFERE WITH EFFICACY OF VACCINATION INTERFERENCE NO VARIABLE STRONG MAREK GUMBORO NEWCASTLE POX REO rhvt-nd

10 The Concept of Vector Vaccine ADVANTAGES OF A rhvt VECTOR VACCINE Very safe Limited interference with MDA Can be applied at the hatchery No Live ND virus Life long persistance Humoral and Cellular Immunity Allows for DIVA «Easy» to work with

11 WHY «F GENE» AS INSERT?

12 A challenging virus NDV Attachment HN F M P NP L RNA Fusion Synthesis Assembling Release

13 A challenging virus NDV Attachment HN F M P NP L RNA Fusion Synthesis Assembling Release

14 HN A challenging virus NDV (Haemagglutinin Neuraminidase) Is responsible for the attachment of NDV to the host cells. It is not a factor of virulence : Introduction of an HN gene from a velogenic NDV into a mesogenic NDV (Anhinga virus) failed to increase its virulence. (Estevez C. et al., Virus Research, 129 (2007), )

15 A challenging virus NDV F (Fusion protein) F0, the precursor of the F protein, needs to be cleaved (by a protease) into F1 and F2 before NDV can infect cells and spread. - F0 of velogenic strains can be cleaved by many proteases : systemic infection - F0 of lentogenic strains can be cleaved only by trypsin- like protease only present on the mucosas : local infection

16 F A challenging virus NDV (Fusion protein) is the most important factor of virulence: Introduction of an amino-acid sequence of a velogenic NDV inside the cleavage site of the fusion protein F0 of an avirulent NDV (ICPI = 0) turned it into velogenic (ICPI = 1,28) : GGRQGR L GRRQRR F (Ben Peeters et al., J. of Virology, (1999), )

17 A challenging virus NDV NP, M, P (Nucleo / Matrix / Phospho - proteins) Are not important for protection. Serum containing antibodies against NP, M and P proteins did not induce any protection (Reynolds & Maraqa, Av. Diseases, 44, (2000), )

18 PERFORMANCES OF A rhvt-f ND VACCINE

19 What can we expect from a vaccine? PROTECTION? 1 Infection HIGHER RESISTANCE TO INFECTION 2 - Clinical expression CLINICAL PROTECTION 3 Re-excretion REDUCTION OF RE-EXCRETION

20 What can we expect from a vaccine? Assessment of a vaccine «take» in the presence of MDA Onset of Immunity (OOI) Level of Immunity (LOI) Reduction of re-excretion Spectrum of protection Duration of Immunity (DOI) Resistance to infection Easy to administer

21 Onset of Immunity (OOI) rhvt-f ND vaccine Live Live La Sota (Phylaxia) antigen (Extract from study SCI SSIU Ceva Phylaxia)

22 Disease pressure Onset of Immunity (OOI) rhvt-f ND vaccine: recommended vaccination programs MDA Vectormune ND Low O O Live ND Priming D1 Medium O O O Live ND boost D14-18 High O O O O

23 Level of Immunity (LOI) Lab. trials in Thailand - Layers - rhvt-f injected SQ at d1 - Live ND vaccine by eye drop at d1 - Killed ND vaccine injected SQ at d1 - Challenge : - Thai vvndv strain - at 2, 3 and 4 weeks of age

24 Percentage of protection Level of Immunity (LOI) Lab. trials in Thailand - Layers VECTORMUNE HVT-NDV Laboratory challenge trials in Thailand Vectormune rhvt-f HVT-NDV Cevac Live ND Vitapest vaccine L Cevac Live ND Vitapest + rhvt-f L + Vectormune HVT-NDV Cevac Live ND Vitapest + Killed L + ND Cevac Broiler ND K Controls Age at challenge (days)

25 Reduction of re-excretion - Commercial broilers (20 per group) - rhvt-f SQ or in-ovo (-3d) - Challenge : EID50 Chimalhuacan NDV strain - at 3 or 4 or 6 weeks of age - IN + ON route - Oropharyngeal and Cloacal swabs taken 3 and 7 days post challenge - Virus quantification using RT-PCR Trials at SSIU / Ceva Phylaxia

26 Reduction of re-excretion Clinical protection 56

27 Reduction of re-excretion Challenge at D28 * * * * * * * * Reduction of shedding

28 Reduction of re-excretion Challenge at D40 * * * * * * * * Reduction of shedding

29 Spectrum of Protection Yes! Does it protect against various types of NDV? Yes! Yes! F insert Yes! 59

30 Duration of Immunity (DOI) Registration trials in the USA - Layers - Layer pullets with MDA against MD and ND - rhvt-f injected at d1 SQ. - Challenge : EID 50 Texas GB NDV strain - at 19 weeks of age

31 Duration of Immunity (DOI) % ND protection % Control SQ Vaccinated 20 0% 0 19 Weeks Old

32 Duration of Immunity (DOI) - Lohman Brown layer pullets with MDA (mean HI = 5,5 log 2) - rhvt-f at d1 (SQ) - rhvt-f at d1 (SQ) + Live ND vaccine ED at d1 + Killed ND (w15 IM) - Challenge : EID 50 Malaysian 2010 vvndv viscerotropic isolate (Genotype VII) - IN route (0,1 ml) - at 3, 4, 6, 10, 15, 33 and 72 weeks of age Trials at SSIU / Ceva Phylaxia

33 % Protection Duration of Immunity (DOI) Trials at SSIU / Ceva Phylaxia Protection of layers against vvndv challenge Vectormune HVT-NDV Vectormune HVT-NDV + Cevac Vitapest L + Killed Controls Age at challenge (in weeks) 33 72

34 Duration of Immunity (DOI) Challenge at 33 weeks: egg production ND challenge (w33) * *negative controls not challenged

DIVA serological monitoring Commercial Broilers vaccinated SQ at day 1 Protection / NDV challenge 60% 90% 100% 100% % of positives: «A»ELISA HI «B»ELISA 100%

detected by YG - 150422 HITest as well as «A» ELISA, but poorly by «B» ELISA. Detection by «A»* ELISA is even earlier than detection by HITest.

35 DIVA serological monitoring Commercial Broilers vaccinated SQ at day 1 Protection / NDV challenge 60% 90% 100% 100% % of positives: «A»ELISA HI «B»ELISA 100% 100% 40% 100% 100% 60% HI Test & ELISAs 60% 20% 0% 60% 30% 0% Positivity limits: «A»: 993 «B»: 1159 HIT: 2 Antibody response to rhvt-f vaccination is well detected by YG HITest as well as «A» ELISA, but poorly by «B» ELISA. Detection by «A»* ELISA is even earlier than detection by HITest. Protection against challenge is established before antibody response is detected. (Extract from study SCI SSIU Ceva Phylaxia) *ID Vet ND indirect ELISA for rhvt-f vaccines

36 Conclusions For the control of Newcastle Disease (ND) vaccination with rhvt-f vaccine is a very efficacious tool, if complementary to:

37 Conclusions For the control of Newcastle Disease (ND) vaccination with rhvt-f vaccine is a very efficacious tool, if complementary to: Good animal husbandry Stringent Biosecurity Effective Sanitary Police

38 Conclusions ND vaccination program must match the field disease pressure: Low ND is a potential risk Medium ND is a real risk High ND is a strong risk

39 Conclusions Thanks to molecular biology, it was possible to make a vaccine not neutralized by MDA (HVT), carrying a protective factor (F) from NDV and inducing high level of protection.

40 Conclusions rhvt-f vaccine takes whatever MDA status The take depends only on the quality of administration Administration is to be done at the hatchery (in-ovo or SQ) i.e. in the most reliable way Early immunity is re-inforced by MDA, and, if necessary, by live ND vaccine(s) No live ND vaccine is released inside the house / farm / region / country hence less post vaccination reactions Duration of immunity is outstanding Shedding of challenge virus is reduced

41 THANK YOU FOR YOUR ATTENTION

Vectormune ND a step towards control of Newcastle Disease

Poultry Vaccinology Summit April 28-30, Budapest, Hungary Vectormune ND a step towards control of Newcastle Disease Yannick Gardin 1, Vilmos Palya 1, John Elattrache 1, Marcelo Paniago 2, Christophe Cazaban