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How ND vaccines stimulate bird’s immune system and
immunological specificity of rHVT-F vector vaccine
F.Rauw
Avian Virology and Immunology Unit
28-30th April 2015 CEVA Poultry Vaccinology Sumit_Budapest, Hungary
Primary Lymphoid organs in chicken
o The Bursa of Fabricius (BF) :
• This organ is unique to bird
• Regulates the development of the humoral compartment of the immune system
• Essential for the development of B-cells (≈ bone marrow in mammals) (Ig gene diversification and amplification). Only 5% of the total cell numbers survive to form the mature B-cell population. This minority of cells start to emigrate out of bursa around hatching
o The Thymus :
• Regulates the development of the cellular compartment of the immune system
• Essential for the development of T-cells (mainly TH). In chicken, all T-cells originate in thymus and they emigrate to form the peripheral cell pool
o Bursa of Fabricius and thymus regress with onset of sexual maturity.
Thymus
Bursa
Peripheral Lymphoid organs in chicken
o Chickens have no lymph nodes (≈ the primary site of antigen presentation in mammals).
o Spleen : important for antigen processing and antibody production after hatching. After the involution of the thymus and the bursa, spleen becomes as a primary organ, supplier of lymphoid cells for others peripheral organs.
o Others peripheral organs : lymphoid tissues associated to the harderian gland and to the digestive, respiratory and reproductive tracts
o Day-old chick = fully immunocompetent but not fully immunomature (takes 2 weeks)
Thymus
Bursa
Harderian gland (HG)
Spleen
Two types of immune responses
o Innate - Natural - Non-specific - Not acquired immunity • Epithelial barriers (skin and mucous membranes)
• Phagocytic cells : granulocytes, macrophages, dendritic cells (DTC)
• Natural killer cells (NK )
• Plasma proteins
Very fast, not specific to pathogens and lack of memory
7/05/2015 4
Two types of immune responses
o Innate - Natural - Non-specific - Not acquired immunity • Epithelial barriers (skin and mucous membranes)
• Phagocytic cells : granulocytes, macrophages, dendritic cells (DTC)
• Natural killer cells (NK )
• Plasma proteins
Very fast, not specific to pathogens and lack of memory
o Adaptive - Specific - Acquired immunity • B-cells : immunoglobulins (IgG , IgM and IgA)
• T-cells : cytokines (IFN-g, Il-2, IL-4, IL-13 …)
Specificity , diversity and memory
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Bridging
Adaptative immunity : 5 phases
o Properties : Specificity , diversity and memory
7/05/2015 6
Adaptative immune response in chickens
Chickens respond to vaccination or infection by developing humoral, cellular and mucosal immune responses specific to antigens
At systemic level :
• Humoral immunity
• Cell-mediated immunity
At local level :
• Mucosal immunity : • Antibody-mediated immunity • Cell-mediated immunity
Systemic and local immune response in chickens
Local immunity in the digestive tract
Local immunity in the respiratory tract
Local immunity in the head
Humoral immunity
Cell-mediated immunity
Local immunity in the reproductive tract
Adaptative immune response in chickens
Chickens respond to vaccination or infection by developing humoral, cellular and mucosal immune responses specific to antigens
How is it induced ? How is it acting ? How is it measurable ? Which types of results ?
At systemic level :
• Humoral immunity
• Cell-mediated immunity
At local level :
• Mucosal immunity : • Antibody-mediated immunity • Cell-mediated immunity
Immune response in chickens
Chickens respond to vaccination or infection by developing humoral, cellular and mucosal immune responses specific to antigens
o Humoral immunity : • B-cells
• Measured by detection of specific antibodies in sera (HI, seroneutralisation, ELISAs)
Humoral immunity : antibody production
7/05/2015 11
IgM IgG
B
Antigen-specific binding
Primary response (vaccination /infection )
Plasmocytes
Humoral immunity : antibody production
7/05/2015 12
IgM IgG
B
Secondary response (boost or infection post-vaccination)
Primary response (vaccination /infection )
Antigen-specific binding
Plasmocytes
Humoral immunity : antibody production
7/05/2015 13
IgM IgG
B
Secondary response (boost or infection post-vaccination)
Primary response (vaccination /infection )
Plasmocytes
Antigen-specific binding
Humoral immunity : antiviral activity
7/05/2015 14
Blocking viral entry and fusion
NDV
By macrophage
Antibody-depended cell-mediated cytotoxicity by NK cells, macrophages and monocytes
HN and F-NDV Antibodies against F or HN induced by
vaccination
Humoral immunity : detection
Humoral immunity
Jugular vein
Serum
ELISA
Specific IgG – IgM – IgA - Igtot
HI
HN-specific Ig
Seroneutralisation
Neutralizing Ig
IgG or IgY
IgM IgA
NDV-specific IgG ELISA
Purified whole NDV Saturation
Mouse anti-Chicken IgG - BIOT
NDV-specific IgG
TMB substrat
A450nm
Streptavidine-POD
0,0
0,5
1,0
1,5
2,0
2,5
1 2 3 4 5
Weeks of age
Negative Tracheotropic ND vaccine Enterotropic ND vaccine
Day-old SPF chickens
Occulo/nasal inoculation
1 dose attenuated Tracheotropic or
Enterotropic ND vaccine
Rauw F. et al. (2010). Vaccine, 28 : 823-833
NDV-specific IgM ELISA
NDV-specific IgM
Purified whole NDV
Mouse IgG1 anti-NDV - BIOT
Mouse anti-Chicken IgM Saturation
TMB substrat
A450nm
Streptavidine-POD
0,0
0,5
1,0
1,5
2,0
2,5
1 2 3 4 5
Weeks of age
Negative Tracheotropic ND vaccine Enterotropic ND vaccine
Day-old SPF chickens
Occulo/nasal inoculation
1 dose attenuated Tracheotropic or
Enterotropic ND vaccine
Rauw F. et al. (2010). Vaccine, 28 : 823-833
NDV-specific IgA ELISA
NDV-specific IgA
Purified whole NDV
Mouse IgG1 anti-NDV - BIOT
Mouse anti-Chicken IgA
TMB substrat
A450nm
Streptavidine-POD
Saturation
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1 2 3 4 5
Weeks of age
Negative Tracheotropic ND vaccine Enterotropic ND vaccine
Day-old SPF chickens
Occulo/nasal inoculation
1 dose attenuated Tracheotropic or
Enterotropic ND vaccine
Rauw F. et al. (2010). Vaccine, 28 : 823-833
Immune response in chickens
Chickens respond to vaccination or infection by developing humoral, cellular and mucosal immune responses specific to antigens
o Humoral immunity : • B-cells
• Measured by detection of specific antibodies in sera (HI, seroneutralisation, ELISAs)
o Cell-mediated immunity (CMI) :
• T-cells : T helpers cells (TH cells, CD4 cells ) and cytotoxic T-cells (CTL, CD8 cells)
• Measured by proliferation tests and cytokines production after specific (ex vivo) recall on lymphocytes
Cell-mediated immunity : fonctions
The major goal of cell-mediated immunity (CMI) is to destroy infected cells or extracellular pathogens
CD4
APC
CD8
o Antigen presenting cells (APCs) :
• Macrophages, B-cells and dendritic cells (DC)
• Able to capture pathogens and present them to T-cells, in the context of the major histocompatibility complex (MHC)
Ag Capture
APC APC
Ag presentation
Cell-mediated immunity : fonctions
The major goal of cell-mediated immunity (CMI) is to destroy infected cells or extracellular pathogens
Ag Capture
APC APC
CD4
APC
CD8
o Antigen presenting cells (APCs) :
• Macrophages, B-cells and dendritic cells (DC)
• Able to capture pathogens and present them to T-cells, in the context of the major histocompatibility complex (MHC)
o CD4 TH – T helper cells : recognize foreign antigen expressed by the APCs in the context of MHC-II and in response, release cytokines (TH1-TH2)
Ag presentation
CD4
MHC-II on APCs
Ag
Cell-mediated immunity : fonctions
The major goal of cell-mediated immunity (CMI) is to destroy infected cells or extracellular pathogens
Ag Capture
APC APC
CD4
APC
CD8
o Antigen presenting cells (APCs) :
• Macrophages, B-cells and dendritic cells (DC)
• Able to capture pathogens and present them to T-cells, in the context of the major histocompatibility complex (MHC)
o CD4 TH – T helper cells : recognize foreign antigen expressed by the APCs in the context of MHC-II and in response, release cytokines (TH1-TH2)
o CD8 CTL – cytotoxic T cells : recognize foreign antigen presented by infected cells in the context of the MHC-I and are able to lyse infected cells that present viral antigens
Ag presentation
CD4
MHC-II on APCs
Ag
CD8
MHC-I on all cells
Ag
IFN-g, IL-2
Cell-mediated immunity : fonctions
7/05/2015 23
APC
Il-4, IL-13
IFN-g, IL-2
Ag
APC
CD8
MHC-I
Ag
Infected cells
LYSIS Macrophages
activation
Ag
CD8 CTL– cytotoxic T-cells
Perforins, granzyme
CD4
MHC-II
Ag
IFN-g
Antibody production
Memory TH -cells
Memory CTL cells
Memory B-cells
CD8
MHC-I
Ag
CD4 TH - Helper T-cells
Cell-mediated immunity : detection
Cell-mediated immunity
Jugular vein
Spleen
Spleen
Peripheral blood
Cell-mediated immunity : detection
Cytokines production IFN-g
In vivo Specific T-lymphocytes
(CD4 - CD8 T-cells)
Viral proteins
+ Antigen recall activation
Isolation
Lymphocytes
• Spleen
• Blood
Antigens
(pathogens, vaccines)
T-cells
Specific T-lymphocytes (CD4 - CD8 T-cells)
Memory immunity
IFN-g MEASUREMENT
ELISA Enzyme-linked
immunosorbent assay
Ex vivo
Cytokines production Proliferation
IFN-g
NDV-specific CMI TH1 : ChIFNg production
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IFN
-g
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Alsever Heparin Alsever Heparin Alsever Heparin Alsever Heparin Alsever Heparin Alsever Heparin
1 µg/ml 5 µg/ml 10 µg/ml 1 µg/ml 5 µg/ml 10 µg/ml
48h 72h
Anticoagulant, gp-NDV concentration and culture conditions
O.D
.
Negative
Enterotrocpic live ND vaccine
Enterotropic live ND vaccine and challenge
0.0
0.5
1.0
1.5
2.0
2.5
3.0
2 % ChSi 10 % ChSi
PMA/Iono (1 µg/ml)
IFN
-g
Day-old SPF chickens
Occulo/nasal inoculation
1 dose attenuated Tracheotropic or
Enterotropic ND vaccine
In the spleen
In the peripheral blood
Rauw F. et al. (2010). Vaccine, 28 : 823-833
IFN-g, IL-2
Cell-mediated immunity : fonctions
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APC
Il-4, IL-13
Macrophages activation
Ag
CD4
MHC-II
Ag
Antibody production
Memory B-cells
CD4 TH - Helper T-cells
CMI TH2 : ChIL-4 & ChIL-13
Monoclonal Ab specific to ChIL-4 and to ChIL-13
Polyclonal Ab specific to ChIL-4
and to ChIL-13
rChIL-4 and rChIL-13 produced in E.coli
Bac-ChIL-4 and Bac-ChIL-13 produced in Sf9 insect cells
ELISA Enzyme-linked
immunosorbent assay
ELISPOT Enzyme-linked
immunosorbent spot
pCIneo-ChIL-4 and pCIneo-ChIL-13 produced in
mammalian COS cells
(kindly provided by P.Kaiser)
Cell-mediated immunity : fonctions
29
Ag
APC
CD8
MHC-I
Ag
Infected cells
LYSIS
CD8 CTL– cytotoxic T-cells
Perforins, granzyme
Memory CTL cells
CD8
MHC-I
Ag
Immune response in chickens
Chickens respond to vaccination or infection by developing humoral, cellular and mucosal immune responses specific to antigens
o Humoral immunity : • B-cells
• Measured by detection of specific antibodies in sera (HI, seroneutralisation, ELISAs)
o Cell-mediated immunity (CMI) :
• T-cells : T helpers cells (TH cells, CD4 cells ) and cytotoxic T-cells (CTL, CD8 cells)
• Measured by proliferation tests and cytokines production after specific (ex vivo) recall on lymphocytes
o Mucosal immunity :
• Immunity at mucosal surfaces : major route of entry for many infectious agents into the body : local immunity in the head, the digestive, respiratory and reproductive tracts
• Local antibody- and cell-mediated immunity = first line of defense against pathogens
• Poorly investigated in poultry so far
• Difficulties and labor-intensiveness of collecting samples
Mucosal response induction
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Lumen
Epithelium
Lamina propria
Specific antibody response
Specific cellular
response
Local immunity in the head
Local immunity in the head
Harderian gland (HG)
Conjunctiva
Tears
Antibody
NDV-specific lachrymal antibody-mediated immunity
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
1 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 IgA NDV specific IgM
Time post-vaccination and Ig isotypes
O.D
.
Negative
Tracheotropic live ND vaccine
Enterotropic live ND vaccine
Centrifugation -20°C NDV-specific IgG/M/A ELISA
Rauw F. et al. (2010). Vaccine, 28 : 823-833
Tears
Day-old SPF chickens
Occulo/nasal inoculation
1 dose attenuated Tracheotropic or
Enterotropic ND vaccine
Local immunity in the digestive tract
Local immunity in the digestive tract
Ileum Jejunum
Caecal tonsils (CT)
Local immunity in the digestive tract
Local immunity in the digestive tract
Duodenum
Antibody CMI
Local NDV-specific antibody-mediated immunity in the digestive tract
Rauw F. et al. (2010). Vaccine, 28 : 823-833
Ex vivo tissues culture (72h)
Centrifugation -20°C NDV-specific IgG/M/A ELISA
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
1 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 IgA NDV specific IgM
Time post-vaccination and Ig isotypes
O.D
.
Negative
Tracheotropic live ND vaccine
Enterotropic live ND vaccine
Day-old SPF chickens
Occulo/nasal inoculation
1 dose attenuated Tracheotropic or
Enterotropic ND vaccine
Local NDV-specific CMI TH1 (ChIFNg) in the digestive tract
Cuts (5 mm) of duodenal tissues
37 °C
30 min
LPL : Lamina Propria Lymphocytes
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4 5 6 7 4 5 6 7
1 µg/ml gp-NDV 5 µg/ml gp-NDV
Weeks post-vaccination (challenge at 4 weeks p.v.)
O.D
.
Negative
Enterotropic live ND vaccine
Enterotropic live ND vaccine with challenge
Rauw F. et al. (2010). Veterinary Immunology and Immunopathology, 134 : 249-258
Washes and enzymatic digestion
Viral proteins
+ Antigen recall
activation
IFN-g
Local immunity in the respiratory tract
Local immunity in the respiratory tract
Trachea
Lung
Local immunity in the respiratory tract
Local immunity in the respiratory tract
Trachea
Lung
Trachea
Lung
Antibody CMI
Local NDV-specific antibody-mediated immunity in the respiratory tract (lung)
NDV-specific IgG/M/A ELISA Euthanasia by
i.p injection of Nembutal
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
3 5 3 5 3 5
IgG IgA IgM
Isotypes and weeks post-vaccination
O.D
.
Negative
Tracheotropic live ND vaccine
Enterotropic live ND vaccine
Rauw F. et al. (2010). Vaccine, 28 : 823-833
Centrifugation 1 min 20 000 x g
10x concentration
Amicon Ultra-centrifugal filter unit
with ultra-100 kDa membrane
Day-old SPF chickens
Occulo/nasal inoculation
1 dose attenuated Tracheotropic or
Enterotropic ND vaccine
Washes
Local AIV-specific CMI TH1 (ChIFNg) in the respiratory tract (lung)
Cuts (5 mm) of lung
37 °C
2 h
Ficoll HistoPaque
1083 gradient
Erythrocytes
Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : 27-37
10-day-old SPF chickens
106 EID50 LP H5N2 Oculo/nasal inoculation
Washes and enzymatic digestion
Viral proteins
+
Antigen recall
activation
IFN-g
Local AIV-specific CMI TH1 (ChIFNg) in the respiratory tract (trachea)
37 °C
2 h
Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : 27-37
Cuts (5 mm) of trachea
10-day-old SPF chickens
106 EID50 LP H5N2 Oculo/nasal inoculation
Washes and enzymatic digestion
Viral proteins
+
Antigen recall
activation
IFN-g
Local immunity in the reproductive tract
Local immunity in the reproductive tract
Local NDV-specific immunity in the reproductive tract
2-week-old 28 weeks
of age
Day-old 11-week-old
Attenuated ND vaccine
17-week-old
Inactivated ND vaccine
32 weeks of age
Local antibody-mediated immunity
Local CMI
Immune response in chickens
Chickens respond to vaccination or infection by developing humoral, cellular and mucosal immune responses specific to antigens
o Humoral immunity :
• Development of virus specific ELISAs to detect IgG, IgM and IgA specific [Rauw et al., 2009, 2010]
o Cell-mediated immunity (CMI) :
• Tools to measure TH CMI :
• TH1 : ChIFNg production after antigen-specific (ex vivo) recall on lymphocytes :
• In the spleen [Lambrecht et al., 2004, Rauw et al., 2009, 2010]
• In the peripheral blood [Rauw et al., 2010]
• TH2 : ChIL-4 and ChIL-13 production after antigen-specific (ex vivo) recall on lymphocytes : in progress
• Tools to measure CTL : in progress
o Mucosal immunity :
• Tools to measure lachrymal, respiratory and digestive immunity [Rauw et al., 2009, 2010a, 2010b, 2011]
• Reproductive tract : in progress NDV – AIV – IBDV
Transfert of passive immunity in chickens
o Maternal Derived Antibodies (MDA) or Passive Immunity is the naturally transfer of antibodies from immunized breeder hens to the progeny through the egg
o To protect young chicks against pathogens during a period (1-2 weeks and less than 4 weeks) where their immune system is not fully mature.
How MDA are transferred from the hen to the progeny ?
1. MDA transfer from the hen to the egg
• IgG(Y) are secreted by the chicken ovarian into the developing ova or egg yolk
• IgA and IgM are produced locally in the magnum in the oviduct and transfer to the albumen
Transfert of passive immunity in chickens
How MDA are transferred from the hen to the progeny ?
1. MDA transfer from the hen to the egg
2. MDA transfer from the egg to the progeny
IgG(Y) :
Are transferred from the egg yolk to the offspring via the embryonnic circulation
Only IgGs are able to cross the intestinal barrier through the embryonic circulation
The transfer starts from 7 ED and reaches its maximum rate at 17-18 ED (3 to 4 days before hatching)
Transfert of passive immunity in chickens
How MDA are transferred from the hen to the progeny ?
1. MDA transfer from the hen to the egg
2. MDA transfer from the egg to the progeny
IgG(Y) :
Are transferred from the egg yolk to the offspring via the embryonnic circulation
Only IgGs are able to cross the intestinal barrier through the embryonic circulation
The transfer starts from 7 ED and reaches its maximum rate at 17-18 ED (3 to 4 days before hatching)
IgA and IgM in albumen are ingested by chick and protect the intestine.
MDA interference with vaccination
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40
% p
rote
ctio
n
Time post vaccination (days)
Efficiency of attenuated ND vaccine administered to day-old chicks
Control (SPF)
Control (NDV MDA)
Vaccinated (SPF)
Vaccinated (NDV MDA)
Bennejean et al., 1978, Avian Pathology, 15-27
SPF chickens
Conventional chickens
Early NDV vaccination
Rauw F. et al. (2009). Vaccine, 27 : 3631-3642
To study the impact of MDA
o Development of an artificial model mimicking natural passive immunity in SPF chicken by injection of a NDV- and/or AIV-specific polyserum :
• Production of NDV- and AIV-specific polyserum :
7/05/2015 50
Attenuated vaccine (106 EID50) by oculo-nasal route
Day-old 4-week-old
Inactivated vaccine
2-week-old
NDV-specific polyserum
Serum HI titer = 12 log2
3 weeks
Lardinois. et al. (2014). Avian Pathology, 143 : 118-124
To study the impact of MDA
o Development of an artificial model mimicking natural passive immunity in SPF chicken by injection of a NDV- and/or AIV-specific polyserum :
• Production of NDV- and AIV-specific polyserum :
• Model of artificial passive immunity induction :
7/05/2015 51
Attenuated vaccine (106 EID50) by oculo-nasal route
Day-old 4-week-old
Inactivated vaccine
2-week-old
NDV-specific polyserum
Serum HI titer = 12 log2
3 weeks
AIV-specific polyserum
6-week-old
Inactivated purified virus
with CFA
8-week-old
Inactivated purified virus
with IFA
11-week-old
HPAIV (106 EID50) by oculo-nasal
route
Serum HI titer = 8 log2
10 days
200 µl in egg yolk at 14 ED
200 µl at day-old by im route
Lardinois. et al. (2014). Avian Pathology, 143 : 118-124
Research on NDV specific immunity
WHY ?
o By improving immunological parameters and further characterizing the chicken immune response elicited by current ND vaccines, a correlation with clinical protection and shedding reduction could be done
o Good parameters of protection could be established
o Challenge experiments could be reduced
To improve the efficacy of ND vaccination programs
Live vector vaccines
o Definition :
• A live vector vaccine is a vaccine that uses an attenuated virus (vector) to transport pieces of the virus (pathogen) in order to stimulate an immune response against this virus
o Development and production :
• To determine the protective gene to introduce in the virus vector
• To insert the protective gene into the genome of the vector (herpesvirus, poxvirus, adenovirus…)
• To produce the recombinant vector = live vector vaccine
NDV – AIV – IBDV
ND Live vector vaccine : rHVT-F
NDV F
gene
Homologous HVT
sequence
Pec promoter
SV40 polyA
Homologous HVT sequence
HVT genome
Live vector vaccines
o Definition :
• A live vector vaccine is a vaccine that uses an attenuated virus (vector) to transport pieces of the virus (pathogen) in order to stimulate an immune response against this virus
o Development and production :
• To determine the protective gene to introduce in the virus vector
• To insert the protective gene into the genome of the vector (herpesvirus, poxvirus, adenovirus…)
• To produce the recombinant vector = live vector vaccine
o Validation :
• Being intracellular, live vector vaccine should be able to prime a humoral response, to induce a strong cellular and mucosal immune responses against the inserted gene and strong B & T-cell memory immunity
NDV – AIV – IBDV
1 2 IL-4, IL13
Immunity induced by live vector vaccine
7/05/2015 56
B cells
First in vivo replication of live vector vaccine by
targeted cells
Priming: Viral antigen capture and
presentation by APCs - Th cells activation - Cytokine production - CTL and B activation
IL-2, IFN-g
Effective responses: - Ab production - CTL activity
Induction of memory immunity
Vector vaccine
IL-4
IL-13
1
1
2
Viral antigen
2
1 2 IL-4, IL13
Immunity induced by live vector vaccine
7/05/2015 57
First in vivo replication of live vector vaccine by
targeted cells
Priming : Viral antigen capture and
presentation by APCs - TH cells activation - Cytokine production - CTL activation - B cells activation
B cells
IL-2, IFN-g
Effective responses: - Ab production - CTL activity
Induction of memory immunity
Vector vaccine
IL-4
IL-13
1
1
2
Viral antigen
2
1 2 IL-4, IL13
Immunity induced by live vector vaccine
7/05/2015 58
First in vivo replication of live vector vaccine by
targeted cells
Priming : Viral antigen capture and
presentation by APCs - TH cells activation - Cytokine production - CTL activation - B cells activation
IL-2, IFN-g
Effective responses : - Ab production - CTL activity
Induction of memory immunity
Vector vaccine
IL-4
IL-13
1
1
2
Viral antigen
2
B cells
Immunity induced by rHVT-ND vaccine
Day-old SPF chickens
One dose of rHVT-ND Subcutaneous inoculation
Every 2-3 days or every week
6-10 weeks
Serum
Humoral immunity
HI and ELISA-IgG/M/A tests
CMI
Spleen
ChIFNg production
Local antibody-mediated immunity
Duodenum
ELISA-IgG/M/A tests
Lung
HVTIMMUNOVAC-ND-1,2,3,4,5,6,7,8
NDV-specific IgG ELISA
*
*
* * *
* * *
Day-old SPF chickens
1 dose of rHVT-ND Subcutaneous inoculation
*
* * * *
* *
NDV-specific IgM ELISA
*
* *
*
* * *
*
*
Day-old SPF chickens
1 dose of rHVT-ND Subcutaneous inoculation
*
* *
*
NDV-specific IgA ELISA
*
* *
* * * * *
* * *
* *
*
*
Day-old SPF chickens
1 dose of rHVT-ND Subcutaneous inoculation
ND vaccination schedule on conventional layer chickens
7/05/2015 63
Oculo-nasale inoculation
Day-old conventionnal layer chickens
Live ND vaccine 1 dose
ND vaccination schedules on conventional layer chickens
7/05/2015 64
+ Oculo-nasale
inoculation
Day-old conventionnal layer chickens
Live ND vaccine 1 dose
rHVT-F vaccine 1 dose
Subcutaneous inoculation
• protection : • 90% to 100 % (challenge at
5 weeks) • virus shedding
• OP swabs: 60% to 50% • Cl. swabs: 80% to 40%
ND vaccination schedules on conventional layer chickens
rHVT-ND/Live ND vaccines
Live ND vaccine
ND vaccination schedules on conventional layer chickens
7/05/2015 66
Oculo-nasale inoculation
Day-old conventionnal layer chickens
Live ND vaccine 1 dose
rHVT-F vaccine 1 dose
Inactivated ND vaccine 1 dose
Subcutaneous inoculation
Subcutaneous inoculation
ND vaccination schedules on conventional layer chickens
7/05/2015 67
+ Oculo-nasale
inoculation
Day-old conventionnal layer chickens
Live ND vaccine 1 dose
rHVT-F vaccine 1 dose
Inactivated ND vaccine 1 dose
Subcutaneous inoculation
Subcutaneous inoculation
ND vaccination schedules on conventional layer chickens
7/05/2015 68
+ Oculo-nasale
inoculation
Day-old conventionnal layer chickens
Live ND vaccine 1 dose
rHVT-F vaccine 1 dose
Inactivated ND vaccine 1 dose
Subcutaneous inoculation
Subcutaneous inoculation
+
• protection : • 90% to 100 % (challenge at
5 weeks) • virus shedding
• OP swabs: 60% to 50% • Cl. swabs: 80% to 40%
ND vaccination schedules on conventional layer chickens
• protection : • 90% to 100 % (challenge at
10 weeks) • virus shedding :
• OP swabs: 90% to 40% • Cl. swabs: 20% to 0%
rHVT-ND/Live ND vaccines
Live ND vaccine
rHVT-ND/Live ND vaccines Inactivated ND/Live ND
vaccines
Thanks !
2003 2007 2012
CELOVAC project MUCOVAC project HVTIMMUNOVAC project
2015
12 years
CEVA-CERVA collaboration
Vilmos Palya
Yannick Gardin
Scientific promoters
Technical staff Eva Ngabirano
Sophie Lemaire
Martine Gonze
Fiona Ingrao (PhD)
PUBLICATIONS : • Rauw F. et al. (2007). Journal of Interferon and Cytokines, 27 : 11-118. • Rauw F. et al. (2009). Vaccine, 27 : 3631-3642. • De Vriese et al. (2010). Avian diseases, 54, 246-252. • Rauw F. et al. (2010). Veterinary Immunology and Immunopathology, 134 : 249-258. • Rauw F. et al. (2010). Vaccine, 28 : 823-833. • Rauw F. et al. (2011). Vaccine, 29 ; 2590-2600. • Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : 27-37 • Rauw F. et al. (2012). Avian Diseases, 56: 913-922 • Rauw F. et al, (2014). Avian Pathology, 43:26-36 • Rauw F. et al. (in press). Avian Pathology
Bénédicte Lambrecht
Thierry van den Berg
Publications
o Rauw F., Gardin Y., Palya V., Gonze M., Lemaire S., van Borm S., van den Berg T. & Lambrecht B. (2009). « Humoral, cell-mediated and mucosal immunity induced by oculo-nasal vaccination of one-day-old SPF and conventional layer chicks with live Newcastle Disease vaccines ». Vaccine, 27 : 3631-3642.
o Rauw F., Gardin Y., Palya V., Anbari S., Gonze M., Lemaire S., van den Berg T. & Lambrecht B. (2010). « The positive adjuvant effect of chitosan on antigen-specific cell-mediated immunity after chickens vaccination with live Newcastle disease vaccine ». Veterinary Immunology and Immunopathology, 134, 249-258.
o Rauw F., Gardin Y., Palya V., Anbari S., Lemaire S., Boschmans M., van den Berg T. & Lambrecht B. (2010). « Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old ». Vaccine, 28 : 823-833.
o Rauw F., Anbari S., van den Berg T. & Lambrecht B. (2011). « Measurement of systemic and local respiratory cell-mediated immunity after influenza infection in chickens ». Veterinary Immunology and Immunopathology, 143, 27-37.
o Rauw F., Palya V., Van Borm S., Welby S., Tatar-Kis T., Gardin Y., Moore Dorsey K., Aly M.M., Hassan M.K., Soliman, M.A., Lambrecht B. & van den Berg T. (2011). « Further evidence of antigenic drift and protective efficacy afforded by a recombinant HVT-H5 vaccine against challenge with two antigenically divergent Egyptian clade 2.2.1. HPAI H5N1 strains ». Vaccine, 29, 2590-2600.
o Rauw F., Palya V., Gardin Y., Tatar-Kis T., Moore Dorsey K., Lambrecht B. & van den Berg T. (2012). « Efficacy of rHVT-AI vector vaccine in broilers with passive immunity against challenge with two antigenically divergent egyptian clade 2.2.1 HPAI H5N1 strains ». Avian Diseases, 56, 913-922.
o Rauw F., Gardin Y., Palya V., van den Berg T. & Lambrecht B. (2014). « Combination of attenuated vaccine at day-old with rHVT is more protective against NDV challenge than combination with inactivated vaccine ». Avian Pathology, 43 (1), 26-36.
o Rauw F., Van Borm S., Welby S., Ngabirano E., Gardin Y., Palya V. & Lambrecht B. (in press). « Quantification of rHVT-F genome load in feather follicles by specific real-time qPCR as indicator of NDV-specific humoral immunity induced by day-old vaccination in SPF chickens ». Avian Pathology,.
