Axis magazine Vectormune ND

INSTALL THE ULTIMATE ANTIVIRUS

Vectormune ND速 reduces Newcastle Disease virus shedding, with maximum protection and no side effects.

CONTENTS I. INTRODUCTION................................................................................................................... 2 II. VECTORMUNE® ND........................................................................................................ 3 III. ONSET OF IMMUNITY................................................................................................ 4 IV. DURATION OF IMMUNITY.................................................................................... 5 V. REDUCTION OF SHEDDING............................................................................. 6 VI. SPECTRUM OF PROTECTION......................................................................... 7 VII. COMPATIBILITY STUDIES....................................................................................... 8 VIII. COMPARISON BETWEEN VECTORMUNE® ND AND OTHER rHVT-F VACCINE................................................................... 10 IX. REFERENCES......................................................................................................................... 11

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I. INTRODUCTION Newcastle Disease (ND) is a highly contagious disease which varies widely in the type and severity of symptoms. It is one of the main barriers for international trade of poultry and poultry products and the economic impact of this disease is enormous. However, despite being recognized for almost 90 years, being caused by one single serotype of paramyxovirus and having commercially available very efficient vaccines, ND still challenges veterinarians and farmers all around the world. In Asia, particularly, this disease is a constant threat for the profitability of the poultry industry and its proper prevention has become a crucial part of the biosecurity program of any farm.

Vaccination is an important part of the prevention program against this disease. However, in ND endemic countries, day old chicks carry high levels of maternally derived antibodies that interfere with live as well as killed ND vaccines given at the hatchery up to a point where neutralization is complete and prevent any vaccine take. Furthermore, vaccinations conducted in the farms, either to avoid this early interference, or to boost previous immunizations, are very often very poorly conducted. Finally, live attenuated vaccines that are the backbone of a broiler ND vaccination program may be responsible for lesions of the upper respiratory tract, post vaccination and rolling reactions that slow down the growth, worsen flock’s uniformity and leave the chickens susceptible to other pathogens.

Within this context, a new generation of vaccines was needed and the vector technology with rHVT-F vaccines, such as Vectormune® ND, overcomes all these aforementioned hurdles.

In fact, a very significant investment was made by Ceva Animal Health to know and understand the potentialities of this new product. Scientific work inside as well as outside the company, in collaboration with independent research centers, was designed, organized and conducted. The information related to the vaccine induced immunity as well as tangible protection results far exceeded our expectations.

Today, we know that Vectormune® ND is much more than just a new ND vaccine, and it is the objective of this edition of the Axis Magazine to summarize the key studies that helped us to realize how innovative and potent this product is. But before going ahead with the reading of these studies, it is probably important to briefly present the product and summarize the most striking associated features and benefits.

II. VECTORMUNE® ND

NDV D26 PEC promotor

HVT FC 126 p/3

VECTOR

DONNOR

Vectormune® ND is a vaccine, using HVT as vector in the genome of which, the “F” gene extracted from a genotype I NDV (D26 strain) has been inserted. The HVT strain used (FC 126), its origin, the low number of passages it has undergone, the “F” insert, the insertion site, the promoter selected to ensure the expression of the F gene are all key elements explaining the uniqueness of this vaccine with most of them patented and proprietary to Ceva Animal Health so that Vectormune® ND is actually unique and cannot be compared to other rHVT-F vaccines.

The “F” (for “fusion”) protein is the epitope present on the surface of NDV, allowing it to attach to and penetrate inside of targeted cells. One can easily understand that if immunity is built up against “F”, then NDV can no longer infect cells and create damage, and this probably explains the incredibly high efficacy of Vectormune® ND. Vaccinated chickens are not only protected against clinical and economical consequences of the infection, but replication of NDV inside the chicken’s body is also hampered as indicated by a significant reduction of the shedding of the challenge virus as well as a more limited increase in antibody titer following infection.

Following several years of investigation work with Vectormune® ND, we do believe that this vaccine is more a revolution than a simple evolution. Vectormune® ND will most probably change the approach of Newcastle disease prevention in the field and soon be considered as a strong tool toward the long-term control of this important poultry disease.

III. ONSET OF IMMUNITY The onset of immunity induced by Vectormune® ND depends directly on the replication of the HVT virus (vector) and consequently on the expression of the fusion (F) gene of Newcastle Disease virus.

One-day old commercial broilers, with MDANDV measured by HI test of 3.4 log2 were vaccinated with one full dose of Vectormune® ND by subcutaneous route. Another group of day-old chicks remained unvaccinated to serve as a control group. The challenges were carried out at 2, 3, 4, and 6 weeks of age with a Malaysian isolate of NDV, genotype VII, 5 log10 EID50 /bird, nasal route (200 µl).

THE RESULTS of clinical protection are summarized in the following graphic:

Clinical protection against NDV challenge 100

% of protection

80 60 40 20 0 2

3

4

6

Age of challenges (weeks) Control

Vectormune® ND

The protection progressively increases with time reaching full protection at around 3 to 4 weeks of age. In fact, immunity can be detected as early as two weeks after vaccination. However, in order to strengthen up the protection during the first weeks of age in areas with high ND pressure, it is necessary to spray a live ND vaccine at day of age (in the hatchery) and, depending on the case, add a field booster at around 10-15 days of age is recommended.

The benefits of the association of Vectormune® ND with a live ND vaccine applied in the hatchery have already been demonstrated.

Day-old commercial broilers with MDANDV measured by HI test of 5.3 log2 were divided in two groups. The first one received Vectormune ND by subcutaneous route and Cevac Vitapest L by eye-drop route. The second group remained unvaccinated to serve as control.

All groups were challenged at 3, 4, 5 and 6 weeks of age with the Chimalhuacan velogenic NDV (Mexican reference NDV strain – genotype V) by mucosal route at the dose 5 log10 EID50 /bird.

THE RESULTS of clinical protection are summarized in the following graphic:

Clinical protection against NDV challenge 100 80

% of protection

p/4

Extracted from Study SCI-137-2011

60 40 20 0 3

4

5

6

Age of challenges (weeks) Vectormune® ND + Vitapest L

The clinical protection against early challenges was reinforced when Vectormune® ND was associated to a live ND vaccine applied at day-of-age.

Control

Extracted from DV-055-2007

As shown in these trials, the onset of immunity induced by Vectormune® ND depends on replication of the herpesvirus of turkey (HVT) hence the expression of the F gene. Single vaccination with Vectormune® ND at one day of age provided complete clinical protection against NDV challenge from 3 weeks of age. In regions where ND pressure is high, it is recommended to associate Vectormune® ND with a live ND vaccine (by spray) in the hatchery and a live booster in the farm.

IV. DURATION OF IMMUNITY One of the trends of the egg industry is to keep the layers longer in the farms. In fact, this approach has a positive economic impact on the cost of production, but the duration of immunity induced by the vaccines has being questioned. In fact, Vectormune® ND is made of Herpesvirus of Turkey as the vector. The HVT remains in the birds for the whole life and its replication “constantly boosts” the ND protection.

In order to assess the duration of immunity induced by Vectormune® ND, commercial day-old chicks of layer type with MDA titre against NDV measured by HI test of 5.5 log2 were vaccinated with Vectormune® ND by subcutaneous route. The other group remained unvaccinated to serve as control. The groups were kept in isolated rooms.

Chickens from both groups were challenged with a Malaysian isolate (D1524/1/1,2/MY/10) of NDV (genotype VII) at 3, 4, 6, 10, 15, 25, 33, 40, 55 and 72 weeks of age. All challenges were performed via the intra-nasal route with a dose of 5.0 log10 ELD50 /bird.

THE RESULTS of clinical protection are summarized in the following graphic:

Clinical protection against NDV challenge 100 100

60 60 40 40 20 20 0 2 0 3

3

4

5

Age of challenges (week) ® ND 10 4 Vectormune 6

15

25

Control 33

40

Age of challenges (weeks) Vectormune® ND

55

72

Protection from egg drop production has been as well evaluated at 33 weeks of age challenge. While control group died rapidly, no mortality, nor drop in egg production or egg shell quality default were observed in the vaccinated hens with Vectormune® ND.

Control

Results extracted from the Study SCI-159-2010

A single vaccination with Vectormune® ND at one day of age, after reaching complete clinical protection against NDV challenge at 4 weeks of age, induces 100% of clinical protection up to 72 weeks of age.

Egg drops are as well better controlled than with conventional live & killed ND vaccination program.

It allows to reduce (or even remove) the boosters with live ND vaccines commonly done during production and a better focus on infectious bronchitis control.

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% of protection % of protection

80 80

V. REDUCTION OF SHEDDING VECTORMUNE® ND OFFERS BETTER CONTROL THAN KILLED VACCINATION PROGRAM When birds carrying MDANDV are used in the trials, the differences between these technologies become even clearer.

Commercial broilers with MDANDV of 7.4 log2 mean HI titre were vaccinated with three different vaccination programs: GROUP 1: Vectormune® ND (SQ route) + Vitabron (Spray route) GROUP 2: Cevac Broiler ND K (SQ route) + Vitabron (Spray route) GROUP 3: Genotype VII killed vaccine (SQ route) + Vitabron (Spray route) The challenges were carried out at 25 days of age with a recent isolate of NDV from Indonesia (D1675/11/ID - genotype VIIa) at a dose of 5.0 lgELD50 / chick by intranasal route.

THE RESULTS of clinical protection are summarized in the following graphic:

Clinical protection against NDV challenge Extracted from Study SCI-155-2012

100

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% of protection

80

60

40

20

0 25

Age of challenges (days) Vectormune® ND

Cevac Broiler ND K

Genotype VII killed vaccine

Control

THE RESULTS of the shedding reduction are summarized in the following graphic: Each bar with different letter means that results were statistically different for a day post challenge

titer equivalent unit (lg EID 50/0,2 ml)

Shedding reduction after 25 days challenge 8 B

7

B

6 5

B

A A

A

A

4

A

3 2 1 0 Vectormune® ND

4 days post challenge oropharyngeal

Age of challenges (days)

4 days post challenge cloaca

Control

7 days post challenge oropharyngeal

7 days post challenge cloaca

As the level of MDANDV was high (7.4 log2 mean HI titre), there was a clear interference with the active immunity induced by both inactivated vaccines inducing low protection level. Shedding reduction was not monitored due to low level of protection observed in these 2 groups. On the other hand, the group vaccinated with Vectormune® ND showed good level of protection and a reduction of virus shedding compared to control birds. Therefore, the cell associated presentation of Vectormune® ND enables the vaccine to stimulate immunity despite high MDA level.

VECTORMUNE® ND OFFERS BETTER CONTROL THAN LIVE ND VACCINATION PROGRAM Commercial broilers with MDANDV of 5.75 log2 mean HI titre were vaccinated with two different vaccination programs: GROUP 1: Vectormune® ND (SQ route) GROUP 2: live enterotropic ND vaccine (Eye drop route) at D1 and D18.

The challenges were carried out at 21, 28 and 35 days of age with a t isolate of NDV from south Africa “Goose Paramyxovirus”: GPMV 171/06/ genotype VIId at a dose of 5.0 lgELD50 / chick by intranasal route.

THE RESULTS of clinical protection are summarized in the following graphic:

THE RESULTS of the shedding reduction are summarized in the following graphic:

Clinical protection against NDV challenge

Shedding reduction after 28 days challenge titer equivalent unit (lg EID 50/0,2 ml)

% of protection

100 80 60 40 20 0 21 days

28 days

35 days

Age of challenges (days) Vectormune® ND

Live ND

Control

Extracted from Study SCI-007-2011

8 7 6 5 4 3 2 1 0 Vectormune® ND

Live ND

Control

Age of challenges (days) 4 days post challenge oropharyngeal

4 days post challenge cloaca

7 days post challenge oropharyngeal

7 days post challenge cloaca

Extracted from Study SCI-007-2011

Vectormune ND induces a strong control of clinical symptoms and mortality and a better reduction of shedding of challenge virus compared to live Newcastle vaccination program. p/7

®

VI. SPECTRUM OF PROTECTION All ND viruses belong to a single serotype (PMV1), therefore by definition any NDV strain utilized to prepare a vaccine should induce protection against morbidity and mortality following challenge with any virulent NDV strains. However, with the improvement and “popularization” of molecular techniques, different genotypes of the NDV have been identified in different geographical areas and questions have been raised about the protection induced by vaccines against them. In particular, homologous vaccine strains to the local field isolate were first regarded as a promising option.

The following table summarizes all the isolates against which Vectormune® ND has been tested and protection demonstrated.

GENOTYPE

STRAIN

ICPI

TROPISM

PREVALENCE

II II

B1B1 (Hitchner B1)

0.20

not applicable

vaccine strain

Texas GB/48

1.70-1.74

Neurotropic

US reference

IV

Herts 33/56

1.88-2.00

Viscerotropic

US reference

IV

T-53

1.9

?

Russia

V

Chimalhuacan (D516/1/05 MX)

1.89

Viscerotropic

Mexico, Latin America

VII

Talal/86

1.8-1.9

?

Saudi Arabia

VIIa

D1675/11

?

Viscerotropic

Indonesia

VIIa

D1598/1/11/PH

?

Viscerotropic

Philippines

VIIb

D575/6/05 PE

?

Viscerotropic

Peru, Pakistan

VIId

Lopburi

1.86

Viscerotropic

Thailand

VIId

D

1.98-2.00

Viscerotropic

Thailand Middle East

VIId

D1435/3/3/SA/10

?

Neurotropic

VIId

D1524/1/1,2/MY/10

?

Viscerotropic

Malaysia

VIId

Goose PMV (171/06)

1.85

Viscerotropic

South Africa

VIId

D1500/2/1/10/CN

?

Viscerotropic

China, Venezuela

VIII

RB Daagstam (ND/01/ZA)

?

Viscerotropic

South Africa

VII. COMPATIBILITY STUDIES VECTORMUNE® ND & TRANSMUNE Gumboro and Newcastle Disease are among the most important disease to poultry producers. Moreover, Transmune is the most used vaccine against Gumboro Disease in broilers in the world.

Therefore, in order to assess the compatibility between Vectormune® ND and Transmune, a very comprehensive trial was conducted. Commercial broiler chickens with MDANDV of 3.4 log2 mean HI titre and MDAIBD of 6089 mean VN titre were divided in three groups: GROUP 1: Vectormune® ND (SQ route) GROUP 2: Vectormune® ND + Transmune (SQ route) GROUP 3: Not vaccinated (Control group) At 8 days of age, chickens from all groups were challenged with vvMDV RB1B strain by SQ route (0.2 ml) and the observation period (lesions verification) was up to 76 days post-challenge.

At 14 and 21 days of age, ten (10) spleens per group were collected for real-time PCR for HVT vaccine take detection.

At 2, 3, 4, and 6 weeks of age, challenges with a Malaysian isolate NDV strain, genotype VII, 5 log10 EID50 /bird, nasal route (200 µl) were also carried out in birds from each group. Moreover, pharyngeal and cloacal swabs were collected 4 and 7 dpch after the challenges performed at 3 and 6 wk.

From D18 to D35, 10 bursa and blood samplings were collected twice weekly per group. When histology confirmed bursa changes, PCR was run in 2 consecutive samplings to confirm the identity of the virus.

The compatibility was assessed based on the following criteria: detection of Vectormune® ND in the spleen, detection of the W2512 strain in the bursa of Fabricius, protection against ND challenge and protection against Marek’s Disease challenge.

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THE RESULTS of clinical protection are summarized in the following graphic:

Clinical protection against vvNDV challenge 100

This graphic illustrates clearly that Transmune does not interfere with the ability of Vectormune® ND to protect the birds against a heavy ND challenge.

% of protection

80

60

40

20

0 21 days

28 days

35 days

Vectormune ND + Transmune

Control

®

Vectormune ND ®

Extracted from Study SCI-137-2011

The detection of Transmune take in the bursa was evaluated from day 18 to day 35 by histology.

THE FOLLOWING table summarizes the results: GROUPS D18

Histopathological bursal lesions D21 D25 D28 D32

D35

Vectormune ND + Transmune

-

-

+

+

+

+

Transmune

-

+

+

+

+

+

Extracted from Study SCI-137-2011

The first signs of vaccine take were detected between 21 and 25 days of age. There was no marked difference between the two groups. Furthermore, RT-PCR & RFLP confirmed the identity of W2512 in the two groups.

FINALLY, the protection against Marek’s Disease is shown in the following graphic:

Clinical protection against MDV-1 (RB1B) challenge 100

The group of birds vaccinated with the combination of Vectormune® ND and Transmune had even less attributable signs to Marek’s Disease observed up to 76 days post challenge than the group vaccinated with Vectormune® ND alone. It clearly shows that Transmune does not interfere with the ability of Vectormune® ND to protect against Marek’s Disease challenge.

Ratio of protected birds

80

60

40

20

0 Vectormune® ND + Transmune

Vectormune® ND

Control

Extracted from Study SCI-137-2011

Based on the results of the detection of the W2512 strain in the bursa of Fabricius, protection against ND challenge and protection against Marek’s Disease challenge, it is clear that Vectormune® ND and Transmune are fully compatible.

Several trials have been carried out to assess the compatibility between Vectormune® ND and Vectormune® AI as these vaccines are against the most devastating diseases for the poultry industry (these two diseases are the only ones affecting poultry that were classified as belonging to the former A list of notifiable diseases to the OIE).

In one of them, SPF chickens were divided into four groups and vaccinated as follows: GROUP 1 - Vaccinated with one dose of Vectormune® ND by SQ route GROUP 2 - Vaccinated with one dose of Vectormune® AI by SQ route GROUP 3 - Vaccinated with one dose of Vectormune® ND and one dose of Vectormune® AI (mixed in the same diluents) by SQ route GROUP 4 - Not vaccinated (Control group) At 14, 21, 28, 42 and 56 days of age, chickens from groups 1, 3 and 4 were challenged with 5.0 lgEID50 /0.1 ml of a Malaysian NDV isolate (D1524/1/1,2/MY/10) by nasal route.

Likewise, at 28 and 56 days of age, birds form the groups 2, 3 and 4 were challenged with 106 EID50 / chick of a HPAIV Egyptian 2008 H5N1 strain, clade 2.2.1.

THE FOLLOWING graphic summarizes the clinical protection against NDV challenge.

Clinical protection against vvNDV challenge 100

% of protection

80

60

40

20

0 W2 Vectormune® ND

W3

W4

W6

Vectormune® ND +Vectormune® AI

Extracted from Study SCI-101-2011

W8 Control

The results of NDV challenge showed that Vectormune ® ND vaccine administered either alone or in combination with Vectormune® AI provided high level of clinical protection. Therefore, interference of Vectormune® AI with Vectormune® ND based on the clinical protection was not observed.

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VECTORMUNE® ND & VECTORMUNE® AI

THE RESULTS of clinical protection against AI are summarized in the following graphic:

Clinical protection against HPAIV (H5N1) challenge 100

% of protection

80

60

40

20

0 W4

W8 Vectormune® ND + Vectormune® AI

Vectormune® AI

Control

Extracted from Study SCI-101-2011

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Based on the challenge results carried out with velogenic NDV and HPAIV, Vectormune® ND and Vectormune® AI are compatible and therefore these vaccines can be used together.

VIII. COMPARISON BETWEEN VECTORMUNE® ND AND OTHER rHVT-F VACCINE Although Vectormune® ND and the other commercially available rHVT-F vaccine express the F protein of the ND virus, there are marked differences in their construction such as the HVT strain used as the vector, the insertion site and the promoter. These differences certainly have an impact on their efficacy.

Vaccine

HVT strain

Donor virus

Donor gene

Insertion site into HVT

Promoter

Vectormune® ND

FC 126

D26 strain

Fusion

Between UL 45 & 46 genes

Pec

rHVT-F vaccine

PB1

Clone 30 strain

Fusion

Into US 10 gene

RSV LTR

In order to compare these two vector rHVT-F vaccines, several laboratory and field trials have been conducted.

Commercial broilers with MDANDV (HI titre at 3 days of age: log2 HI 3.4) were divided into 3 groups as follows: GROUP 1: Vaccinated with Vectormune® ND by SQ route GROUP 2: Vaccinated with a rHVT-F vaccine by SQ route GROUP 3: Not vaccinated (control group) Challenges were performed at 3, 4 and 6 weeks of age with a viscerotropic velogenic NDV isolate (D1524/1/1,2/MY/10), genotype VII, with a dose of 5.0 ELD50 /chicken applied via intra-nasal route.

THE RESULTS of clinical protection against NDV challenge are summarized in the following graphic:

Clinical protection against NDV challenge 100

% of protection

80

60

40

20

0 3

4

6

Age of challenges (weeks) Vectormune® ND

rHVT-F vaccine

Control

These results show that, at 3 weeks of age, the group vaccinated with Vectormune® ND already reached 75% clinical protection while the group vaccinated with the other rHVT-F vaccine had none.

At the challenge carried out at 4 weeks of age, the group which received Vectormune® ND showed very high level (95%) of clinical protection. The clinical protection induced by the other rHVT-F vaccine was still delayed.

Extracted from Study SCI-136-2011

Finally, by 6 weeks of age, both vector vaccines were able to induce 100% of clinical protection.

The experience of more than 10 billion birds vaccinated around the world in different epidemiological context has demonstrated the strong benefits of usage of Vectormune® ND to better control mortality and virus shedding, and to bring better profit and peace of mind to the poultry industry.

IX. REFERENCES • Palya, V.; Tatár-Kis, T.; Mató, T.; Felföldi, B.; Kovács, E. and Gardin, Y. Onset and long-term duration of immunity provided by a single vaccination with a turkey herpesvirus vector ND vaccine in commercial layers. Veterinary Immunology and Immunopathology 158, 105–115, 2014.

• Palya, V.; Tatar-Kis, T.; Mato, T. and Gardin, Y. Interference between Different HVT Vectored Vaccines Applied Subcutaneously at Day-Old in Commercial Broilers. AAAP meeting, 2010.

• Rauw, F. ; Gardin, Y.; Palya, V.; Anbari, S. ; Lemaire, S. ; Boschmans, M. ; van den Berg, T. and Lambrecht, B. 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, 2010.

• Rauw, F. ; Gardin, Y.; Palya, V.; Ngabirano E.; van Borm S.; van den Berg, T. and Lambrecht, B. Distribution and humoral immunity induced by a rHVT-ND vaccine in SPF chickens. Poster Berlin.

• Study SCI-137-2011 – Compatibility of Vectormune ND vaccine with Cevac Transmune IBD in day-old broilers, Ceva-Phylaxia, 2011.

• Study SCI-155-2012 – Efficacy test of different ND vaccination programs in commercial broiler chickens against genotype VIIa NDV challenge, Ceva-Phylaxia, 2012.

• Study DV-055-2007- Efficacy of Recombinant HVT-ND alone or in Combination with live NDV Vaccines, Ceva-Phylaxia, 2007.

Yannick Gardin Director Innovation Strategy Department Marcelo Paniago Director Global Veterinary Services Poultry Ceva Animal Health, Libourne, France.

Pascal Paulet Poultry Corporate Product Manager Christophe Cazaban Global Veterinary Services Manager - Knowledge Manager

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There are marked differences between these new generation vaccines, even if of the same type.

Several trials have shown that immunity to ND induced by Vectormune® ND developed markedly faster compared to the other rHVT-F vaccine.

Photos : Ceva - Langlois

www.vectormune-nd.com - www.vectormune.com CEVA SANTE ANIMALE 10, Avenue de la Ballastière - BP 126 - 33500 LIBOURNE Cedex (France) Tel : + 33 5 5 7 55 40 40 - Fax : + 33 5 57 55 41 92 - www.ceva.com - contact@ceva.com