Infectious laryngotracheitis virus (ILTV) vaccine intake evaluation by detection of virus amplification in feather pulps of vaccinated chickens

Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus, ILTV. The live vaccine is applied worldwide by drinking water or by the respiratory route, and by the vent application in Israel. No system of direct evaluation of the efficacy of vaccination exists today, except of antibody elicitation, which is an indirect indication of vaccination intake and might happen due to environment exposure. We suggest for the first time an assay for evaluating the accuracy of the vaccination process by spotting the spread of the live vaccine systemically, namely by virus detection in the feather shafts of the vaccinated birds. The feathers are particularly beneficial as they are easy to collect, non-lethal for the bird, therefore advantageous for monitoring purposes. Moreover, the continuous survey of the vaccine virus unveiled the different kinetics of viremia by the different vaccination routes; while after the vent vaccination the systemic viremia peaks during the first week afterwards, after two consecutive vaccine administration by drinking water with 6 day interval, the vireamia peaks only after the second administration. A robust amplification was needed because the vaccine ILTV was present in the bird in minute quantities compared to the wild-type virus. For the vaccine virus identification in feather shafts a nested real-time PCR for the TK ILTV gene was developed. The sensitivity of detection of the nested rtPCR was greater by 1000 compared to conventional nested PCR and 10 times that real-time PCR.     

Uptake and spread of infectious laryngotracheitis vaccine virus within meat chicken flocks following drinking water vaccination

Vaccination against infectious laryngotracheitis virus (ILTV) in commercial broiler flocks in the field, which is only undertaken in the face of a local outbreak, requires mass administration techniques, usually via drinking water. This is often fraught with difficulties such as variable vaccination “reactions” and sometimes, vaccination failure. Laboratory testing of the outbreak strains however invariably shows the vaccines in use to be protective. To investigate this paradox, the dynamics of an ILT vaccine virus was examined within broiler flocks during a natural outbreak. In an initial flock, 70 birds were individually identified and had tracheal swabs collected sequentially at intervals from 1 to 26 days after vaccination and submitted for ILTV detection using qPCR. This evaluation was extended by collection of tracheal swabs from 40 to 45 random birds at 4, 7–8, 12–13 and 25–26 days post vaccination (pv) across a further 7 flocks. The results showed a very variable early uptake of vaccine virus from the drinking water (between 3% and 52% of tested birds with detectable virus in trachea at 4 days pv) and revealed that actual vaccination of the flocks relied on bird to bird transmission of the vaccine virus. In flocks with very low (<10%) initial bird uptake, successful exposure of vaccine virus to the majority of the flock can be delayed, leaving a large proportion of birds as susceptible at the likely time of possible exposure to wild virus. This may explain the cases of apparent failure of vaccination in the field. The variable bird to bird spread can be associated with reversion to virulence, this may explain the rolling vaccine reactions often observed. The variation in initial vaccine uptake may be affected by some factors involved with the administration technique and this requires further study in a larger sample size.     

Glycoprotein G deficient infectious laryngotracheitis virus is a candidate attenuated vaccine

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is currently controlled by vaccination with conventionally attenuated virus strains. These vaccines have limitations because of residual pathogenicity and reversion to virulence, suggesting that a novel vaccine strain that lacks virulence gene(s) may enhance disease control. Glycoprotein G (gG) has recently been identified as a virulence factor in ILTV. In this study the immunogenicity and relative pathogenicity of gG deficient ILTV was investigated in SPF chickens. Birds vaccinated with gG deficient ILTV were protected against clinical signs of disease following challenge with virulent ILTV and gG deficient ILTV was also shown to be less pathogenic than currently available commercial vaccine strains. Thus gG deficient ILTV appears to have potential as a vaccine candidate.     

Replication-independent reduction in the number and diversity of recombinant progeny viruses in chickens vaccinated with an attenuated infectious laryngotracheitis vaccine

Recombination is closely linked with virus replication and is an important mechanism that contributes to genome diversification and evolution in alphaherpesviruses. Infectious laryngotracheitis (ILTV; Gallid alphaherpesvirus 1) is an alphaherpesvirus that causes respiratory disease in poultry. In the past, natural (field) recombination events between different strains of ILTV generated virulent recombinant viruses that have caused severe disease and economic loss in poultry industries. In this study, chickens were vaccinated with attenuated ILTV vaccines to examine the effect of vaccination on viral recombination and diversity following subsequent co-inoculation with two field strains of ILTV. Two of the vaccines (SA2 and A20) prevented ILTV replication in the trachea after challenge, but the level of viral replication after co-infection in birds that received the Serva ILTV vaccine strain did not differ from that of the mock-vaccinated (control) birds. Even though the levels of viral replication were similar in the two groups, the number of recombinant progeny viruses and the level of viral diversity were significantly lower in the Serva-vaccinated birds than in mock-vaccinated birds. In both the mock-vaccinated and Serva-vaccinated groups, a high proportion of recombinant viruses were detected in naïve in-contact chickens that were housed with the co-inoculated birds. Our results indicate that vaccination can limit the number and diversity of recombinant progeny viruses in a manner that is independent of the level of virus replication. It is possible that immune responses induced by vaccination can select for virus genotypes that replicate well under the pressure of the host immune response.     

Genetic and antigenic relatedness of H3 subtype influenza A viruses isolated from avian and mammalian species

In 2004, we isolated triple reassortant H3N2 influenza viruses from turkey breeder hens in Ohio and Illinois. The Illinois flock was vaccinated twice with an inactivated H3N2 vaccine containing a swine origin virus before the outbreak. Additionally, a commercial inactivated vaccine containing an H3N4 virus of duck origin is being used in some turkey breeders. This prompted us to initiate a comparative study on the antigenic and genetic relatedness of various H3 subtype influenza viruses isolated from turkeys, ducks, pigs and humans. The antigenic relatedness between the different viruses was evaluated with the Archetti and Horsfall formula, while nucleotide genetic similarities were calculated using pairwise alignments. Results obtained indicated a high degree of antigenic (>90%) and genetic (>99%) similarities among the turkey-origin H3N2 viruses. However, the turkey viruses were antigenically distantly related to the swine-origin vaccine virus (<30%), although they had approximately 95% genetic similarity in the HA1 gene. Additionally, major genetic and antigenic changes were observed between the turkey viruses and the H3N4 duck vaccine virus as well as the H3N2 human virus. Such genetic and antigenic differences between the turkey-origin viruses and other H3 subtype viruses including vaccine strains could be the reason for the failure in protection in the Illinois turkey breeders vaccinated with swine origin virus. This also emphasizes the importance of using viruses for vaccines that are antigenically similar to the field strains.     

Safety and vaccine efficacy of a glycoprotein G deficient strain of infectious laryngotracheitis virus delivered in ovo

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is commonly controlled by vaccination with conventionally attenuated vaccines. Glycoprotein G (gG) is a virulence factor in ILTV and a gG deficient strain of ILTV (ΔgG-ILTV) has shown potential for use as a vaccine. In the poultry industry vaccination via drinking water is common, but technology is now available to allow quicker and more accurate in ovo vaccination of embryos at 18 days of incubation. In this study ΔgG-ILTV was delivered to chicken embryos at three different doses (10², 10³ and 10⁴ plaque forming units per egg) using manual in ovo vaccination. At 20 days after hatching, birds were challenged intra-tracheally with wild type ILTV and protection was measured. In ovo vaccination was shown to be safe, as there were no developmental differences between birds from hatching up to 20 days of age, as measured by weight gain. The highest dose of vaccine was the most efficacious, resulting in a weight gain not significantly different from unvaccinated/unchallenged birds seven days after challenge. In contrast, birds vaccinated with the lowest dose showed weight gains not significantly different from unvaccinated/challenged birds. Gross pathology and histopathology of the trachea reflected these observations, with birds vaccinated with the highest dose having less severe lesions. However, qPCR results suggested the vaccine did not prevent the challenge virus replicating in the trachea. This study is the first to assess in ovo delivery of a live attenuated ILTV vaccine and shows that in ovo vaccination with ΔgG-ILTV can be both safe and efficacious.     

Comparative efficacy of North American and antigenically matched reverse genetics derived H5N9 DIVA marker vaccines against highly pathogenic Asian H5N1 avian influenza viruses in chickens

Highly pathogenic (HP) H5N1 avian influenza has become endemic in several countries in Asia and Africa, and vaccination is being widely used as a control tool. However, there is a need for efficacious vaccines preferably utilizing a DIVA (differentiate infected from vaccinated animals) marker strategy to allow for improved surveillance of influenza in vaccinated poultry. Using a reverse genetics approach, we generated Asian rgH5N9 vaccine strain deriving the hemagglutinin gene from A/chicken/Indonesia/7/2003 (H5N1) with modification of the cleavage site to be low pathogenic (LP) and N9 neuraminidase gene from the North American LP A/turkey/Wisconsin/1968 (H5N9) virus. The recombinant rgH5N9, A/turkey/Wisconsin/1968 (H5N9) A/chicken/Hidalgo/232/1994 (H5N2), and wild type HP A/chicken/Indonesia/7/2003 (H5N1) viruses were used to prepare inactivated oil-emulsified whole virus vaccines. Two weeks after vaccination, chickens were challenged with either Asian HP H5N1 viruses, A/chicken/Indonesia/7/2003 (W.H.O. clade 2.1) or A/chicken/Supranburi Thailand/2/2004 (W.H.O. clade 1.0). The H5 HA1 of the North American vaccine strains exhibited 12% amino acid differences including amino acid changes in the major antigenic sites as compared to the Asian HP H5N1 challenge viruses, serologically exhibited substantial antigenic difference, but still provided 100% protection from mortality. However, challenge virus shedding was significantly higher in chickens immunized with antigenically distinct American lineage vaccines as compared to the antigenically matched Asian rgH5N9 and the wild type Asian H5N1 vaccine. The antibody response to the heterologous subtype neuraminidase proteins were discriminated in vaccinated and infected chickens using a rapid fluorescent 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid sodium salt as substrate for neuraminidase inhibition assay. This study demonstrates the value of using a vaccine containing antigenically matched H5 hemagglutinin for control of HP H5N1 avian influenza in poultry and the potential utility of a heterologous neuraminidase as a DIVA marker.     

A subunit vaccine candidate derived from a classic H5N1 avian influenza virus in China protects fowls and BALB/c mice from lethal challenge

In recent years, numerous human infections with avian influenza viruses in Asia have raised the concern that the next influenza pandemic is imminent. The most effective way to combat human avian influenza is through vaccination of the public. In this study, we developed an influenza A recombinant protein (rH5HA) directed against the hemagglutinin (HA) of a classic H5N1 high pathogenic avian influenza virus isolated in South China in 1996. Following purification of the recombinant protein expressed from a baculovirus expression system, we evaluated the efficiency of rH5HA on specific pathogen free (SPF) chicken, commercial chicken, and in BALB/c mice in an infection-protection model. The results demonstrated that rH5HA induced antibody responses and provided full protection in both SPF chickens and commercial chickens. Protective immunity was generated within 2 weeks in chickens as young as 7-day post-hatch using a minimum amount of rH5HA protein (2 μg/bird/vaccination). The serum antibody generated from rH5HA immunization was protective and lasted more than 6 months. Our data also demonstrated that rH5HA immunization protected BALB/c mice from a lethal challenge with pathogenic avian influenza virus. These results suggested that vaccination with rH5HA could be a vaccine candidate for the control of H5N1 avian influenza in poultry, in mice, and potentially in other mammals including human.     

Horizontal transmission dynamics of a glycoprotein G deficient candidate vaccine strain of infectious laryngotracheitis virus and the effect of vaccination on transmission of virulent virus

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. The virus is horizontally transmitted and causes large outbreaks of disease. Recent studies have shown that a glycoprotein G deficient candidate vaccine strain of ILTV (ΔgG ILTV) is safe and protects birds from disease following challenge with virulent virus. This study examined the transmission dynamics of this candidate vaccine and of ILTV in field and experimental settings. The reproduction ratio (R₀, average number of secondary infectious cases from a typical infectious case) was calculated from the growth rate of disease epidemics in broiler flocks. Assuming a latent period of 2 days and an infectious period of 4 days R₀ was estimated to be 2.43 (95% CI 2.25-2.69). In experimental settings the transmission characteristics of ΔgG ILTV were similar to those of wildtype virus, and importantly ΔgG ILTV remained safe following one in vivo passage and subsequent infection via contact-exposure. There was minimal transmission of wildtype virus in vaccinated birds. The findings from this study further demonstrate the suitability of ΔgG ILTV for use as a live attenuated vaccine. Knowledge of the basic reproduction ratio of ILTV will be valuable for future studies that aim to improve disease control using vaccination programs.     

Superinfection and recombination of infectious laryngotracheitis virus vaccines in the natural host

Infectious laryngotracheitis virus (ILTV, Gallid alphaherpesvirus 1) causes severe respiratory disease in chickens and has a major impact on the poultry industry worldwide. Live attenuated vaccines are widely available and are administered early in the life of commercial birds, often followed by one or more rounds of revaccination, generating conditions that can favour recombination between vaccines. Better understanding of the factors that contribute to the generation of recombinant ILTVs will inform the safer use of live attenuated herpesvirus vaccines. This study aimed to examine the parameters of infection that allow superinfection and may enable the generation of recombinant progeny in the natural host. In this study, 120 specific-pathogen free (SPF) chickens in 8 groups were inoculated with two genetically distinct live-attenuated ILTV vaccine strains with 1–4 days interval between the first and second vaccinations. After inoculation, viral genomes were detected in tracheal swabs in all groups, with lowest copies detected in swabs collected from the groups where the interval between inoculations was 4 days. Superinfection of the host was defined as the detection of the virus that was inoculated last, and this was detected in tracheal swabs from all groups. Virus could be isolated from swabs at a limited number of timepoints, and these further illustrated superinfection of the birds as recombinant viruses were detected among the progeny. This study has demonstrated superinfection at host level and shows recombination events occur under a very broad range of infection conditions. The occurrence of superinfection after unsynchronised infection with multiple viruses, and subsequent genomic recombination, highlight the importance of using only one type of vaccine per flock as the most effective way to limit recombination.     

Vaccine-induced host responses against very virulent Marek's disease virus infection in the lungs of chickens

The aim of this study was to investigate the kinetics of virus replication and cellular responses in the lungs following infection with Marek's disease virus (MDV) and/or vaccination with herpesvirus of turkey (HVT) via the respiratory route. Chickens vaccinated with HVT and challenged with MDV had a higher accumulation of MDV and HVT genomes in the lungs compared to the chickens that received HVT or MDV alone. This increase in virus load was associated with augmented expression of interferon (IFN)-γ and interleukin (IL)-10, and increased T cell infiltration. These findings shed more light on the immunological events that occur in the lungs after vaccination or infection with MDV.     

A recombinant field strain of Marek's disease (MD) virus with reticuloendotheliosis virus long terminal repeat insert lacking the meq gene as a vaccine against MD

Marek's disease virus (MDV) GX0101, which is a field strain with a naturally occurring insertion of the reticuloendotheliosis virus (REV) long terminal repeat (LTR) fragment, shows distinct biological activities. Deletion of the meq gene in GX0101 contributes to its complete loss of pathogenicity and oncogenicity in SPF chickens, but this virus has a kanamycin resistance gene (kan^r) residual at the site of meq gene. In the present study, the kan^r was knocked out and a meq-null virus with a good replicative ability termed SC9-1 was selected. In vivo studies showed that SC9-1 had no pathogenicity or tumorigenicity to chickens. There were no obvious impacts on chicken weight, immune organ index or antibody levels induced by avian influenza virus (AIV)/newcastle disease virus (NDV) inactivated vaccines compared with the control group. The SC9-1 virus provided superior protection than CVI988/Rispens vaccine in both SPF chickens and Hy-line brown chickens when challenged with a very virulent MDV (rMd5 strain). There was no obvious change in SC9-1 protection against MDV rMd5 in SPF chickens after 20 passages in chicken embryonic fibroblast cells (CEFs). In conclusion, SC9-1 is a safe and effective vaccine candidate for the prevention of Marek's disease.     

Long lasting immunity in chickens induced by a single shot of influenza vaccine prepared from inactivated non-pathogenic H5N1 virus particles against challenge with a highly pathogenic avian influenza virus

An influenza vaccine was prepared from inactivated whole particles of the non-pathogenic strain A/duck/Hokkaido/Vac-1/04 (H5N1) virus using an oil adjuvant containing anhydromannitol-octadecenoate-ether (AMOE). The vaccine was injected intramuscularly into five 4-week-old chickens, and 138 weeks after vaccination, they were challenged intranasally with 100 times 50% chicken lethal dose of the highly pathogenic avian influenza (HPAI) virus A/chicken/Yamaguchi/7/04 (H5N1). All 5 chickens survived without exhibiting clinical signs of influenza, although 2 days post-challenge, 3 vaccinated chickens shed limited titres of viruses in laryngopharyngeal swabs.     

A recombinant Newcastle disease virus (NDV) expressing infectious laryngotracheitis virus (ILTV) surface glycoprotein D protects against highly virulent ILTV and NDV challenges in chickens

Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). Currently, modified live ILTV vaccines are used to control ILT infections. However, the live ILTV vaccines can revert to virulence after bird-to-bird passage and are capable of establishing latent infections, suggesting the need to develop safer vaccines against ILT. We have evaluated the role of three major ILTV surface glycoproteins, namely, gB, gC, and gD in protection and immunity against ILTV infection in chickens. Using reverse genetics approach, three recombinant Newcastle disease viruses (rNDVs) designated rNDV gB, rNDV gC, and rNDV gD were generated, each expressing gB, gC, and gD, respectively, of ILTV. Chickens received two immunizations with rNDVs alone (gB, gC, and gD) or in combination (gB + gC, gB + gD, gC + gD, and gB + gC + gD). Immunization with rNDV gD induced detectable levels of neutralizing antibodies with the magnitude of response greater than the rest of the experimental groups including those vaccinated with commercially available vaccines. The birds immunized with rNDV gD showed complete protection against virulent ILTV challenge. The birds immunized with rNDV gC alone or multivalent vaccines consisting of combination of rNDVs displayed partial protection with minimal disease and reduced replication of challenge virus in trachea. Immunization with rNDV gB neither reduced the severity of the disease nor the replication of challenge virus in trachea. The superior protective efficacy of rNDV gD vaccine compared to rNDV gB or rNDV gC vaccine was attributed to the higher levels of envelope incorporation and infected cell surface expression of gD than gB or gC. Our results suggest that rNDV expressing gD is a safe and effective bivalent vaccine against NDV and ILTV.     

Protection of chickens against H5N1 highly pathogenic avian influenza virus infection by live vaccination with infectious laryngotracheitis virus recombinants expressing H5 hemagglutinin and N1 neuraminidase

Attenuated vaccine strains of the alphaherpesvirus causing infectious laryngotracheitis of chickens (ILTV, gallid herpesvirus 1) can be used for mass application. Previously, we showed that live virus vaccination with recombinant ILTV expressing hemagglutinin of highly pathogenic avian influenza viruses (HPAIV) protected chickens against ILT and fowl plague caused by HPAIV carrying the corresponding hemagglutinin subtypes [Lüschow D, Werner O, Mettenleiter TC, Fuchs W. Protection of chickens from lethal avian influenza A virus infection by live-virus vaccination with infectious laryngotracheitis virus recombinants expressing the hemagglutinin (H5) gene. Vaccine 2001;19(30):4249–59; Veits J, Lüschow D, Kindermann K, Werner O, Teifke JP, Mettenleiter TC, et al. Deletion of the non-essential UL0 gene of infectious laryngotracheitis (ILT) virus leads to attenuation in chickens, and UL0 mutants expressing influenza virus haemagglutinin (H7) protect against ILT and fowl plague. J Gen Virol 2003;84(12):3343–52]. However, protection against H5N1 HPAIV was not satisfactory. Therefore, a newly designed dUTPase-negative ILTV vector was used for rapid insertion of the H5-hemagglutinin, or N1-neuraminidase genes of a recent H5N1 HPAIV isolate. Compared to our previous constructs, protein expression was considerably enhanced by insertion of synthetic introns downstream of the human cytomegalovirus immediate-early promoter within the 5′-nontranslated region of the transgenes. Deletion of the viral dUTPase gene did not affect in vitro replication of the ILTV recombinants, but led to sufficient attenuation in vivo. After a single ocular immunization, all chickens developed H5- or N1-specific serum antibodies. Nevertheless, animals immunized with N1-ILTV died after subsequent H5N1 HPAIV challenge, although survival times were prolonged compared to non-vaccinated controls. In contrast, all chickens vaccinated with either H5-ILTV alone, or H5- and N1-ILTV simultaneously, survived without showing any clinical signs. Real-time RT-PCR indicated limited challenge virus replication after vaccination with H5-ILTV only, which was completely blocked after coimmunization with N1-ILTV. Thus, chickens can be protected from H5N1 HPAIV-induced disease by live vaccination with an attenuated hemagglutinin-expressing ILTV recombinant, and efficacy can be further increased by coadministration of an ILTV mutant expressing neuraminidase. Furthermore, chickens vaccinated with ILTV vectors can be easily differentiated from influenza virus-infected animals by the absence of serum antibodies against the AIV nucleoprotein.     

Novel in-ovo chimeric recombinant Newcastle disease vaccine protects against both Newcastle disease and infectious bursal disease

Development of a safe and efficient in-ovo vaccine against Newcastle disease (NDV) and very virulent infectious bursal disease virus (vvIBDV) is of great importance. In this study, a chimeric NDV LaSota virus with the L gene of Clone-30 (rLaC30L) was used to generate a recombinant chimeric virus expressing the VP2 protein of vvIBDV (rLaC30L-VP2). The safety and efficacy of rLaC30L-VP2 in-ovo vaccination was then evaluated in 18-day-old special pathogen free (SPF) chicken embryos and commercial broiler embryos for prevention of NDV and vvIBDV. Hatchability and global survival rate of the hatched birds was not affected by in-ovo rLaC30L-VP2 vaccination. However, rLaC30L-VP2 in-ovo vaccination induced significant anti-IBDV and anti-NDV antibodies in SPF birds and commercial broilers, and 100% of vaccinated chickens were protected against a lethal NDV challenge. In-ovo rLaC30L-VP2 vaccination also provided resistance against vvIBDV challenge in a significant amount of animals. These results suggest that rLaC30L-VP2 is a safe and efficient bivalent live in-ovo vaccine against NDV and vvIBDV.     

Generation of a reassortant avian influenza virus H5N2 vaccine strain capable of protecting chickens against infection with Egyptian H5N1 and H9N2 viruses

BackgroundAvian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells.MethodsTo investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens.ResultsWe observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding.DiscussionOur findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production.     

Assessment of viral replication in eggs and HA protein yield of pre-pandemic H5N1 candidate vaccine viruses

H5N1 infection and the potential for spread from human to human continue to pose a severe public health concern. Since vaccination remains the most effective way to prevent a potential H5N1 pandemic, the World Health Organization (WHO) Collaborating Centers (CCs) and Essential Regulatory Laboratories (ERLs) engineered and developed a panel of H5N1 pre-pandemic vaccine viruses for pandemic vaccine preparedness as well as production of antigen potency testing reagents (reference antigen and reference anti-serum) for vaccine standardization. To develop a strategy utilizing a number of biochemical methods for the characterization of the viral growth properties and protein yield in eggs, we have selected eight H5N1 pre-pandemic viruses and determined the viral Egg Infectious Dose 50 (EID₅₀), total protein yield, hemagglutinin (HA) to nucleoprotein (NP) ratios (HA:NP), and HA1 content of each virus. Our results showed that all the tested H5N1 vaccine viruses grew to high titers in eggs. The total viral protein yield varies within a narrow range, whereas there were greater differences in the HA:NP protein ratios among the eight viruses. The RP-HPLC based HA1 content analysis demonstrated that the viruses A/Anhui/1/2010, A/Hubei/1/2005, and A/goose/Guiyang/337/2006 contained higher HA contents than other five viruses including A/Vietnam/1203/2003. Our approach for analyzing virus growth and protein yield will allow us identify optimal vaccine virus in a timely manner. In addition, we successfully purified the HA proteins of H5N1 vaccine viruses by optimizing bromelain cleavage conditions. Our studies on the HA protein purification may improve the quality control of the production of influenza vaccine test reagent.     

Recombinant infectious laryngotracheitis virus expressing Newcastle disease virus F protein protects chickens against infectious laryngotracheitis virus and Newcastle disease virus challenge

In this study, we isolated and identified an infectious laryngotracheitis virus (ILTV) that was naturally avirulent in specific pathogen-free (SPF) chickens, with the aim of developing a more efficacious vaccine against ILTV and Newcastle disease virus (NDV). We constructed a US9-deleted ILTV mutant based on this avirulent ILTV, and then constructed a recombinant ILTV (designated ILTV-ΔUS9-F) expressing the fusion protein (F) of the genotype VII NDV based on the US9-deleted ILTV mutant. Expression of the F protein in ILTV-ΔUS9-F-infected cells was confirmed by indirect immunofluorescence assay and western blotting. The inserted F gene was stably expressed in ILTV-ΔUS9-F. The growth kinetics of ILTV-ΔUS9-F were comparable to those of the wild-type ILTV strain. Vaccination of SPF chickens with ILTV-ΔUS9-F produced no clinical signs but did induce low levels of NDV-specific enzyme-linked immunosorbent assay and neutralizing antibodies. A single vaccination with 10⁴ plaque-forming units (PFU) of ILTV-ΔUS9-F provided good protection against both genotype VII and IX NDVs based on clinical signs, similar to the protection provided by the commercial live La Sota vaccine. Notably, ILTV-ΔUS9-F limited the replication and shedding of genotype VII NDV from oropharyngeal swabs more efficiently than the La Sota vaccine. In addition, vaccination with lower doses (10³ and 10² PFU) of ILTV-ΔUS9-F also provided sufficient clinical protection. These results indicated that ILTV-ΔUS9-F may be a bivalent vaccine candidate against both ILTV and NDV.     

Heterologous prime-boost immunization of Newcastle disease virus vectored vaccines protected broiler chickens against highly pathogenic avian influenza and Newcastle disease viruses

Avian Influenza virus (AIV) is an important pathogen for both human and animal health. There is a great need to develop a safe and effective vaccine for AI infections in the field. Live-attenuated Newcastle disease virus (NDV) vectored AI vaccines have shown to be effective, but preexisting antibodies to the vaccine vector can affect the protective efficacy of the vaccine in the field. To improve the efficacy of AI vaccine, we generated a novel vectored vaccine by using a chimeric NDV vector that is serologically distant from NDV. In this study, the protective efficacy of our vaccines was evaluated by using H5N1 highly pathogenic avian influenza virus (HPAIV) strain A/Vietnam/1203/2004, a prototype strain for vaccine development. The vaccine viruses were three chimeric NDVs expressing the hemagglutinin (HA) protein in combination with the neuraminidase (NA) protein, matrix 1 protein, or nonstructural 1 protein. Comparison of their protective efficacy between a single and prime-boost immunizations indicated that prime immunization of 1-day-old SPF chicks with our vaccine viruses followed by boosting with the conventional NDV vector strain LaSota expressing the HA protein provided complete protection of chickens against mortality, clinical signs and virus shedding. Further verification of our heterologous prime-boost immunization using commercial broiler chickens suggested that a sequential immunization of chickens with chimeric NDV vector expressing the HA and NA proteins following the boost with NDV vector expressing the HA protein can be a promising strategy for the field vaccination against HPAIVs and against highly virulent NDVs.