Vaccinal efficacy of molecularly cloned Gallid alphaherpesvirus 3 strain 301B/1 against very virulent Marek's disease virus challenge.

Marek's disease virus (MDV), a causative agent of Marek's disease, has evolved its virulence partly because the current control strategies fail to provide sterilizing immunity. Gallid alphaherpesvirus 3 (GaHV-3) and turkey herpesvirus have been developed as bivalent vaccines to improve upon the level of protection elicited by single formulations. Since the in vitro passage of vaccines can result in attenuation, a GaHV-3 strain 301B/1 was cloned as a bacterial artificial chromosome (BAC) by inserting the mini-F replicon into the virus genome. A fully infectious virus, v301B-BAC, was reconstituted from the 301B/1 BAC clone and had similar growth kinetics comparable to that of the parental 301B/1 virus with strong reactivity against anti-301B/1 chicken sera. Protective efficacies of v301B-BAC, parental 301B/1, and SB-1 vaccine were evaluated against a very virulent MDV Md5 challenge. Clinical signs were significantly lower in the v301B-BAC vaccinated groups (24-25 %), parental 301B/1 (29 %) compare to that of non-vaccinated control (100%) and the removal of BAC sequences from v301B-BAC genome further reduced this to 17 %. The protective indices of v301B-BACs (75-76 %) were comparable with those of both the 301B/1 and the SB-1 vaccine (71%). Removal of the mini-F replicon resulted in a reconstituted virus with a protective index of 83 %. The shedding of challenge virus was notably lower in the v301B-BAC, and v301B-delBAC vaccinated groups. Overall, the protective efficacy of the 301B-BAC-derived vaccine virus against a very virulent MDV challenge was comparable to that of the parental 301B/1 virus as well as the SB-1 vaccine virus.

Transcriptional Analyses of Innate and Acquired Immune Patterning Elicited by Marek's Disease Virus Vaccine Strains: Turkey Herpesvirus (HVT), Marek's Disease Virus 2 (strain SB1), and Bivalent Vaccines (HVT/SB1 and HVT-LT/SB1).

Marek's disease (MD) is a complex pathology of chickens caused by MD virus (MDV) 1 and is observed as paralysis, immune suppression, neurologic signs, and the rapid formation of T-cell lymphomas. The incidence of MD in commercial broilers is largely controlled via vaccination, either in ovo or at hatch with live attenuated vaccines, i.e., turkey herpesvirus (HVT) or a bivalent combination of HVT with the MDV 2 strain (SB1). To further extend the protection conferred by bivalent HVT/SB-1, recombinant HVTs encoding transgenes of other avian viruses have similarly been used for in ovo administration. Despite decades of use, the specific mechanisms associated with vaccine-induced protection remain obscure. Additionally, the mechanistic basis for vaccine synergism conferred by bivalent HVT/SB-1, compared with HVT or SB-1 administered alone, is largely unknown. In the present study, we report on temporal changes in innate and acquired immune-patterning gene expression by using ex vivo splenocyte infection and in ovo vaccination models. We report that in the ex vivo splenocyte infection model, by 72 hr postinfection, vaccines induced IFN and IFN-stimulated gene expression, with lesser proinflammatory cytokine induction. For several genes (TLR3, IFN-γ, OASL, Mx1, NOS2A, and IL-1ß), the effects on gene expression were additive for HVT, SB1, and HVT/SB1 infection. We observed similar patterns of induction in in ovo-vaccinated commercial broiler embryos and chicks with HVT/SB-1 or recombinant HVT-based bivalent combination (HVT-LT/SB-1). Furthermore, HVT/SB-1 or HVT-LT/SB-1 in ovo vaccination appeared to hasten immune maturation, with expression patterns suggesting accelerated migration of T and natural killer cells into the spleen. Finally, HVT/SB-1 vaccination resulted in a coordinated induction of IL-12p40 and downregulation of suppressors of cytokine signaling 1 and 3, indicative of classical macrophage 1 and T-helper 1 patterning.

Análisis transcripcionales de patrones inmunes innatos y adquiridos inducidos por cepas vacunales del virus de la enfermedad de Marek: virus herpes del pavo (HVT), virus de Marek 2 (cepa SB1) y vacunas bivalentes (HVT/SB1 y HVT-LT/SB1). La enfermedad de Marek (MD) es una patología compleja de los pollos causada por el virus de Marek (MDV) 1 y se observa como parálisis, depresión inmune, signos neurológicos y la formación rápida de linfomas de células T. La incidencia de la enfermedad de Marek en pollos de engorde comerciales se controla en gran medida a través de la vacunación, ya sea in ovo o al momento de la eclosión con vacunas vivas atenuadas, por ejemplo, herpesvirus de pavo (HVT) o una combinación bivalente de HVT con la cepa SB1. Para ampliar aún más la protección conferida por la vacuna bivalente HVT/SB-1, los HVT recombinantes que codifican transgenes de otros virus aviares se han utilizado de forma similar para la administración in ovo. A pesar de décadas de uso, los mecanismos específicos asociados con la protección inducida por la vacuna siguen sin ser esclarecidos completamente. Además, el mecanismo para la sinergia de la vacuna conferida por la vacuna bivalente HVT/SB-1, en comparación con la administración de la cepa HVT o de la cepa SB-1 por sí solas, es en gran medida desconocida. En el presente estudio, se informa sobre los cambios temporales en la expresión genética de patrones inmunes innatos y adquiridos mediante la infección de esplenocitos ex vivo y en modelos de vacunación in ovo. Se reporta que en el modelo de infección de esplenocitos ex vivo, por 72 horas después de la infección, las vacunas indujeron IFN y la expresión de genes estimulada por IFN, con menor inducción de citocinas proinflamatorias. Para varios genes (TLR3, IFNc, OASL, Mx1, NOS2A e IL-1ß), los efectos sobre la expresión de genes fueron aditivos para la infección por HVT, SB1 y HVT/SB1. Se Observaron patrones de inducción similares en embriones de pollo y pollos de engorde comerciales vacunados in ovo con HVT/SB-1 o con la combinación bivalente recombinante basada en HVT (HVT-LT/SB-1). Además, la vacunación in ovo con HVT/SB-1 o HVT-LT/SB-1 parecen acelerar la maduración inmune, con patrones de expresión que sugieren una migración acelerada de células T y células asesinas naturales en el bazo. Finalmente, la vacuna HVT/SB-1 dio como resultado una inducción coordinada de IL-12p40 y una regulación a la baja de supresores de las señales de citocinas 1 y 3, indicativas de los patrones clásicos de macrófagos 1 y células cooperadoras tipo 1.

Towards a mechanistic understanding of the synergistic response induced by bivalent Marek's disease vaccines to prevent lymphomas.

BACKGROUND: Marek's disease (MD) is a lymphoproliferative disease of chickens caused by Marek's disease virus (MDV), an oncogenic α-herpesvirus. Since 1970, MD has been controlled by widespread vaccination; however, more effective MD vaccines are needed to counter more virulent MDV strains. The bivalent vaccine combination of SB-1 and herpesvirus of turkey (HVT) strain FC126 has been widely used. Nonetheless, the mechanism(s) underlying this synergistic effect has not been investigated. METHODS: Three experiments were conducted where SB-1 or HVT were administered as monovalent or bivalent vaccines to newly hatched chickens, then challenged five days later with MDV. In Experiment 1, levels of MDV replication in PBMCs were measured over time, and tumor incidence and vaccinal protection determined. In Experiment 2, MDV and vaccine strains replication levels in lymphoid organs were measured at 1, 5, 10, and 14 days post-challenge (DPC). In Experiment 3, to verify that the bursa was necessary for HVT protection, a subset of chicks were bursectomized and these birds plus controls were similarly vaccinated and challenged, and the levels of vaccinal protection determined. RESULTS: The efficacy of bivalent SB-1 + HVT surpasses that of either SB-1 or HVT monovalent vaccines in controlling the level of pathogenic MDV in PBMCs until the end of the study, and this correlated with the ability to inhibit tumor formation. SB-1 replication in the spleen increased from 1 to 14 DPC, while HVT replicated only in the bursa at 1 DPC. The bursa was necessary for immune protection induced by HVT vaccine. CONCLUSION: Synergy of SB-1 and HVT vaccines is due to additive influences of the individual vaccines acting at different times and target organs. And the bursa is vital for HVT to replicate and induce immune protection.

A Recombinant Turkey Herpesvirus Expressing F and HN Genes of Avian Avulavirus-1 (AAvV-1) Genotype VI Confers Cross-Protection against Challenge with Virulent AAvV-1 Genotypes IV and VII in Chickens.

Newcastle disease (ND) is responsible for significant economic losses in the poultry industry. The disease is caused by virulent strains of Avian avulavirus 1 (AAvV-1), a species within the family Paramyxoviridae. We developed a recombinant construct based on the herpesvirus of turkeys (HVT) as a vector expressing two genes: F and HN (HVT-NDV-F-HN) derived from the AAvV-1 genotype VI ("pigeon variant" of AAvV-1). This recombinant viral vaccine candidate was used to subcutaneously immunize one group of specific pathogen-free (SPF) chickens and two groups of broiler chickens (20 one-day-old birds/group). Humoral immune response was evaluated by hemagglutination-inhibition test and enzyme-linked immunosorbent assay (ELISA). The efficacy of the immunization was assessed in two separate challenge studies performed at 6 weeks of age with the use of virulent AAvV-1 strains representing heterologous genotypes IV and VII. The developed vaccine candidate elicited complete protection in SPF chickens since none of the birds became sick or died during the 2-week observation period. In the broiler groups, 90% and 100% clinical protection were achieved after challenges with AAvV-1 of IV and VII genotypes, respectively. We found no obvious relationship between antibody levels and protection assessed in broilers in the challenge study. The developed recombinant HVT-NDV-F-HN construct containing genes from a genotype VI AAvV-1 offers promising results as a potential vaccine candidate against ND in chickens.

Evaluation of Protective Efficacy When Combining Turkey Herpesvirus-Vector Vaccines.

Turkey herpesvirus (HVT) is widely used as a vaccine against Marek's disease in chickens and recently as a vector for foreign genes from infectious bursal disease virus, Newcastle disease (ND) virus, infectious laryngotracheitis (ILT) virus, and avian influenza virus. Advantages of HVT-vector vaccines are that the vaccines do not contain live respiratory viruses or live infectious bursal disease virus able to replicate and cause disease or embryo mortality, they can be administered at hatch or in ovo, and they are relatively insensitive to interference from maternally derived antibodies. As producers have tried to combine HVT-vector vaccines to protect against additional diseases, reports have indicated that applying two vectored vaccines using the same HVT vector is reported to reduce the efficacy of one or both vaccines. To confirm this interference, we evaluated commercial vaccines from multiple companies, including products with inserts designed to protect against ND, infectious ILT, and infectious bursal disease (IBD). Using a standard dosage, we found that the ILT product was most severely affected by the addition of other vaccines, as demonstrated by a significant increase in clinical signs, significant decrease in weight gain, and increase in quantity of challenge virus observed from tracheal swabs collected from Days 3-5 postchallenge. The ND and IBD products were also affected by the addition of other vaccines, although in most cases differences compared to vaccination with the vector alone were not statistically significant. This study demonstrates the importance of following manufacturer guidelines and the need for validating alternative strategies to benefit from the high level of protection offered by vector vaccines.

Evaluación de la eficacia de la protección cuando se combinan vacunas recombinantes con base en el virus herpes del pavo como vector. El virus herpes de los pavos (HVT) se usa ampliamente como una vacuna contra la enfermedad de Marek en pollos y recientemente como un vector para genes externos como del virus de la enfermedad infecciosa de la bolsa, del virus de la enfermedad de Newcastle (ND), del virus de la laringotraqueítis infecciosa (ILT) y del virus de la influenza aviar. Las ventajas de las vacunas con vector de HVT consisten en que las vacunas no contienen virus vivos respiratorios o virus de la enfermedad infecciosa de la bolsa, no son capaces de replicarse y causar enfermedad o mortalidad embrionaria, pueden administrarse en el momento de la eclosión o in ovo y son relativamente insensibles a la interferencia de anticuerpos de origen materno. A medida que los productores han intentado combinar las vacunas con el vector HVT para inducir protección contra enfermedades adicionales, los informes han indicado que la aplicación de dos vacunas vectorizadas utilizando el mismo vector HVT reduce la eficacia de una o de ambas vacunas. Para confirmar esta interferencia, se evaluaron las vacunas comerciales de múltiples compañías, incluidos los productos con inserciones diseñadas para proteger contra la enfermedad de Newcastle, la laringotraqueítis infecciosa aviar y contra la enfermedad infecciosa de la bolsa. Utilizando una dosis estándar, se encontró que el producto para la laringotraqueítis infecciosa se vio más afectado por la adición de otras vacunas, como lo demuestra un aumento significativo en los signos clínicos, una disminución significativa en el aumento de peso y un aumento en la cantidad de virus de desafío observados en los hisopos traqueales recolectados de tres a cinco días después del desafío. Los productos para la enfermedad de Newcastle y para la enfermedad de Gumboro también se vieron afectados por la adición de otras vacunas, aunque en la mayoría de los casos las diferencias en comparación con la vacunación únicamente con el vector no fueron estadísticamente significativas. Este estudio demuestra la importancia de seguir las pautas del fabricante y la necesidad de validar estrategias alternativas para beneficiarse del alto nivel de protección ofrecido por las vacunas con vectores.

Study of Efficacy and Replication of Recombinant Vector Vaccines by Using Turkey Herpesvirus Combined with Other Marek's Disease Vaccines.

Several recombinant turkey herpesviruses (rHVTs) have been developed within the past decades, and they are now used commercially worldwide. In broiler chickens, rHVTs are usually administered alone, but in long-living birds they are used in combination with Marek's disease (MD) vaccines of other serotypes (i.e., CVI988). The objectives of this work were to 1) evaluate protection against MD conferred by HVT and two rHVTs when combined with CVI988 and 2) optimize the use of rHVT in combination with CVI988 to maximize replication of rHVT without compromising MD protection. Various vaccine protocols, all using rHVT or HVT at the recommended dose (RD), were evaluated. Protocols evaluated included in ovo vaccination with HVT+CVI988 or rHVT+CVI988 (using either the double dose [DD] or the RD of CVI988), day of age vaccination of rHVT+CVI988 at DD, and revaccination protocols using rHVT in ovo followed by CVI988 at DD at day of age. Our results show that, when combined with CVI988, HVT and rHVTs confer a similar level of protection against MD (>90%) regardless of whether CVI988 was used at RD or at DD. However, the combination of rHVT with CVI988 at DD resulted in reduced replication rates of rHVT (60%-76% vs. 95%-100%). Our results show that such a negative effect could be avoided without jeopardizing MD protection by administering CVI988 at RD (if combined in ovo with rHVT) or administered rHVT first in ovo followed by CVI988 at DD at day of age.

Estudio de la eficacia y replicación de vacunas con vectores recombinantes mediante el uso del virus del herpes del pavo combinado con otras vacunas contra la enfermedad de Marek. Varios herpesvirus de pavo recombinantes (rHVT) se han desarrollado en las últimas décadas y ahora se utilizan comercialmente en todo el mundo. En pollos de engorde, los rHVT generalmente se administran solos, pero en aves de vida larga se usan en combinación con vacunas contra la enfermedad de Marek (MD) de otros serotipos (especialmente, CVI988). Los objetivos de este trabajo fueron 1) evaluar la protección contra la enfermedad de Marek conferida por herpesvirus de pavo (HVT9 y por dos rHVT cuando se combinan con la cepa CVI988 y 2) optimizar el uso de rHVT en combinación con la cepa CVI988 para maximizar la replicación de rHVT sin comprometer la protección contra la enfermedad de Marek. Se evaluaron varios protocolos de vacunas, todos con rHVT o con HVT a la dosis recomendada (RD). Los protocolos evaluados incluyeron la vacunación in ovo con HVT + CVI988 o rHVT + CVI988 (usando la dosis doble o la dosis recomendada de la cepa CVI988), la vacunación al día de la edad con rHVT + CVI988 con dosis doble, y los protocolos de revacunación usando rHVT seguido por la cepa CVI988 con dosis doble al día de edad. Los resultados muestran que cuando se combinan con CVI988, HVT y rHVT confieren un nivel de protección similar contra la enfermedad de Marek (> 90%) independientemente de que la cepa CVI988 se haya usado a la dosis recomendada o con dosis doble. Sin embargo, la combinación de rHVT con la cepa CVI988 con doble dosis produjo una reducción en las tasas de replicación de rHVT (60% ­76% vs. 95% ­100%). Estos resultados muestran que dicho efecto negativo podría evitarse sin poner en peligro la protección contra la enfermedad de Marek administrando la cepa CVI988 a la dosis recomendada (si se combina in ovo con rHVT) o administrando rHVT primero in ovo, seguido de CVI988 con dosis doble al día de la edad.

Impact of HVT Vaccination on Splenic miRNA Expression in Marek's Disease Virus Infections.

Marek's Disease is a lymphoproliferative disease of chickens caused by Marek's Disease Virus. Similar to other herpesviruses, Marek's Disease Virus (MDV) encodes its own small non-coding regulatory RNAs termed microRNAs (miRNAs). We previously found that the expression profile of these viral miRNAs is affected by vaccination with Herpesvirus of Turkeys (HVT). To further characterize miRNA-mediated gene regulation in MDV infections, in the current study we examine the impact of HVT vaccination on cellular miRNA expression in MDV-infected specific-pathogen-free (SPF) chickens. We used small RNA-seq to identify 24 cellular miRNAs that exhibited altered splenic expression in MDV infected chickens (42 dpi) compared to age-matched uninfected birds. We then used Real Time-quantitative PCR (RT-qPCR) to develop expression profiles of a select group of these host miRNAs in chickens receiving the HVT vaccine and in vaccinated chickens subsequently infected with MDV. As was seen with viral miRNA, host miRNAs had unique splenic expression profiles between chickens infected with HVT, MDV, or co-infected birds. We also discovered a group of transcription factors, using a yeast one-hybrid screen, which regulates immune responses and cell growth pathways and also likely regulates the expression of these cellular miRNAs. Overall, this study suggests cellular miRNAs are likely a critical component of both protection from and progression of Marek's Disease.

A double recombinant herpes virus of turkeys for the protection of chickens against Newcastle, infectious laryngotracheitis and Marek's diseases.

A double recombinant strain of herpes virus of turkeys (HVT) was constructed that contains the fusion (F) gene from Newcastle disease virus (NDV) and the gD plus gI genes from infectious laryngotracheitis virus (ILTV) inserted into a non-essential region of the HVT genome. Expression of the F protein was controlled by a human cytomegalovirus promoter, whereas expression of gD plus gI was driven by an ILTV promoter. The double recombinant vaccine virus (HVT-NDV-ILT) was fully stable genetically and phenotypically following extended passage in cell culture and infection of chickens. Safety of the vaccine virus was confirmed by overdose and backpassage studies in specific-pathogen-free chickens. Chickens vaccinated with a single dose of HVT-NDV-ILT administered by the in ovo route were highly protected from challenge with the velogenic NDV (GB Texas), ILTV (LT 96-3) and Marek's disease virus (GA 5) strains (97%, 94% and 97%, respectively). Similarly, chickens vaccinated with a single dose by subcutaneous (SC) route at 1 day of age were highly protected from challenge with the same three viruses (100%, 100%, and 88%, respectively). The protection level of a single dose given by in ovo or SC route against challenge with a virulent Marek's disease virus strain demonstrates that insertion of multiple genes from two different pathogens within the HVT genome had no adverse effect on the capacity of HVT to protect against Marek's disease. These results demonstrate that HVT-NDV-ILT is a safe and efficacious vaccine for simultaneous control of NDV, ILTV and Marek's diseases.

The efficacy of recombinant turkey herpesvirus vaccines targeting the H5 of highly pathogenic avian influenza virus from the 2014-2015 North American outbreak.

The outbreak of highly pathogenic avian influenza virus in North American poultry during 2014 and 2015 demonstrated the devastating effects of the disease and highlighted the need for effective emergency vaccine prevention and control strategies targeted at currently circulating strains. This study evaluated the efficacy of experimental recombinant turkey herpesvirus vector vaccines with three different inserts targeting the hemagglutinin gene of an isolate from the recent North American influenza outbreak. White leghorn chickens were vaccinated at one day of age and challenged with A/Turkey/Minnesota/12582/2015 H5N2 at 4 weeks of age. Birds were analyzed for survival, viral shedding at two and four days after infection, and specific antibody prior to challenge and from surviving birds. The three experimental vaccines demonstrated 100%, 45% and 15% survival with the most effective vaccine significantly reducing oral and cloacal viral shedding compared to all other groups and generated specific antibody prior to challenge with highly pathogenic avian influenza virus. More studies are needed using diverse H5Nx highly pathogenic virus isolates to fully determine the breadth of coverage against possible exposure strains, as well as possible impact of maternally derived antibody on protection and vaccine efficacy.

Immunity Elicited by a Turkey Herpesvirus-Vectored Newcastle Disease Vaccine in Turkey Against Challenge With a Recent Genotype IV Newcastle Disease Virus Field Strain.

Newcastle disease (ND) is still a major poultry disease worldwide. Vaccination remains the principal method of controlling ND in endemic countries. Various vaccination strategies, including the use of recently developed recombinant vaccines, have been used to control it. Recombinant vaccines that use the herpesvirus of turkey (HVT) as a vector to express one of the key antigens of Newcastle disease virus (NDV) have been developed to overcome some of the drawbacks related to the use of conventional vaccines. HVT as a vector appears to have unique beneficial characteristics: it is extremely safe, it is not affected by the presence of maternally derived antibodies, and therefore can be applied in the hatchery either in ovo or to day-old chicks. Due to its persistence in the bird, the HVT vector can be expected to induce life-long immune stimulation. In the present study, the efficacy of an HVT-based vector vaccine expressing the F gene of NDV (rHVT-F) was tested against a velogenic genotype IV NDV challenge in commercial turkeys with high levels of maternal antibodies (8.7 ± 0.8 log2 hemagglutination inhibition titer). The birds were vaccinated on the day of hatch by the subcutaneous route. Development of a humoral immune response to vaccination was detectable from 4 weeks of age by ELISA. The challenge strain used represents recent NDV genotype IV field strains from Morocco. Challenge with this strain induced ND-specific clinical signs and stunting without subsequent mortality in the non-vaccinated birds, whereas the vaccinated turkey poults showed protection as early as 3 weeks of age based on lack of clinical signs, better body weight gain, and reduction of challenge virus shedding. This is the first reported efficacy study of an HVT-vectored ND vaccine against a velogenic NDV challenge in commercial turkeys.

Modified live infectious bursal disease virus (IBDV) vaccine delays infection of neonatal broiler chickens with variant IBDV compared to turkey herpesvirus (HVT)-IBDV vectored vaccine.

Chickens are commonly processed around 35-45days of age in broiler chicken industry hence; diseases that occur at a young age are of paramount economic importance. Early age infection with infectious bursal disease virus (IBDV) results in long-lasting immunosuppression and profound economic losses. To our knowledge, this is the first study comparing the protection efficacy of modified live (MdLV) IBDV and herpesvirus turkey (HVT)-IBDV vaccines against early age variant IBDV (varIBDV) infection in chicks. Experiments were carried out in IBDV maternal antibody (MtAb) positive chicks (n=330), divided into 6 groups (n=50-60/group), namely Group 1 (saline), Group 2 (saline+varIBDV), Group 3 (HVT-IBDV), Group 4 (HVT-IBDV+varIBDV), Group 5 (MdLV) and Group 6 (MdLV+varIBDV). HVT-IBDV vaccination was given via the in ovo route to 18-day-old embryonated eggs. MdLV was administered via the subcutaneous route in day-old broilers. Group 2, Group 4 and Group 6 were orally challenged with varIBDV (SK-09, 3×103 EID50) at day 6 post-hatch. IBDV seroconversion, bursal weight to body weight ratio (BBW) and bursal histopathology were assessed at 19 and 35days of age. Histopathological examination at day 19 revealed that varIBDV-SK09 challenge caused severe bursal atrophy and lower BBW in HVT-IBDV but not in MdLV vaccinated chicks. However by day 35, all challenged groups showed bursal atrophy and seroconversion. Interestingly, RT-qPCR analysis after varIBDV-SK09 challenge demonstrated an early (9days of age) and significantly high viral load (∼5744 folds) in HVT-IBDV vaccinated group vs unvaccinated challenged group (∼2.25 folds). Furthermore, flow cytometry analysis revealed inhibition of cytotoxic CD8+ T-cell response (CD44-downregulation) and decreased splenic lymphocytes counts in chicks after HVT-IBDV vaccination. Overall, our data suggest that MdLV delays varIBDV pathogenesis, whereas, HVT-IBDV vaccine is potentially immunosuppressive, which may increase the risk of early age varIBDV infection in broilers.

Turkey herpesvirus with an insertion in the UL3-4 region displays an appropriate balance between growth activity and antibody-eliciting capacity and is suitable for the establishment of a recombinant vaccine.

We constructed turkey herpesvirus (HVT) vector vaccines in which the VP2 gene of infectious bursal disease virus (IBDV) was inserted into the HVT genome in the following regions: UL3-4, UL22-23, UL45-46, and US10-SORF3. We then evaluated the relationship between the gene insertion site and the capacity of the virus to elicit antibodies. rHVT/IBD (US10) showed good growth activity in vitro, with growth comparable to that of the parent HVT. On the other hand, rHVT/IBD (UL3-4), rHVT/IBD (UL22-23), and rHVT/IBD (UL45-46) exhibited decreased growth activity in chicken embryo fibroblast (CEF) cells compared to the parent HVT. However, the rHVT/IBD (US10) elicited lower levels of virus-neutralizing (VN) antibodies compared to the other constructs. rHVT/IBD (UL3-4) and rHVT/IBD (UL45-46) appeared to be similar in their ability to elicit VN antibodies. Based on the results of in vitro and in vivo assays, rHVT/IBD (UL3-4) was selected for further testing. In a challenge assay, rHVT/IBD (UL3-4) protected chickens from challenge with virulent Marek's disease virus serotype 1 and IBDV. In conclusion, the site of gene insertion may have a strong effect on the growth of the vector virus in vitro and its antibody-eliciting capacity. Insertions in the UL3-4 region permitted a balance between growth activity and VN-antibody-eliciting capacity, and this region might therefore be an appropriate insertion site for IBDV VP2.

Characterization of two recombinant HVT-IBD vaccines by VP2 insert detection and cell-mediated immunity after vaccination of specific pathogen-free chickens.

Infectious bursal disease (IBD) is an avian viral disease that causes severe economic losses in the poultry industry worldwide. The live IBD virus (IBDV) has a potential immunosuppressive effect. Currently available IBDV vaccines have shortcomings, prompting the development of safer and more effective vaccination approaches, including the use of the recombinant turkey herpesvirus vaccine expressing the immunogenic structural VP2 protein of IBDV (recombinant HVT (rHVT)-IBD). The objectives of this study were twofold: (i) to develop in vitro assays and molecular tools to detect the VP2 protein and gene and (ii) to evaluate cell-mediated immunity (CMI) induced by rHVT-IBD vaccination of day-old specific pathogen-free chickens. The VP2 protein expressed by rHVT-IBD-infected chicken embryo fibroblasts was detected using the enzyme-linked immunosorbent assay and immunofluorescence. Using molecular techniques, the VP2 gene was detected in various organs, providing a method to monitor vaccine uptake. rHVT-IBD vaccination induced CMI responses in specific pathogen-free chickens at 5 weeks. CMI was detected by measuring chicken interferon-gamma after ex vivo antigenic stimulation of splenocytes. Moreover, our results showed that the enzyme-linked immunospot approach is more sensitive in detecting chicken interferon-gamma than enzyme-linked immunosorbent assay. The tools developed in this study may be useful in the characterization of new-generation recombinant vaccines and the cellular immune response they induce.

Efficacy of Recombinant HVT-IBD Vaccines Administered to Broiler Chicks from a Single Breeder Flock at 30 and 60 Weeks of Age.

The efficacy of commercially available recombinant herpesvirus of turkeys-infectious bursal disease (rHVT-IBD) virus vaccines was studied in broiler chickens derived from an IBDV-vaccinated breeder flock at 30 wk of age (Trial 1) and 60 wk of age (Trial 2). In parallel, specific-pathogen-free (SPF) white leghorn chickens were used to evaluate vaccine efficacy to control for the effects of maternally derived antibodies (MDA) associated with the broiler chickens. Broilers and SPF leghorns were vaccinated subcutaneously in the neck at 1 day of age with Vaxxitek® HVT+IBD or Vectormune® HVT-IBD vaccines and were placed in isolators. On 10, 14, 18, 22, and 26 days postvaccination (DPV), vaccinated and nonvaccinated broilers and SPF leghorns were bled prior to challenge via the oral-nasal route with infectious bursal disease (IBD) reference strains ST-C, Delaware variant E (Del E), or contemporary field isolates DMV/5038/07 or FF6. Microscopic lesion assessment of the bursa was useful for assessing IBDV challenge in both rHVT-IBD-vaccinated broiler and SPF leghorn chickens. In general, rHVT-IBD vaccines induced greater protection as the time between vaccination and challenge increased. Based on incidence of microscopic lesions (IML) of bursa tissue, Vaxxitek HVT+IBD vaccination of SPF leghorns induced protection by 18 DPV and continued to protect 22 DPV and 26 DPV in Trials 1 and 2. Vectormune HVT-IBD vaccine induced protection of SPF leghorns by 18 or 22 DPV in Trial 1, depending upon the IBDV challenge strain. However, the onset of protection was delayed until 22 or 26 DPV in Trial 2. With either commercial vaccine, rHVT-IBD vaccination of broiler chickens was not as effective as was observed in SPF leghorns, based on IML of bursa tissue. However, Vaxxitek HVT+IBD vaccination protected broilers following challenge with ST-C in both Trial 1 (30-wk-old breeder progeny) and Trial 2 (60-wk-old breeder progeny). Partial protection against FF6 (Trial 1) and DMV/5038/07 (Trial 2) challenges was observed. Vectormune HVT-IBD vaccination protected broilers vs. FF6 challenge in Trial 1. In Trial 2, the vaccine did not offer protection on the basis of IML of bursa tissue. The results indicate that 1) bursa/body weight ratios were not consistently useful as a tool for assessing IBDV challenge in broiler chickens with anti-IBDV MDA compared to assessment by IML of bursa tissue, though were useful for assessing protection in SPF leghorns; and 2) both vaccines may offer some protection to older broilers; however, a window of susceptibility exists between the waning of MDA and the development of vaccine-induced antibodies. The SPF studies showed that some vaccinated chickens were not protected from an IBDV challenge earlier than 14 DPV while broiler studies showed that MDA was not fully protective beyond 10 DPV. Because these vaccines did not protect chickens from an IBDV challenge during this window of susceptibility, our data show that breeder vaccination programs for IBDV must aim to maximize anti-IBDV MDA in progeny to protect against early IBDV challenge.

Development and Evaluation of the Protective Efficacy of Novel Marek's Disease Virus Rispens Vector Vaccines Against Infectious Bursal Disease.

Infectious bursal disease (IBD) is a major disease affecting the poultry industry and is caused by infection with IBD virus (IBDV). To develop a novel vaccine to prevent IBD in chickens, recombinant Marek's disease virus Rispens viruses carrying the VP2 gene of IBDV driven by five different promoters (Rispens/IBD) were constructed using homologous recombination and a bacterial artificial chromosome (BAC). Rispens/IBD driven by the chicken beta-actin (Bac) promoter (Rispens/Bac-IBD), Rous sarcoma virus promoter, or simian virus 40 promoter were administered to 1-day-old SPF chicks, and the protective efficacy against IBDV was evaluated by challenging chicks with virulent IBDV. As a result, Rispens/Bac-IBD showed the best protection (87%). Next, we constructed the virus driven by the Bac-derived Coa5 promoter (Rispens/Coa5-IBD) for a secondary in vivo trial using commercial layer chickens since Rispens/Bac-IBD was thought to be genetically unstable. Rispens/Coa5-IBD showed stability in vitro and exhibited better antibody production and protection during challenge against virulent IBDV at both 5 (95%) and 7 wk of age (91%) compared with that of Rispens/Bac-IBD (90% at 5 wk of age and 84% at 7 wk of age). Thus, Rispens/Coa5-IBD may be a novel promising vaccine against IBD and virulent Marek's disease.

Efficacy of Various HVT Vaccines (Conventional and Recombinant) Against Marek's Disease in Broiler Chickens: Effect of Dose and Age of Vaccination.

Herpesvirus of turkeys (HVT) has been successfully used as a Marek's disease (MD) vaccine for more than 40 yr. Either alone (broiler chickens) or in combination with vaccines of other serotypes (broilers, broiler breeders, and layers), HVT is used worldwide. In recent years, several vector vaccines based on HVT (rHVT) have been developed. At present, there are both conventional HVT and rHVTs in the market, and it is unknown if all of them confer the same level of protection against MD. The objective of this study was to further characterize the protection conferred by two conventional HVTs (HVT-A and HVT-B) and three recombinant HVTs (rHVT-B, rHVT-C, and rHVT-D) against MD in broiler chickens. In a first study we evaluated the efficacy of two conventional HVTs (HVT-A and HVT-B) administered at different doses (475, 1500, and 4000 PFU) at day of age on the ability to protect against an early challenge with very virulent plus strain 645. In a second experiment we evaluated the protection ability of several HVTs (both conventional and recombinant) when administered in ovo at a dose of 1500 PFU using the same challenge model. Our results show that each HVT product is unique, regardless of being conventional or recombinant, in their ability to protect against MD and might require different PFUs to achieve its maximum efficacy. In Experiment 1, HVT-A at 4000 PFU conferred higher protection (protection index [PI] = 63) than any of the other vaccine protocols (PI ranging from 36 to 47). In Experiment 2, significant differences were found among vaccine protocols with PI varying from 66 (HVT-A) to 15 (rHVT-D). Our results show that each HVT is unique and age at vaccination and vaccine dose greatly affected vaccine efficacy. Furthermore, they highlight the need of following manufacturer's recommendations.

Protective Efficacy of Recombinant Turkey Herpes Virus (rHVT-H5) and Inactivated H5N1 Vaccines in Commercial Mulard Ducks against the Highly Pathogenic Avian Influenza (HPAI) H5N1 Clade 2.2.1 Virus.

In Egypt, ducks kept for commercial purposes constitute the second highest poultry population, at 150 million ducks/year. Hence, ducks play an important role in the introduction and transmission of avian influenza (AI) in the Egyptian poultry population. Attempts to control outbreaks include the use of vaccines, which have varying levels of efficacy and failure. To date, the effects of vaccine efficacy has rarely been determined in ducks. In this study, we evaluated the protective efficacy of a live recombinant vector vaccine based on a turkey Herpes Virus (HVT) expressing the H5 gene from a clade 2.2 H5N1 HPAIV strain (A/Swan/Hungary/499/2006) (rHVT-H5) and a bivalent inactivated H5N1 vaccine prepared from clade 2.2.1 and 2.2.1.1 H5N1 seeds in Mulard ducks. A 0.3ml/dose subcutaneous injection of rHVT-H5 vaccine was administered to one-day-old ducklings (D1) and another 0.5ml/dose subcutaneous injection of the inactivated MEFLUVAC was administered at 7 days (D7). Four separate challenge experiments were conducted at Days 21, 28, 35 and 42, in which all the vaccinated ducks were challenged with 106EID50/duck of H5N1 HPAI virus (A/chicken/Egypt/128s/2012(H5N1) (clade 2.2.1) via intranasal inoculation. Maternal-derived antibody regression and post-vaccination antibody immune responses were monitored weekly. Ducks vaccinated at 21, 28, 35 and 42 days with the rHVT-H5 and MEFLUVAC vaccines were protected against mortality (80%, 80%, 90% and 90%) and (50%, 70%, 80% and 90%) respectively, against challenges with the H5N1 HPAI virus. The amount of viral shedding and shedding rates were lower in the rHVT-H5 vaccine groups than in the MEFLUVAC groups only in the first two challenge experiments. However, the non-vaccinated groups shed significantly more of the virus than the vaccinated groups. Both rHVT-H5 and MEFLUVAC provide early protection, and rHVT-H5 vaccine in particular provides protection against HPAI challenge.

Experimental and Field Results Regarding Immunity Induced by a Recombinant Turkey Herpesvirus H5 Vector Vaccine Against H5N1 and Other H5 Highly Pathogenic Avian Influenza Virus Challenges.

Vaccination against H5N1 highly pathogenic avian influenza (AI) virus (HPAIV) is one of the possible complementary means available for affected countries to control AI when the disease has become, or with a high risk of becoming, endemic. Efficacy of the vaccination against AI relies essentially, but not exclusively, on the capacity of the vaccine to induce immunity against the targeted virus (which is prone to undergo antigenic variations), as well as its capacity to overcome interference with maternal immunity transmitted by immunized breeding hens to their progeny. This property of the vaccine is a prerequisite for its administration at the hatchery, which assures higher and more reliable vaccine coverage of the populations than vaccination at the farm. A recombinant vector vaccine (Vectormune® AI), based on turkey herpesvirus expressing the hemagglutinin gene of an H5N1 HPAIV as an insert, has been used in several experiments conducted in different research laboratories, as well as in controlled field trials. The results have demonstrated a high degree of homologous and cross protection against different genetic clades of the H5N1 HPAIV. Furthermore, vaccine-induced immunity was not impaired by the presence of passive immunity, but on the contrary, cumulated with it for improved early protection. The demonstrated levels of protection against the different challenge viruses exhibited variations in terms of postchallenge mortality, as well as challenge virus shedding. The data presented here highlight the advantages of this vaccine as a useful and reliable tool to complement biosecurity and sanitary policies for better controlling the disease due to HPAIV of H5 subtypes, when the vaccination is applied as a control measure.

Combination of Two Marek's Disease Virus Vectors Shows Effective Vaccination Against Marek's Disease, Infectious Bursal Disease, and Newcastle Disease.

Herpesvirus of turkeys (HVT) is a widely used vector for poultry vaccines. However, different HVTs expressing different foreign antigens cannot always be used simultaneously because of the risk of recombination and interference. In this study, we inoculated a mixture of an HVT-expressing the antigen of Newcastle disease virus (NDV; HVT/ND) and Marek's disease virus (MDV) serotype 1 Rispens virus expressing the antigen of infectious bursal disease virus (IBD; Ripens/IBD) into chickens. This mixture showed 94%, 100%, or 94% protection against MDV, IBDV, or NDV challenge, respectively. In conclusion, the combination of Rispens/IBD and HVT/ND is effective for vaccination against MDV, IBDV, and NDV without significant interference.

Effects of T-2 Toxin on Turkey Herpesvirus-Induced Vaccinal Immunity Against Marek's Disease.

T-2 toxin, a very potent immunotoxic Type A trichothecene, is a secondary metabolite produced primarily by Fusarium spp., which grows on cereal grains and can lead to contaminated livestock feed. Repeated exposure to T-2 toxin has been shown to cause immunosuppression and decrease the resistance of exposed animals to a variety of infectious diseases; however, the effects of T-2 toxin on Marek's disease (MD) vaccinal immunity have not been reported. Four trials were conducted to determine the effects of T-2 toxin on vaccinal immunity against MD. Day-old, white leghorn chicks of Avian Disease and Oncology Laboratory line 15I5 × 71 were treated daily for 7 days via crop gavage with T-2 toxin at a sublethal dose of 1.25 mg/kg body weight. Treated and untreated chicks were also vaccinated with turkey herpesvirus (HVT) at hatch and were challenged with the JM strain of MD virus (MDV) at 8 days of age. Chickens were tested for HVT viremia at 1 wk postvaccination immediately before challenge, and for HVT and MDV viremia at 3 wk postchallenge. Chickens were observed for the development of MD lesions and mortality within 8 wk of age. T-2 toxin significantly reduced body weight and titers of HVT viremia within 7 days after hatch. T-2 toxin shortened the incubation period for the development of MD lesions and mortality, but only in unvaccinated chickens. The percent MD protection in T-2-toxin-treated, HVT-vaccinated chickens ranged from 82% to 96% and was comparable to that in HVT-vaccinated untreated control chickens (89%-100%). The data suggest that exposure of chickens to sublethal doses of T-2 toxin for 7 consecutive days after hatch may influence the development of 1) HVT viremia; and 2) MD lesions and mortality, but only in unvaccinated chickens.