Vector vaccines and immunity. Thierry Van den Berg Coda Cerva, Belgium

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1 Vector vaccines and immunity Thierry Van den Berg Coda Cerva, Belgium

2 Overview of the presentation o Theory Background Classical vaccines New technology vaccines Chicken immunology o Laboratory Tools for measurement of chicken immunity Humoral Cell-mediated Local (or mucosal): HLT, BLT, GLT & RLT o Practice NDV o Conclusions 17/04/2013 2

3 Industrial poultry vaccination = Mass application Up to birds in a single space Need for effective methods im = vaccinating high enough proportion of birds in the flock Proportion will depend on Type of infectious agent involved Current epidemiological situation Epizootic diseases (I-ND): maximized protection Many other situations: minimize economic impact of disease 3

4 S F E T Y P O T E N C Y MD

5 Transfert of passive immunity in birds

6 Pros and cons of classical vaccines ttenuated Inactivated dministration Varied routes (mass) SC, IM Safety Immunity Residual virulence Risk of reversion Broad (hum., cell., muc.) Quick onset Lower < 2 wk-old OK >2 wk-old Humoral mainly Slow onset Field strains match Not always Yes Efficacy Sometimes too attenuated MD interference Low < 2wk-old Production Low dose High dose Quality risk Extraneous agents Bad inactivation

7 Why new types of vaccines? To further improve classical vaccines New technology vaccines need to show clear advantages over classical vaccines nd fill the gaps!! e.g. attenuation impossible or large scale production difficult, interference of MD, mass vaccination, DIV Need to correlate protection parameters with vaccine performances

8 Evolution to reach the «ideal vaccine» Quality Safety Efficacy Ideal vaccine Ease of production Ease of administration Duration Interference of MD 8

Live vector vaccine definition Recombinant vector vaccines are bioengineered vaccines similar to DN vaccines, but they use an attenuated virus to introduce DN to cells of the body where it will be

9 Live vector vaccine definition Recombinant vector vaccines are bioengineered vaccines similar to DN vaccines, but they use an attenuated virus to introduce DN to cells of the body where it will be expressed. 3 steps: o o o Clone the protective gene Insert the protective gene into the genome of the vector (herpesvirus, poxvirus, adenovirus ) Produce the vector = vaccine Being intracellular, they are able to induce a strong cellular immunity Slower immune responses against the inserted product and Low/late humoral immunity prime a humoral response! Strong B & T cell memory 9

10 dvantages of live vector vaccines o Safety = vector dependent Live attenuated (no reversion to virulence) No need for adjuvant Early age (day-old or in ovo) o Efficacy = insert and vector dependent Broad immunity (cellular and humoral) Rapid onset after one administration Interference with maternal antibodies Bivalent vaccine if vector is a vaccine o Production = vector dependent o Differential diagnostic (DIV) = insert dependent

11 What is in ovo vaccination? ED0 ED18 Hatching Incubator regularly turning In ovo vaccination Transport Hatching tray no turning Opportunity to immunise each individual chick without additional stress of manipulation t ED18 : - development of the immune system is nearly complete - active immune response possible.

12 NDV + IBDV + ED16 ED18 ED19 ED20 ED21 Day 1 Day 2 Day 3 NDV-specific IgG IBDV-specific Ig

13 Transfer of maternal antibodies from the yolk sac to the chick window of opportunity exists for avoiding interference of maternal antibodies with vaccination In ovo vaccination : low titres of maternal antibodies virus replication possible low vaccination doses evoke strong, long-lasting immune responses provided that the vaccine is not pathogenic for the embryo IBDV NDV

Lymphoïd organs in chickens Harderian glands Thymus o

regulate the development of the humoral and cellular

Caecal tonsils o Peripheral organs : spleen, gut-,

14 Lymphoïd organs in chickens Harderian glands Thymus o Primary organs : the bursa of Fabricius and the thymus regulate the development of the humoral and cellular compartments of the immune system, respectively. Caecal tonsils o Peripheral organs : spleen, gut-, bronchus- and head-associated lymphoid tissue (GLT, BLT and HLT, respectively). Spleen Meckel diverticulum Bursa of Fabricius o Chickens do not have lymph nodes, the primary site of antigen presentation in mammals.

15 Immune response in chickens Local immunity in the head Local immunity in the respiratory tract Local immunity in the reproductive tract Humoral immunity Cell-mediated immunity Local immunity in the digestive tract

16 Immune response in chickens Chickens respond to infection or vaccination by developing humoral, cellular and mucosal immune responses o Humoral immunity : B-cells Measured by detection of specific antibodies in sera (ELIS, HI, SN) o Cell-mediated immunity : T-cells (Th1 and Th2 responses) Measured by proliferation tests ( 3 H uptake, WST1) and cytokines production (ChIFNg) after specific recall (ex vivo) on splenic and peripheral blood lymphocytes o Mucosal immunity : Immunity at mucosal surfaces : major route of entry for many infectious agents into the body : HLT, BLT and GLT + reproductive tract Local antibody- and cell-mediated immunity = first line of defence against pathogens Viral excretion is reduced by local b Difficulties and labor-intensiveness of collecting samples Poorly investigated in poultry

, 2009, 2010] In the peripheral blood [Rauw et al., 2010] In the digestive tract [Rauw et al., 2010] In the respiratory tract [Rauw et al.

17 ssessment of the immune responses Humoral immunity : Development of virus specific ELISs to detect IgG, IgM and Ig specific [Rauw et al., 2009, 2010] Cell mediated immunity: New tools to measure CMI: production of ChIFNg after recall stimulation In the spleen [Lambrecht et al., 2004, Rauw et al., 2009, 2010] In the peripheral blood [Rauw et al., 2010] In the digestive tract [Rauw et al., 2010] In the respiratory tract [Rauw et al., 2011] Local (mucosal) immunity : Live vaccines are inoculated by mucosal route (spray, drinking water) New tools to measure lachrymal, respiratory and digestive immunity [Rauw et al., 2009, 2010a, 2010b] NDV IV IBDV 17

18 Immune response in the chickens Humoral immunity Serum

19 NDV-specific IgG ELIS TMB substrat 450nm Streptavidine-POD Mouse anti- Chicken IgG - BIOT Day-old SPF chickens (n = 5) 1 dose of rhvt-nd Subcutaneous inoculation NDV-specific IgG Purified whole NDV (2 µg/ml) Saturation (2,5 % casein)

20 TMB substrat Streptavidine-POD NDV-specific IgM ELIS 1 dose of rhvt-nd Subcutaneous inoculation 450nm Mouse IgG1 anti-ndv - BIOT Day-old SPF chickens (n = 5) Purified whole NDV Mouse anti- Chicken IgM NDV-specific IgM Saturation

21 TMB substrat NDV-specific Ig ELIS Streptavidine- POD 450nm 1 dose of rhvt-nd Subcutaneous inoculation Mouse IgG1 anti-ndv - BIOT Day-old SPF chickens (n = 5) Purified whole NDV NDV-specific Ig Mouse anti- Chicken Ig Saturation

22 Immune response in chickens Cell-mediated immunity Spleen Peripheral blood

23 Cell-mediated immunity (CMI) Lymphocytes Spleen g recall activation Cytokines production Proliferation Pathogens Blood Digestive tract Respiratory tract + Pathogens proteins RECLL Ex vivo T-lymphocytes T-lymphocytes Specific T-lymphocytes In vivo Isolation CYTOKINES PRODUCTION PROLIFERTION Cytokines production Proliferation ELIS H 3 uptake Specific T-lymphocytes Enzyme-linked immunosorbent assay Cell harvester and cpm assay

24 NDV-specific CMI in the spleen PROTOCOL : Day-old SPF chicks 10 7 c/ml, RPMI 2 % FCSi 72h Flat-bottomed plate prot-ndv : 1 µg/ml BUT : Chickens sacrifice No information about circulation and response of specific T- lymphocytes in periphery Rauw F. et al. (2009). Vaccine, 27 : dose rhvt-nd Subcutaneous inoculation Day-old MD+ chickens (n = 5)

25 NDV-specific CMI in the blood (I) nticoagulant : Heparin lsever solution Blood from jugular vein Sedimentation Low speed centrifugation Ficoll HistoPaque ( ) White blood cells : PBMC PBL ctivation : ChIFNg production by T-lymphocytes 48 or 72h medium (serum %) plate (bottom)

O.D. NDV-specific CMI in the blood (II) 3.0 2.5 2.0 1.5 1.0 0.5 0.

26 O.D. NDV-specific CMI in the blood (II) Negative Enterotrocpic live ND vaccine 3.0 Enterotropic live ND vaccine and challenge % ChSi 10 % ChSi PM/Iono (1 µg/ml) lsever Heparin lsever Heparin lsever Heparin lsever Heparin lsever Heparin lsever Heparin 1 µg/ml 5 µg/ml 10 µg/ml 1 µg/ml 5 µg/ml 10 µg/ml PROTOCOL : Sedimentation (4h) 10 7 c/ml, RPMI 10 % ChSi Heparin 72h Round-bottomed plate prot-ndv : 1 µg/ml 48h nticoagulant, gp-ndv concentration and culture conditions 72h Rauw F. et al. (2010). Veterinary Immunology and Immunopathology, 134 : dose rhvt-nd Subcutaneous inoculation Day-old SPF chickens (n = 5)

27 IV-specific CMI 10 6 EID 50 LP H5N2 Oculo/nasal inoculation Spleen Blood 10-day-old SPF chickens (n = 5) Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : dose rhvt-i Subcutaneous inoculation Very low IV-specific CMI Day-old SPF chickens (n = 5)

28 Immune response in chickens Local immunity in the head Tears ntibody

Lachrymal antibody-mediated immunity specific to NDV O.D. Centrifugation -20 C NDV-specific IgG/M/ ELIS 2.2 2.

2 Negative Tracheotropic live ND vaccine Enterotropic live ND vaccine 1.0 0.8 0.6 Day-old SPF chickens (n = 5) 0.4 0.2 0.

29 Lachrymal antibody-mediated immunity specific to NDV O.D. Centrifugation -20 C NDV-specific IgG/M/ ELIS dose Cevac Vitapest L or Cevac UNI L Oculo/nasal inoculation Negative Tracheotropic live ND vaccine Enterotropic live ND vaccine Day-old SPF chickens (n = 5) week 2 weeks 3 weeks 4 weeks 5 weeks 1 week 2 weeks 3 weeks 4 weeks 5 weeks 1 week 2 weeks 3 weeks 4 weeks 5 weeks NDV specific IgG NDV specific Ig NDV specific IgM Time post-vaccination and Ig isotypes Rauw F. et al. (2010). Vaccine, 28 :

30 Immune response in chickens Local immunity in the digestive tract Duodenum Bile ntibody CMI ntibody

Digestive antibody-mediated immunity specific to NDV O.D. Ex vivo tissues culture (72h) Centrifugation -20 C NDV-specific IgG/M/ ELIS 2.0 1.

31 Digestive antibody-mediated immunity specific to NDV O.D. Ex vivo tissues culture (72h) Centrifugation -20 C NDV-specific IgG/M/ ELIS dose Cevac Vitapest L or Cevac UNI L Oculo/nasal inoculation Negative Tracheotropic live ND vaccine Enterotropic live ND vaccine Day-old SPF chickens (n = 5) week 2 weeks 3 weeks 4 weeks 5 weeks 1 week 2 weeks 3 weeks 4 weeks 5 weeks 1 week 2 weeks 3 weeks 4 weeks 5 weeks NDV specific IgG NDV specific Ig NDV specific IgM Time post-vaccination and Ig isotypes Rauw F. et al. (2010). Vaccine, 28 :

5 Enterotropic live ND vaccine Enterotropic live ND vaccine with challenge 10 7 c/ml, RPMI 10 % FCSi Flat-bottomed plate 72 h prot-ndv : 1 5

32 O.D. GLT CMI specific to NDV 37 C 30 min Washes 37 C Cuts (5 mm) of duodenal tissues HBSS 1 mm EDT HBSS with 0,4 U/ml of Liberase Blendzyme 3 (ROCHE) 30 min Negative PROTOCOL : Enterotropic live ND vaccine Enterotropic live ND vaccine with challenge 10 7 c/ml, RPMI 10 % FCSi Flat-bottomed plate 72 h prot-ndv : 1 5 µg/ml LPL : Lamina Propria Lymphocytes µg/ml gp-ndv 5 µg/ml gp-ndv Weeks post-vaccination (challenge at 4 weeks p.v.) Rauw F. et al. (2010). Veterinary Immunology and Immunopathology, 134 :

33 Immune response in chickens Local immunity in the respiratory tract Trachea Lung ntibody CMI

Respiratory antibody-mediated immunity specific to NDV

p injection of Nembutal -20 C PROTOCOL : Holt et al.

vian Pathology, 34, 396-398 Personnal communications of B.

NDV-specific IgG/M/ ELIS 1 dose Cevac Vitapest L or Cevac

2 Negative Tracheotropic live ND vaccine Enterotropic live

34 Respiratory antibody-mediated immunity specific to NDV O.D. Euthanasia by i.p injection of Nembutal -20 C PROTOCOL : Holt et al. (2005). vian Pathology, 34, Personnal communications of B. Kaspers (Institut fürtierphysiologie, Munich, Germany) NDV-specific IgG/M/ ELIS 1 dose Cevac Vitapest L or Cevac UNI L Occulo/nasal inoculation Negative Tracheotropic live ND vaccine Enterotropic live ND vaccine 1.0 Day-old SPF chickens (n = 5) IgG Ig IgM Isotypes and weeks post-vaccination Rauw F. et al. (2010). Vaccine, 28 :

plate 72 h prot-h5n2 : 1 5 µg/ml Erythrocytes Rauw F. et al. (2011).

35 BLT CMI specific to IV 37 C Cuts (5 mm) of lung 2 h HBSS with 0,2 % (w/v) of Protease XIV (Sigma) Ficoll HistoPaque 1083 gradient PROTOCOL : 10 7 c/ml, RPMI 10 % FCSi Flat-bottomed plate 72 h prot-h5n2 : 1 5 µg/ml Erythrocytes Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : EID 50 LP H5N2 Oculo/nasal inoculation 10-day-old SPF chickens (n = 5)

Flat-bottomed plate 72 h prot-h5n2 : 1 5 µg/ml Rauw F. et al. (2011).

36 Tracheal CMI specific to IV 37 C Cuts (5 mm) of trachea 2 h HBSS with 0,2 % (w/v) of Protease XIV (Sigma) + 0,02 % (w/v) of DNase I (Sigma) PROTOCOL : 10 7 c/ml, RPMI 5 % FCSi Flat-bottomed plate 72 h prot-h5n2 : 1 5 µg/ml Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : EID 50 LP H5N2 Oculo/nasal inoculation 10-day-old SPF chickens (n = 5)

37 Field situation: Goal of vaccination Infection Resistance to infection Clinical signs, mortality, egg drop, growth retardation.. Clinical protection Excretion (oral and cloacal) Reduction of shedding 37

_2_ 3 _2_ 2 _ NDV HI Titre _2 11 _2 10 Inactivated vaccine _2_ 9 _2_ 8 2 7 _2 6 _2_ 5 _2 4 _2_ 3 _2 2 _

38 Shedding reduction Westbury et al. (1984) Excretion 3 days Excretion 10 days Excretion 14 days _2 11 _2 10 2_ 9 _ _2_ 6 _2_ 5 _2_ 4 _2_ 3 _2_ 2 _ NDV HI Titre _2 11 _2 10 Inactivated vaccine _2_ 9 _2_ _2 6 _2_ 5 _2 4 _2_ 3 _2 2 _ Clinical protection llan et al. (1978) 0 % egg drop in layers Egg drop 0 % symptoms (resp., nerv., dig.) Clinical signs 0 % mortality Clinical signs 100 % mortality Mortality 38

39 O.D. O.D. O.D. 1) NDV vaccination: 1st experiment o Day-old o SPF or MD positive o Vitapest or Vitapest + HVT-ND o Follow-up of immune responses from to 2 to 5 week of age 2,5 2,0 1,5 Humoral immunity CMI (spleen) 1,8 1,6 1,4 1,2 1,0 Local immunity in the digestive tract 1,0 0,5 0, Weeks post- vaccination Negative (SPF) Negative (Conv) Cevac Vitapest L ND (SPF) Cevac Vitapest L ND (Conv) rhvt-nd / Cevac Vitapest L ND (Conv) Weeks post- vaccination Negative (SPF) Negative (Conv) Cevac Vitapest L ND (SPF) Cevac Vitapest L ND (Conv) rhvt-nd / Cevac Vitapest L ND (Conv) 0,8 0,6 0,4 0,2 0, Weeks post- vaccination Negative (SPF) Negative (Conv) Cevac Vitapest L ND (SPF) Cevac Vitapest L ND (Conv) rhvt-nd / Cevac Vitapest L ND (Conv)

shedule : Nobilis Clone 30 (Intervet) at

40 2d experiment on conventional layer chickens (MD+) 18 ED (+) One-day-old rhvt-nd vaccine Vectormune ND Live ND vaccine Cevac Vitapest L Eggs from Isa Brown 38-week-old breeder flock which received the following ND vaccination shedule : Nobilis Clone 30 (Intervet) at 4 and 8-week-old Nobilis Newcavac (Intervet) at 16- week-old 4 groups : Negative (non-vaccinated) Live ND rhvt-nd in ovo rhvt-nd/live ND 2 weeks Immunity

41 HI titre (log2) O.D. Humoral immunity HI titre B BC C B 3,0 2,5 2,0 1,5 1,0 0,5 NDV specific IgG ELIS B B B 2 2 weeks postchallenge Times Negative Live ND rhvt-nd rhvt-nd/live ND 0,0 2 weeks postchallenge Weeks post-vaccination Negative Live ND rhvt-nd rhvt-nd/live ND Presence of MD until the 4 th week of age ctive humoral immunity well established : t the 4 th week pv in live ND and rhvt-nd/live ND groups t the 5 th week pv in rhvt-nd group No statistical difference between vaccinated groups at 5 weeks pv

42 S.I. Cell-mediated immunity B B 0 2 weeks postchallenge Weeks post-vaccination Negative ND live rhvt-nd rhvt-nd/live ND Cell-mediated immunity from the 3 rd week pv No statistical difference between vaccinated groups

43 O.D. HLT NDV specific IgG in tears 3,0 2,5 2,0 1,5 B 1,0 0,5 B B 0, Weeks post-vaccination Negative Live ND rhvt-nd rhvt-nd/live ND Presence of MD in tears until the 4 th week of age ctive lachrymal immunity well established : t the 4 th week pv in live ND and rhvt-nd/live ND groups t the 5 th week pv in rhvt-nd group

44 O.D. GLT NDV specific IgG in duodenum 2,0 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 B B 0, Times Negative Live ND rhvt-nd rhvt-nd/live ND Presence of MD in digestive tract until the 4 th week of age Earlier and stronger local antibody-mediated immunity in rhvt- ND/live ND group (P < 0.05) since the 4 th week pv

45 Conclusions Most classical attenuated/inactivated vaccines are satisfactory in laboratory conditions but have some limitations in the field Improved immunological parameters can correlate with improved protection New technology vaccines may solve issues related to classical vaccines w/o negative effect (efficacy, safety, production, mass administration, interference of MD, DIV) This should allow the development on purpose of safer and more efficient vaccination programmes in the future

46 10 years Yannick Gardin Vilmos Palya Thanks! Bénédicte Lambrecht Fabienne Rauw CEV-CERV collaboration Scientific promoters CELOVC project MUCOVC project HVTIMMUNOVC project 2015 Sophie Lemaire Martine Gonze Technical staff Eva Ngabirano PhD PUBLICTIONS : Rauw F. et al. (2007). Journal of Interferon and Cytokines, 27 : Rauw F. et al. (2009). Vaccine, 27 : De Vriese et al. (2010). vian diseases, 54, Rauw F. et al. (2010). Veterinary Immunology and Immunopathology, 134 : Rauw F. et al. (2010). Vaccine, 28 : Rauw F. et al. (2011). Vaccine, 29 ; Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : Rauw F. et al. (2012). vian Diseases, 56:

47 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