YOU THINK INFLUENZA IS FATAL? THINK AGAIN.

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YOU THINK INFLUENZA IS FATAL? THINK AGAIN. HVT vector vaccine for H5 Avian Influenza protection

Avian influenza Avian influenza remains a major threat for the global poultry industry. Among the different avian influenza viruses, the H5 and H7 strains carry the highest risk. They have the capacity to switch from low to high pathogenic types. They are disrupting international trade and are inducing high levels of mortality in infected flocks, moreover they potentially affect human health. Photo : Ceva Indonesia Photo : XXX Photo : Ceva Egypt Ischemic lesions on the comb and the wattles Cyanosis of the shanks Current avian influenza vaccines status Avian influenza control programs are usually based on early diagnosis of the first cases, good bio security measures to limit spreading of the virus, stamping out policy or the implementation of a program using vaccines that preferably allow differentiation between vaccinated and infected birds (DIVA principle). Avian influenza vaccines are usually inactivated vaccines produced from low pathogenic strains or through reverse genetic techniques. However, despite the intensive use of vaccines, the disease is still present in some countries where it has become endemic. The age at injection is one of the critical factors explaining these poor results. Many studies show immunity induced by AI inactivated vaccines is impaired by Maternally Derived Antibody (MDA) when applied at an early age (figure 1). impact of maternally derived antibodies on vaccination A H5N1 inactivated vaccine has been evaluated in SPF chicks that received several levels of H5 antibodies to mimic different maternally derived antibody levels (2). Day-old SPF chickens received different levels of H5N1 antibodies by intraperitoneal injection. One day later they were immunized with reverse genetics H5N1 vaccine, and four weeks later with a second shot of the same vaccine. Three weeks later (seven weeks of age) they were challenged via intranasal, ocular and tracheal routes with a dose of 10 3,5 EID50 of Ck/Qal-Egypt/1/08 H5N1 HPAI virus.

Figure 1: Avian influenza challenge results of chicks with different levels of intraperitoneal anti H5N1 chicken sera (2) 100 80 % Survival 60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 DAYS POST CHALLENGE PBS 1:10 diluted 1:100 diluted Undiluted Unvaccinated High antiboy level at age of injection has interfered with induction of protective immunity by the inactivated vaccine Although widely used, conventional killed vaccines are ineffective in controlling avian influenza. The immunity induced by this type of vaccine is impaired by MDA when applied at day 1, and farm vaccination at an older age is unreliable. These vaccines can quickly lose some or all efficacy if the field virus undergoes any antigenic drift, which is commonly observed. In severely affected countries, where the disease has turned enzootic, there is a real need for new vaccines that are capable of overcoming the problem of interference with MDA and that can be applied at the hatchery where the quality of vaccine administration is much better. There is also a need for vaccines that would be less susceptible to the antigenic variations of the field virus. VECTORMUNE HVT-AIV (Herpes Virus of Turkey-Avian influenza / Vector) Construction of VECTORMUNE HVT AIV vaccine Avian influenza virus belongs to the Orthomyxoviridae family of segmented RNA viruses. The genome is split into eight different gene segments that encode for 10 different viral proteins. The virus particle binds to the cell membrane by the Hemagglutinin (HA) protein. During the cell infection process, the virus particle is endocytosed, then after chemical reaction the HA protein mediates the fusion of the virus and the cell membrane. Later, the RNA of the virus is released inside the host cell (8).

Therefore, immunity against HA protein is a key factor in avian influenza protection for the chicken. Ceva, the leader of poultry vector vaccines, has developed a new vaccine solution to offer the industry a better alternative for preventing avian influenza H5: Vectormune HVT AIV. Vectormune HVT AIV is a genetically engineered live HVT vaccine expressing the HA gene of an H5N1 avian influenza virus and is indicated for use in chickens. HA NA + = 100 to 300 nanometres AI virus HVT VIRUS Vectormune HVT AIV vaccine The vaccine is based on live serotype 3 Marek virus vaccine, HVT: It is not a live avian influenza vaccine The vaccine is applied at the hatchery There is no interference with MDA It induces long-lasting immunity on birds due to the persistency of the HVT strain in chickens The vaccine expresses the HA protein that is the major immunogenic protein for avian influenza protection. is stored in frozen form in a liquid nitrogen dewar. Vectormune HVT AIV is available in: 1000 d 2000 d 4000 d The vaccine has to be thawed and reconstituted with Ceva sterile diluent, a specific diluent for Marek vaccine. At the hatchery: Injection of 0.2 ml per chick at day 1. Ceva sterile diluent is available in: 200 ml, 400 ml, 800 ml 1200 ml, 1600 ml and 2400 ml plastic bags.

Vectormune HVT AIV Efficacy The protection induced by Vectormune HVT AIV has been evaluated in comparison to the protection induced by an inactivated H5N2 vaccine using a recent isolate of H5N1 HPAI virus from Egypt (4). SPF chicks were vaccinated with either Vectormune HVT AIV at day 1, or an inactivated H5N2 vaccine at 10 days of age. Three weeks post-vaccination, the birds were challenged with a 2008 HPAI H5N1 isolate of Egyptian origin. In each vaccine group, 5 chicks were not challenged and were kept as sentinel birds to evaluate the virus shedding from vaccinated and challenged chicks. Symptoms and shedding measured by oropharyngeal swabbing were evaluated three and seven days post challenge. Survival rate (%) Mean number of copy / ml of swabs (log10) 100 90 80 70 60 50 40 30 20 10 0 10 9 8 7 6 5 4 3 2 1 0 Avian influenza disease challenge results 2008 Egypt HP H5N1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Vectormune HVT AIV sentinel Vectormune HVT AIV C D A 3 days H5N1 Shedding Vectormune HVT AIV sentinel Vectormune HVT AIV B C DAYS POST CHALLENGE b challenged inac H5N2 sentinel challenged inac H5N2 a b * 7 days challenged inac H5N2 sentinel challenged inac H5N2 a DAYS POST CHALLENGE challenged unvaccinated challenged unvaccinated Mean at time points with no common superscript (A/B or a/b) differ significantly P<0.05. * All chickens in the challenged unvaccinated group died 3 days after the challenge The Vectormune HVT AIV vaccinated chickens were clinically fully protected from the challenge with a heterologous field virus strain. The H5N2 vaccinated chickens were not. In the Vectormune HVT-AIV vaccinated group, the sentinel birds in contact with the challenged chickens did not show any symptoms after the challenge. Conversely, in the conventional H5N2 vaccinated group, all the sentinel birds in contact with the challenged chickens died. Statistically, the Vectormune HVT AIV vaccine significantly reduced the avian influenza virus shedding compared to the inactivated H5N2 vaccine.

Vectormune HVT AIV protection has also been evaluated against different H5 virus isolates from around the world that belonged to different clades, table 1. Table 1: studies of Vectormune HVT AIV vaccinated chickens challenged with H5 Avian influenza virus strains of different origins (10-11-12 and internal data) Type of Chicken MDA AIV status Challenge type Strain Age (d) Vaccine Survival rate Control SPF Negative H5N1 - HP Broilers Negative H5N1 - HP Broilers Positive (H5N2) H5N1 - HP Broilers Positive (H5N2) H5N1 - HP SPF Negative H5N1 - HP SPF Negative H5N1 - HP Broilers Positive (H5N1) H5N1 - HP Broilers Positive (H5N1) H5N1 - HP A/Viet Nam/1203/04 - Clade 1 A/ duck/ Hungary/ 11804/2006 - Clade 2.2 A/ duck/ Hungary/ 11804/2006 - Clade 2.2 A/ duck/ Hungary/ 11804/2006 - Clade 2.2 Egypt/1709-1VIR08/2007 Egypt/1709-6/2008 Egypt/1709-6/2008 Egypt - 63/2010 28 85% 0% 14 100% 0% 14 100% 20% 21 100% 0% 21 100% 0% 21 100% 0% 28 93% 0% 35 80% 0% SPF Negative H5N2 - HP Queretaro/95 28 95% 0% Broilers Positive (H5N1) H5N1 - HP Ck/Purwakarta cilingga/142/10 28 95% 0% Vectormune HVT AIV has been evaluated against different H5 avian influenza viruses from isolates around the world with consistently high levels of efficacy.

VECTORMUNE HVT-AIV safety VECTORMUNE HVT-AIV has no spreading capacity from vaccinated to non-vaccinated birds (internal study). 18-day-old embryonated SPF eggs were vaccinated with Vectormune HVT AIV. At hatch, the chicks were placed in isolators with non-vaccinated control birds from the same origin. Chickens were bled at 10 days, 14 days and 21 days of age, and Marek HVT virus isolation was conducted from purified white blood cells. Figure 3 : HVT virus isolation results % of birds positive for HVT 100 80 60 40 20 0 10 days of age 14 days of age 21 days of age Vectormune HVT AIV Contact control The vaccine virus did not infect susceptible chickens in contact with the vaccinated VECTORMUNE HVT-AIV chickens. VECTORMUNE HVT-AIV vaccine strain does not spread from vaccinated birds to nonvaccinated birds. Vectormune HVT AIV is an HVT vaccine, strain FC126. This Marek vaccine strain has been widely used for nearly 50 years and has proven to be safe (9). The vector vaccine technology based on the HVT vector has been used with commercial vaccines, vector HVT NDV and vector HVT IBD, on billions of birds since 2007 with no negative impact.

Precautions: Do not administer concomitant vaccination with any other HVT classical or HVT vector vaccine Withdrawal period: 21 days Features and benefits VECTORMUNE HVT AIV Strong protection Demonstrated protection against H5 viruses from several geographical origins Safety Hatchery application with an aqueous adjuvant No spread Convenience Hatchery vaccination at Day 1 Long-lasting protection Based on a HVT vaccine References 1 J. De Vrieese, M. Steensels, V. Palya, Y. Gardin, K. Moore Dorsey. D Lambrecht, S. Van Borm and T V den Berg. Passive protection afforded by maternally derived antibodies in chickens and the antibody interference with the protection elicited by avian influenza inactivated vaccines in progeny. Avian Diseases 2010, 54:246-252 2 Jeong-Ki Kim, Ghazi Kayali, David Walker and coll. Puzzling inefficacy of H5N1 influenza vaccines in Egyptian poultry. PNAS june 15,2010, 17 :24, 11044-11049. 3 Riks Maas, Sigrid Rosema, Diana van Zoelen, Sandra Venema. Maternal immunity against avian influenza H5N1 in chickens : limited protection and interference with vaccine efficacy. Avian Pathology, feb 2011, 40(1), 87-92. 4 F. Rauw, V.Palya, S. Van Borm and coll. Further evidence of antigenic drift and protection efficacy afforded by a recombinant HVT H5 vaccine against challenge with two antigenically divergent Egyptian clade 2;2.1 HPAI H5N1 strains. Vaccine 29 (2011) 2590-2600. 5 Marisa Peyre, Hamid Samaha, Yilma Jobre Malonnen and coll. Avian influenza vaccination in Egypt: limitations of the current strategy. Journal of Molecular and genetic medecine, dec 2009, vol 3 n 2, 198-204. 6 S.Marangon, M. Cecchinato and I. Capua. Use of vaccination in Avian influenza control and eradication. Zoonoses and Public Health. 55 (2008) 65-72. 7 D.E. Swayne, D.L. Suarez. Current developments in avian influenza vaccines, including safety of vaccinated birds as food. EV BIOL. Basel, Karger, 2007, vol 130, 121-131. 8 D.E. Swayne, Avian influenza, 2007, Blackwell publishing. 9 R.L. Witter, K. Nazerian, H.G.Purchase, G.H. Burgoyne, Isolation of turkeys of a cell associated herpes virus antigenically related to Marek s disease virus.am.j.vet.res, vol 31, N 3, march 1970, 525-538. 10 J. De Vriese, Y. Gardin, V. Palya, K. Moore Dorsey, B. Lambrecht, S. Van Borm, T. van den Berg. Efficacy of a rhvt-ai vector vaccine in broilers, with passive immunity against HVT and AIV, against challenge with H5N1 HPAIV challenge. 2009. Proceedings XVI WVPA Congress, Marrakech, Moroco. 11 De Vriese J., Gardin, Y., Palya, V. and van den Berg, T. Passive protection afforded by MDA in chickens and their interference with the protection elicited by AI inactivated vaccines in progeny 2009. Proceedings XVI WVPA Congress, Marrakech, Moroco. 12 J. De Vriese, Y. Gardin, V. Palya, K. Moore Dorsey, B. Lambrecht, S. Van Borm, T. van den Berg. Efficacy of a rhvt-ai vector vaccine in broilers with passive immunity against HVT and AIV, against challenge with H5N1 HPAIV. 2009. Poster. Symposium on Avian Influenza, Athens, Georgia, USA. RC B 403 289 580 - Crédit photo : Thinkstock Composition VECTORMUNE HVT-AIV contains a live frozen serotype 3 Marek (HVT) recombinant vaccine virus indicated for use in chickens The HVT in VECTORMUNE HVT-AIV contains and expresses key protective avian influenza virus antigen. Indications VECTORMUNE HVT-AIV is indicated as an aid in the prevention of avian influenza virus H5 type and Marek s disease in chickens. Vaccinate day-old chicks Administration: Prior to use, thaw the vaccine and reconstitute immediately with sterile diluent. Inject by subcutaneous route: 0.2 ml per day-old chick. Special precautions for use: Vaccinate healthy, susceptible birds only. VECTORMUNE HVT-AIV must be the only HVT vaccine administered to the chickens. Good management practices are recommended to reduce exposure to avian influenza virus and Marek s disease virus for at least two weeks following vaccination. Withdrawal period: 21 days. Storage: Store the vaccine in liquid nitrogen. Warning: Be familiar with all safety and precautionary measures for handling liquid nitrogen to prevent personal injury. CEVA SANTE ANIMALE 10, Avenue de la Ballastière - BP 126-33500 LIBOURNE Cedex (France) Tel : + 33 5 57 55 40 40 - Fax : + 33 5 57 55 41 92 - www.ceva.com - www.vector-vaccines.com