In Ovo Vaccination with Recombinant Herpes Virus of the Turkey-Laryngotracheitis Vaccine Adjuvanted with CpG-Oligonucleotide Provides Protection against a Viral Challenge in Broiler Chickens.

Infectious laryngotracheitis (ILT) is an economically important disease in chickens. We previously showed that an in ovo adjuvantation of recombinant herpesvirus of the turkey-Laryngotracheitis (rHVT-LT) vaccine with CpG-oligonucleotides (ODN) can boost vaccine-induced responses in one-day-old broiler chickens. Here, we evaluated the protective efficacy of in ovo administered rHVT-LT + CpG-ODN vaccination against a wild-type ILT virus (ILTV) challenge at 28 days of age and assessed splenic immune gene expression as well as cellular responses. A chicken-embryo-origin (CEO)-ILT vaccine administered in water at 14 days of age was also used as a comparative control for the protection assessment. The results showed that the rHVT-LT + CpG-ODN or the CEO vaccinations provided significant protection against the ILTV challenge and that the level of protection induced by both the vaccines was statistically similar. The protected birds had a significantly upregulated expression of interferon (IFN)γ or interleukin (IL)-12 cytokine genes. Furthermore, the chickens vaccinated with the rHVT-LT + CpG-ODN or CEO vaccine had a significantly higher frequency of γδ T cells and activated CD4+ or CD8+ T cells, compared to the unvaccinated-ILTV challenge control. Collectively, our findings suggest that CpG-ODN can be used as an effective adjuvant for rHVT-LT in ovo vaccination to induce protective immunity against ILT in broiler chickens.

Prediction of highly pathogenic avian influenza vaccine efficacy in chickens by comparison of in vitro and in vivo data: A meta-analysis and systematic review.

Vaccines for avian influenza (AI) can protect poultry against disease, mortality, and virus transmission. Numerous factors, including: vaccine platform, immunogenicity, and relatedness to the field strain, are known to be important to achieving optimal AI vaccine efficacy. To better understand how these factors contribute to vaccine protection, a systematic meta-analysis was conducted to evaluate efficacy data for vaccines in chickens challenged with highly pathogenic (HP) AI. Data from a total of 120 individual trials from 25 publications were selected and evaluated. Two vaccine criteria were evaluated for their effects on two metrics of protection. The vaccine criteria were: 1) the relatedness of the vaccine antigen and challenge strain in the hemagglutinin 1 domain (HA1) protein sequence; 2) vaccine-induced antibody titers to the challenge virus (VIAC). The metrics of protection were: A) survival of vaccinated chickens vs unvaccinated controls; and B) reduction in oral virus-shedding by vaccinated vs unvaccinated controls 2-4 days post challenge. Three vaccine platforms were evaluated: oil-adjuvanted inactivated whole AI virus, recombinant herpes virus of turkeys (rHVT) vectored, and a non-replicating alpha-virus vectored RNA particle (RP) vaccine. Higher VIAC correlated with greater reduction of virus-shed and vaccine efficacy by all vaccine platforms. Both higher HA1 relatedness and higher VIAC using challenge virus as antigen correlated with better survival by inactivated vaccines and rHVT-vectored vaccines. However, rHVT-vectored and RP based vaccines were more tolerant of variation in the HA1; the relatedness of the HA1 of the vaccine and challenge virus did not significantly correlate with survival with rHVT-vectored vaccines. Protection was achieved with the lowest aa similarity for which there was data, 90-93 % for rHVT vaccines and 88 % for the RP vaccine.

Efficacy of multivalent recombinant herpesvirus of turkey vaccines against high pathogenicity avian influenza, infectious bursal disease, and Newcastle disease viruses.

Vaccines are an essential tool for the control of viral infections in domestic animals. We generated recombinant vector herpesvirus of turkeys (vHVT) vaccines expressing computationally optimized broadly reactive antigen (COBRA) H5 of avian influenza virus (AIV) alone (vHVT-AI) or in combination with virus protein 2 (VP2) of infectious bursal disease virus (IBDV) (vHVT-IBD-AI) or fusion (F) protein of Newcastle disease virus (NDV) (vHVT-ND-AI). In vaccinated chickens, all three vHVT vaccines provided 90-100% clinical protection against three divergent clades of high pathogenicity avian influenza viruses (HPAIVs), and significantly decreased number of birds and oral viral shedding titers at 2 days post-challenge compared to shams. Four weeks after vaccination, most vaccinated birds had H5 hemagglutination inhibition antibody titers, which significantly increased post-challenge. The vHVT-IBD-AI and vHVT-ND-AI vaccines provided 100% clinical protection against IBDVs and NDV, respectively. Our findings demonstrate that multivalent HVT vector vaccines were efficacious for simultaneous control of HPAIV and other viral infections.

A Recombinant Turkey Herpesvirus Expressing the F Protein of Newcastle Disease Virus Genotype XII Generated by NHEJ-CRISPR/Cas9 and Cre-LoxP Systems Confers Protection against Genotype XII Challenge in Chickens.

In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45-UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.

Construction and immune efficacy of a recombinant turkey herpesvirus vaccine strain expressing fusion protein of genotype VII Newcastle disease virus.

Herpesvirus of turkeys (HVT), a commonly used live vaccine against Marek's disease, has proven to be a highly effective viral vector for the generation of recombinant vaccines that deliver protective antigens of other avian pathogens. In this study, a vaccine designated rHVT-NDV-opti F was constructed by inserting a codon-optimized genotype Ⅶ Newcastle disease virus (NDV) fusion (F) gene into the US2 gene of HVT Fc126 vaccine strain using CRISPR/Cas9 gene-editing technology coupled with two single-guide RNAs (sgRNA). The F protein expression of rHVT-NDV-opti F was detectable by western blotting and an indirect immunofluorescence assay. Compared with wildtype HVT, rHVT-NDV-opti F has similar plaque morphology but lower in vitro replication capacity. The F protein of rHVT-NDV-opti F is genetically stable and predominantly expressed in the cell plasma. Immunization of one-day-old specific pathogen-free chickens with 4000 plaque-forming units of rHVT-NDV-opti F induced NDV-specific antibodies and provided 70% protection against a homologous NDV challenge, effectively reducing virus shedding, clinical signs, tissue viral load, and mortality. These results suggest that rHVT-NDV-opti F could be a potential vaccine candidate against Newcastle disease in chickens and that HDR-CRISPR/Cas9 combined with dual sgRNA can rapidly and efficiently construct HVT-vectored vaccine candidates.

The Requirement of Glycoprotein C for Interindividual Spread Is Functionally Conserved within the Alphaherpesvirus Genus (Mardivirus), but Not the Host (Gallid).

Marek's disease (MD) in chickens is caused by Gallid alphaherpesvirus 2, better known as MD herpesvirus (MDV). Current vaccines do not block interindividual spread from chicken-to-chicken, therefore, understanding MDV interindividual spread provides important information for the development of potential therapies to protect against MD, while also providing a natural host to study herpesvirus dissemination. It has long been thought that glycoprotein C (gC) of alphaherpesviruses evolved with their host based on their ability to bind and inhibit complement in a species-selective manner. Here, we tested the functional importance of gC during interindividual spread and host specificity using the natural model system of MDV in chickens through classical compensation experiments. By exchanging MDV gC with another chicken alphaherpesvirus (Gallid alphaherpesvirus 1 or infectious laryngotracheitis virus; ILTV) gC, we determined that ILTV gC could not compensate for MDV gC during interindividual spread. In contrast, exchanging turkey herpesvirus (Meleagrid alphaherpesvirus 1 or HVT) gC could compensate for chicken MDV gC. Both ILTV and MDV are Gallid alphaherpesviruses; however, ILTV is a member of the Iltovirus genus, while MDV is classified as a Mardivirus along with HVT. These results suggest that gC is functionally conserved based on the virus genera (Mardivirus vs. Iltovirus) and not the host (Gallid vs. Meleagrid).

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.

Long-term protection against a virulent field isolate of infectious laryngotracheitis virus induced by inactivated, recombinant, and modified live virus vaccines in commercial layers.

Infectious laryngotracheitis (ILT) is an acute respiratory disease of chickens controlled through vaccination with live-modified attenuated vaccines, the chicken embryo origin (CEO) vaccines and the tissue-culture origin (TCO) vaccines. Recently, novel recombinant vaccines have been developed using herpesvirus of turkey (HVT) and fowl pox virus (FPV) as vectors to express ILTV immunogens for protection against ILT. The objective of this study was to assess the protection efficacy against ILT induced by recombinants, live-modified attenuated, and inactivated virus vaccines when administered alone or in combination. Commercial layer pullets were vaccinated with one or more vaccines and challenged at 35 (35 WCH) or 74 weeks of age (74 WCH). Protection was assessed by scoring clinical signs; and by determining the challenge viral load in the trachea at five days post-challenge. The FPV-LT vaccinated birds were not protected when challenged at 35 weeks; the HVT-LT and TCO vaccines in combination provided protection similar to that observed in chickens vaccinated with either HVT-LT or TCO vaccines when challenged at 35 weeks, whereas protection induced by vaccination with HVT-LT followed by TCO was superior in the 74 WCH group compared with the 35 WCH group. Birds given the inactivated ILT vaccine had fewer clinical signs and/or lower viral replication at 74 WCH when combined with TCO or HVT-LT, but not when given alone. Finally, the CEO-vaccinated birds had top protection as indicated by reduction of clinical signs and viral replication when challenged at 35 weeks (74 weeks not done). These results suggest that certain vaccine combinations may be successful to produce long-term protection up to 74 weeks of age against ILT.

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing.

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Codon pair bias deoptimization of the major oncogene meq of a very virulent Marek's disease virus.

Codon pair bias deoptimization (CPBD) has been successfully used to attenuate several RNA viruses. CPBD involves recoding a viral protein-coding sequence to maximize the number of codon pairs that are statistically underrepresented in the host, which presumably slows protein translation and, hence, causes virus attenuation. However, since recoding preserves the amino acid composition and codon bias, attenuated and parental viruses are antigenically identical. To determine if Marek's disease virus (MDV), a highly oncogenic herpesvirus of the chicken with a large double-stranded DNA genome, can be attenuated by CPBD of its major oncogene meq, we recoded the gene to minimize (meq-D), maximize (meq-O), or preserve (meq-R) the level of codon pairs that are overrepresented in the chicken protein-coding sequences. Unexpectedly, mutants carrying recoded genes produced comparable or increased levels of Meq in the context of viral infection in cultured cells. In addition, parental virus and mutant viruses carrying recoded meq genes replicated with comparable kinetics in vitro and in vivo, and were equally virulent in susceptible chickens. In summary, CPBD of meq failed to produce any quantifiable attenuation of MDV and confirms differences in the complexity of applying CPBD to large DNA viruses versus RNA viruses.

Pentapeptide-insertion scanning mutational analysis of turkey herpesvirus HVT063 reveals residues important for its RNA silencing suppression activity.

HVT063, an RNA-binding protein encoded by turkey herpesvirus, has been shown previously to suppress RNA silencing. Here, a scanning library produced by pentapeptide-insertion scanning mutagenesis was used to identify key residues associated with its RNA silencing suppressor (RSS) activity. Forty-two in-frame insertion mutants of HVT063 protein were evaluated for their RSS activity using the dual-luciferase transient expressing assay system. Sixteen mutations resulted in a loss of RSS activity, 20 mutations resulted in decreased RSS activity, and six mutations exhibited high RSS activity similar to wild-type HVT063. Based on a three-dimensional structure prediction, most of the loss-of-function mutations were located around a predominantly α-helical region at the C-terminal end of HVT063. In particular, a conserved domain in this region, named herpes_UL69, showed low tolerance for five-amino-acid insertions. Combined with the results of our previous studies, basic amino acids could play a key role in RSS activity. These results also demonstrate that pentapeptide-insertion scanning mutagenesis combined with dual-luciferase assays is an effective method to functionally characterize RSSs.

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.

A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system.

Herpesvirus of turkeys (HVT) has been successfully used as live vaccine against Marek's disease (MD) worldwide for more than 40 years either alone or in combination with other serotypes. HVT is also widely used as a vector platform for generation of recombinant vaccines against a number of avian diseases such as infectious bursal disease (IBD), Newcastle disease (ND) and avian influenza (AI) using conventional recombination methods or recombineering tools on cloned viral genomes. In the present study, we describe the application of CRISPR/Cas9-based genome editing as a rapid and efficient method of generating HVT recombinants expressing VP2 protein of IBDV. This approach offers an efficient method to introduce other viral antigens into the HVT genome for rapid development of recombinant vaccines.

Homologous and heterologous antigenic matched vaccines containing different H5 hemagglutinins provide variable protection of chickens from the 2014 U.S. H5N8 and H5N2 clade 2.3.4.4 highly pathogenic avian influenza viruses.

From December 2014 to June 2015, a novel H5 Eurasian A/goose/Guangdong (Gs/GD) lineage clade 2.3.4.4 high pathogenicity avian influenza (HPAI) virus caused the largest animal health emergency in US history resulting in mortality or culling of greater than 48 million poultry. The outbreak renewed interest in developing intervention strategies, including vaccines, for these newly emergent HPAI viruses. In these studies, several existing H5 vaccines or vaccine seed strains with varying genetic relatedness (85-100%) to the 2.3.4.4 HPAI viruses were evaluated for protection in poultry. Chickens received a single dose of either an inactivated whole H5 AI vaccine, or a recombinant fowl poxvirus or turkey herpesvirus-vectored vaccines with H5 AI hemagglutinin gene inserts followed by challenge with either a U.S. wild bird H5N8 (A/gyrfalcon/Washington/40188-6/2014) or H5N2 (A/northern pintail/Washington/40964/2014) clade 2.3.4.4 isolate. Results indicate that most inactivated H5 vaccines provided 100% protection from lethal effects of H5N8 or H5N2 challenge. In contrast, the recombinant live vectored vaccines only provided partial protection which ranged from 40 to 70%. Inactivated vaccine groups, in general, had lower number of birds shedding virus and at lower virus titers then the recombinant vaccine groups. Interestingly, prechallenge antibody titers using the HPAI challenge viruses as antigen in heterologous vaccine groups were typically low (≤2 log2), yet the majority of these birds survived challenge. Taken together, these studies suggest that existing vaccines when used in a single immunization strategy may not provide adequate protection in poultry against the 2.3.4.4 HPAI viruses. Updating the H5 hemagglutinin to be genetically closer to the outbreak virus and/or using a prime-boost strategy may be necessary for optimal protection.

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.

Protection Afforded by a Recombinant Turkey Herpesvirus-H5 Vaccine Against the 2014 European Highly Pathogenic H5N8 Avian Influenza Strain.

A highly pathogenic avian influenza (HPAI) H5N8 (clade 2.3.4.4) virus, circulating in Asia (South Korea, Japan, and southern China) since the beginning of 2014, reached the European continent in November 2014. Germany, the Netherlands, the United Kingdom, Italy, and Hungary confirmed H5N8 infection of poultry farms of different species and of several wild bird species. Unlike the Asian highly pathogenic (HP) H5N1, this HP H5N8 also went transatlantic and reached the American West Coast by the end of 2014, affecting wild birds as well as backyard and commercial poultry. This strain induces high mortality and morbidity in Galliformes, whereas wild birds seem only moderately affected. A recombinant turkey herpesvirus (rHVT) vector vaccine expressing the H5 gene of a clade 2.2 H5N1 strain (rHVT-H5) previously demonstrated a highly efficient clinical protection and reduced viral excretion against challenge with Asian HP H5N1 strains of various clades (2.2, 2.2.1, 2.2.1.1, 2.1.3, 2.1.3.2, and 2.3.2.1) and was made commercially available in various countries where the disease is endemic. To evaluate the protective efficacy of the rHVT-H5 vaccine against the first German H5N8 turkey isolate (H5N8 GE), a challenge experiment was set up in specific-pathogen-free (SPF) chickens, and the clinical and excretional protection was evaluated. SPF chickens were vaccinated subcutaneously at 1 day old and challenged oculonasally at 4 wk of age with two viral dosages, 10(5) and 10(6) 50% egg infective doses. Morbidity and mortality were monitored daily in unvaccinated and vaccinated groups, whereas viral shedding by oropharyngeal and cloacal routes was evaluated at 2, 5, 9, and 14 days postinoculation (dpi). Serologic monitoring after vaccination and challenge was also carried out. Despite its high antigenic divergence of the challenge H5N8 strain, a single rHVT-H5 vaccine administration at 1 day old resulted in a full clinical protection against challenge and a significant reduction of viral shedding in the vaccinated birds.

Recombinant Turkey Herpesvirus-AI Vaccine Virus Replication in Different Species of Waterfowl.

Waterfowl play a key role in the epidemiology of the H5N1 subtype of highly pathogenic avian influenza (HPAI) virus; therefore, efficient immunization of domesticated ducks and geese to maximize the impact of other control measures is of great importance. A recombinant (r)HVT-AI, expressing the HA gene of a clade 2.2 H5N1 HPAI strain had been developed and proved to be efficient against different clades of H5N1 HPAI virus in chickens after a single vaccination at 1 day old and could provide long-term immunity. We investigated whether rHVT-AI applied at 1 day old is able to replicate in different species and crossbreeds of ducks and in geese with the aim of collecting data on the possible application of rHVT-AI vaccine in different species of waterfowl for the control of H5N1 HPAI. We tested the possible differences among different waterfowl species, i.e., between geese (Anser anser, domesticated greylag goose), Muscovy ducks (Cairina moschata forma domestica), Pekin ducks (Anas platyrhynchos forma domestica), and mule ducks (Muscovy duck × Pekin duck), in their susceptibility to support the replication of rHVT-AI. Vaccine virus replication was followed by real-time PCR in spleen, bursa, and feather tip samples. Humoral immune response to vaccination was tested using the hemagglutination inhibition (HI) test and H5-specific commercial ELISA. Significant differences among the different waterfowl species regarding the rate of rHVT-AI replication was detected that were not reflected by the same difference in the immune response to vaccination. Replication of the rHVT-AI vaccine was very limited in Pekin ducks, somewhat better in mule ducks, and the vaccine virus was replicating significantly better in Muscovy ducks and geese, reaching 100% detectability at certain time points after administration at 1 day old. Results indicated that the vaccine virus could establish different levels of persistent infection in these species of waterfowl. No humoral immune response could be detected either by HI test or ELISA during the tested postvaccination period (5 wk).

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.

Vaccine Protection of Turkeys Against H5N1 Highly Pathogenic Avian Influenza Virus with a Recombinant Turkey Herpesvirus Expressing the Hemagglutinin Gene of Avian Influenza.

Outbreaks of H5 highly pathogenic avian influenza (HPAI) in commercial poultry are a constant threat to animal health and food supplies. While vaccination can enhance protection and reduce the spread of disease, there is considerable evidence that the level of immunity required for protection varies by subtype and virulence of field virus. In this study, the efficacy of a recombinant turkey herpesvirus (rHVT) vector vaccine expressing the hemagglutinin gene from a clade 2.2 AI virus (A/Swan/Hungary/4999/2006) was evaluated in turkeys for protection against challenge with A/Whooper Swan/Mongolia/L244/2005 H5N1 HPAI clade 2.2. One-day-old turkeys received a single vaccination and were challenged at 4 wk postvaccination with 2 × 10(6) 50% embryo infectious dose per bird. The results demonstrate that following H5N1 HPAI challenge 96% protection was observed in rHVT-AI vaccinated turkeys. The oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared with sham-vaccinated birds. From respiratory and gastrointestinal tracts, there was a greater than 6 log10 reduction in shedding in vaccinated birds as compared with the controls. This study provides support for the use of a commercially available rHVT-AI vaccine to protect turkeys against H5N1 HPAI.

Efficacy of a Recombinant Turkey Herpesvirus H5 Vaccine Against Challenge With H5N1 Clades 1.1.2 and 2.3.2.1 Highly Pathogenic Avian Influenza Viruses in Domestic Ducks (Anas platyrhynchos domesticus).

Domestic ducks are the second most abundant poultry species in many Asian countries and have played a critical role in the epizootiology of H5N1 highly pathogenic avian influenza (HPAI).In this study, the protective efficacy of a live recombinant vector vaccine based on a turkey herpesvirus (HVT) expressing the H5 gene from a clade 2.2 H5N1 HPAI strain (A/Swan/Hungary/4999/ 2006) (rHVT-H5/2.2), given at 3 days of age, was examined in Pekin ducks (Anas platyrhynchos domesticus). The vaccine was given alone or in combination with an inactivated H5N1 clade 2.3.2.1 reverse genetic (rgGD/2.3.2.1) vaccine given at 16 days of age, either as a single vaccination or in a prime-boost regime. At 30 days of age, ducks were challenged with one of two H5N1 HPAI viruses: A/duck/Vietnam/NCVD-2721/2013 (clade 1.1.2) or A/duck/Vietnam/NCVD-1584/2012 (clade 2.3.2.1.C). These viruses produced 100% mortality in less than 5 days in nonvaccinated control ducks. Ducks vaccinated with the rgGD/2.3.2.1 vaccine, with or without the rHVT-H5/2.2 vaccine, were 90%-100% protected against mortality after challenge with either of the two H5N1 HPAI viruses. The rHVT-H5/2.2 vaccine alone, however, conferred only 30% protection against mortality after challenge with either H5N1 HPAI virus; the surviving ducks from these groups shed higher amount of virus and for longer than the single-vaccinated rgGD/2.3.2.1 group. Despite low protection, ducks vaccinated with the rHVT-H5/2.2 vaccine and challenged with the clade 1.1.2 Vietnam virus had a longer mean death time than nonvaccinated controls (P = 0.02). A booster effect was found on reduction of virus shedding when using both vaccines, with lower oropharyngeal viral titers at 4 days after challenge with either HPAI virus (P < 0.05). Neither rHVT-H5/2.2 nor standard HVT vaccine could be detected in samples collected from multiple tissues at different time points, indicting minimal levels of viral replication. In conclusion, although a minor effect on survival was observed, this study demonstrates the suboptimal protection with the rHVT-H5/2.2 vaccine given alone in Pekin ducks against H5N1 HPAI viruses and only a minor additive effect on virus shedding reduction when used with an inactivated vaccine in a prime-boost regime.