The Construction,Safety And Efficacy Of A Recombinant Herpesvirus Of Turkey Vaccine Strain Expressing Hemagglutinin Of Subtype H9 Avian Influenza Virus

Subtype H9 of avian influenza viruses(AIV),classified as low pathogenic avian influenza(LPAI),causes mild respiratory disease and short-term drops in egg production,however,could also result in higher morbidity and mortality due to co-infection with secondary pathogens.H9 subtype of AIV is extremely harmful to the poultry industry,and vaccination is still one of the main strategies to control the disease.At present,all the avian influenza vaccines used in production are inactivated whole-virus vaccines,which can only induce high levels of circulating antibodies slowly but fail to effectively stimulate the cellular and mucosal immune response.Therefore,novel live H9 avian influenza vaccines,which could be combined with existing inactivated vaccines to provide more comprehensive immune protection,is in an urgent need.The use of herpesvirus of turkey(HVT)for expression of AIV immunogenic protein has become a research trend in recent years.On the one hand,HVT has more non-essential regions to facilitate insertion of heterologous genes,and on the other hand it is a commonly used poultry vaccine for early immunization,such as 1-day-old chicks injected subcutaneously or 18-day-old embryos in ovo,with earlier induction of antibody.In this study,we intend to generate recombinant HVT vectors stably expressing haemagglutinin(HA)of H9 subtype avian influenza virus.1.Construction,genetic stability and safety of rHVT-YBF003Phylogenetic analysis of HA sequences of 21 recent H9 subtype AIV isolates showed that these viruses belong to the clade h9.4.2.5 and are predominant circulating strains in recent years.We then selected the YBF003 to generate a recombinant HVT construct.The HA gene of YBF003 was amplified by RT-PCR,and inserted into the vector containing HVT FC-126 vaccine strain homologous arm and CMV promoter to assemble the transfer vector pFR-H9,which was then co-transfected with the recombinant HVT(rHVT-EGFP)expressing green fluorescent protein(GFP)into chicken embryo fibroblasts(CEF).The EGFP gene of rHVT-EGFP was subsequently replaced with the HA gene expression cassette by homologous recombination,resulting in the recombinant virus rHVT-YBF003.Then rHVT-YBF003 was plaque purified for several rounds on CEF until fluorescent green was not detected.The HA gene cassette insertion site into HVT genome and HA expression was verified by PCR,Western-blot and indirect immunofluorescence assay(IFA),respectively.There was no significant difference in growth characteristics on CEF between the rHVT-YBF003 and parental HVT FC-126 strains.Furthermore,after the rHVT-YBF003 was grown on CEF sequentially for 20 passages,HA gene cassette were confirmed by PCR from 20 randomly selected typical plaques,and similarly,stable expression of HA was detected by IFA.Genetic stability,on D0,the one-day-old SPF chickens were injected subcutaneously in the neck with the rHVT-YBF003 vaccine at 5.0 × 103 PFU/dose.On D21,lymphocytes were isolated from the collected blood,and re-inoculated in another group of one-day-old SPF chickens(3.0×106 lymphocytes/dose).After 6 serial passages in birds,PCR and IFA analysis confirmed presence of HA gene and HA protein expression in all passages.One-day-old SPF chickens were injected subcutaneously with rHVT-YBF003 at 5.0 × 103 PFU/dose.Lymphocytes were isolated from blood every 7 days,and then inoculated on CEF for virus isolation and quantification.The results showed that the viral load of recombinant virus in chickens increased with time,reached the peak at 9 weeks of age,and then gradually declined to a very low level at 20 weeks of age,indicating that the viruses are persistent in chickens for a long period of time.For safety assessment,the rHVT-YBF003 vaccine was inoculated into one-day-old SPF chickens at 5.0 × 104 PFU/dose,clinical signs and growth performance were recorded for 8 weeks.The results showed normal food and water intake with no gross pathological changes and other abnormal observations.Similar observations were obtained in 4-week-old ICR mice inoculated with rHVT-YBF003 at 5.0 ×104 PFU/dose.In addition,the internal organs of the mice were examined and weighed 14 days post inoculation(dpi),indicating no adverse effects.Taken together,these results suggest that the recombinant virus is safe to both susceptible and unsusceptible animals.2.Efficacy evaluation of rHVT-YBF003The efficacy of rHVT-YBF003 was assessed in a vaccination-challenge experiment in SPF chickens,which were randomly assigned four groups.There are in-ovo vaccination group,1-day-old vaccination group,inactivated vaccine vaccination group and mock vaccination group.The dose of vaccination in in-ovo vaccination group was 5.0 ×103 PFU(rHVT-YBF003)per 18-day-old SPF chicken embryos.In the 1-day-old vaccination group,chickens were vaccinated via subcutaneous route.Inactivated H9 AIV vaccine(YBF003 strain)was used in the 14-day-old SPF chickens via intramuscular route and those chickens were designed as the inactivated vaccine vaccination group.The other one-day-old SPF chickens received a mock vaccination of 0.5 mL PBS/chicken by subcutaneous injection.At time point of challenge,all chickens were intravenously injected with 1.0 ×107.5 EID50 of the virulent YBF003 strain at 28 days dpi.The average HI antibody titers of in-ovo vaccination group was significantly higher than 1-day-old vaccination group(4.7 ± 0.60 vs 4.5 ± 0.30),and the protection rates were almost 50%in both groups.However,the protection rate of chickens vaccinated with inactivated vaccine was 50%and the HI antibody titers was 6.5±0.75.Then,the average HI antibody titers of control group was 1.3 ±0.12 and the protection rate was 0%.The protective efficacy of the recombinant virus was evaluated on commercial chickens with the same vaccine strategies as described above.In addition,the boost vaccination group was added.Prime-boost vaccination strategywas employed in this group,which priming with the rHVT-YBF003 in the 1-day-old chickens and boost with the H9 subtype AIV inactivated vaccine at 14 days post priming.At 28 dpi,the average HI antibody titers of in-ovo vaccination group,1-day-old vaccination group and inactivated vaccine vaccination group are 4.3 ± 0.49,4.1 ± 0.89 and 6.2 ±0.34,respectively.All these three groups provided at least 50%protection against challenge.Moreover,the prime-boost vaccination strategy provided 70%protection in challenge test and the average HI antibody titers is 7.8±0.45.The HI antibody titers of control group is from 1.0 ±0.74 to 1.1 ±0.24,and the protection rate is below 30%.3.Large-scale culture process of rHVT-YBF003To increase the yield of the recombinant virus rHVT-YBF003,10-layer flasks with 6335 cm2 growth surface area,contains 1200 mL culture medium,were used for scaling up.The cell density was 2.5×106/mL of CEF were inoculated with 3.0×106 PFU and harvest at 72 hours post inoculation.The resulting inoculums could be dispensed into 50 vials with the antigen amount of 5.0×106PFU/1.8mL per vial.According to the passage level,the harvested virus could be divided into master seeds(F1-F6 generations),working seeds(F7?F12 generations)and production seeds(F13?F17 generations).One generation from each level of virus seeds was randomly selected and the three selected generations were subjected to the following tests,including purity test,mycoplasma test,identity test,safety test,efficacy test,and extraneous agents detection.The results of purity and mycoplasma tests showed that the seeds were free of bacterial and mycoplasma.The results of identity test show that both the HVT virus and HA expression were detected by IFA and Western blotting.To evaluate the vaccine safety,1-day-old SPF chickens were inoculated with 3.0×105 PFU of each selected generations.The results showed that none of the chickens had any clinical signs during the 90 days of observation.To detect the exogenous agents in vaccine,the SPF chickens were primed with each selected generations via nasal and ocular routes at 1 day of age and boosted via intramuscular injection at 21 days of age.Then,the serum were collected to detect antibodies against several common antigens in chicken at 42 days post priming.The result shows that only antibodies against HVT and H9 subtype AIV were detected which suggested that the virus seeds were free of any exogenous agents.In summary,we could conclude that the virus seeds of rHVT-YBF003 are completely meet the quality standards of national regulations.In this study,we have constructed a HVT recombinant strain(rHVT-YBF003)expressing HA protein of H9 subtype AIV.The inserted HA gene remained intact after several in vitro and in vivo passages.The rHVT-YBF003 exhibit good safety profiles and no clinical signs and pathological changes were seen in the tissues of susceptible and the less susceptible animals.In the efficacy tests,the rHVT-YBF003 virus could provide early and enough protection against AIV H9 infection after in-ovo or subcutaneous vaccination in 1-day-old SPF chickens.Besides,rHVT-YBF003 virus could confer same protection as the traditional inactivated vaccine with lower HI antibody.This finding suggested that the rHVT-YBF003 virus could not only activate the humoral immune response,but also induce the cellular and mucosal immunity.With the advantage of in-ovo vaccination,the administration of rHVT-YBF003 virus could be laborsaving.Particularly,the prime-boost vaccination strategy in this study shows the synergistic protection effect against the H9 subtype AIV in commercial chickens.In addition,qualify master and working seeds were established,then the large-scale culture process was also optimized for further application.