Potentiation of cell-mediated immune responses against recombinant HN protein of Newcastle disease virus by recombinant chicken IL-18

In this study, recombinant fowlpox viruses (rFPV/HN) expressing Newcastle disease virus (NDV) HN protein and rFPV/HN/chIL-18 co-expressing chicken IL-18 (chIL-18) and HN protein have been constructed and characterized. The co-expressed rHN/chIL-18 antigen or rchIL-18, expressed by our previous construct rFPV/chIL-18 and co-administered with NDV rHN, was assessed for its immunostimulatory activities and protection against NDV challenge in 2-week-old chickens. Chickens were vaccinated, intramuscularly, with various amounts of rHN or rHN/chIL-18 mixed with mineral oil. Production of hemagglutination-inhibition (HI) antibody depended on the concentration of the injected rHN or rHN/chIL-18. The lower HI antibody titers were obtained in chickens group rHN/chIL-18/6 and rHN/chIL-18/7, receiving 50 ng rHN/16.5 ng chIL-18 with mineral oil and 20 ng rHN/6.6 ng chIL-18 with mineral oil, respectively, compared to those in chickens rHN/6 and rHN/7, respectively receiving 50 ng and 20 ng rHN with mineral oil alone. However, the same protection rates were obtained from chickens in groups rHN/chIL-18/6 and rHN/6. Chicken groups rHN/chIL-18/7 and rHN/chIL-18/8 showed higher protective achievements than those in groups rHN/7 and rHN/8, respectively. When rchIL-18 was co-injected with 20ng rHN plus mineral oil, low level of HI antibody titer was produced; whereas, higher level of IFN-γ production and full protection rates were obtained. On the other hand, lower levels of IFN-γ production and lower protection rate (67%) were obtained in chickens injected with the same amount of rHN with mineral oil alone. Similar results were obtained when 10 ng rHN was used. Thus, when the concentration of rHN decreased to 50 ng or less, rchIL-18 reduced HI antibody production. The increase in IFN-γ production suggested that the enhancement of the cell-mediated immunity might confer the protection from NDV challenge, even accompanied with low HI antibody induction.     

Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens

A recombinant fowlpox virus (rFPV/VP2) expressing infectious bursal diseases virus (IBDV) VP2 gene has been constructed. After purification and identification of rFPV/VP2, the adjuvant activity of the recombinant chicken IL-12 (rchIL-12), synthesized by our previous construct of rFPV/chIL-12, in rFPV/VP2-expressed rVP2 antigen was assessed in one-week-old specific-pathogen free chickens. The results indicated that rchIL-12 alone or rchIL-12 plus mineral oil (MO) co-administered with rVP2 antigen significantly enhanced the production of serum neutralization (SN) antibody against IBDV, compared to those with MO alone. The SN titers in groups receiving rVP2 antigen with MO alone were more inconsistent after vaccination. On the other hand, rchIL-12 significantly stimulated IFN-γ production in serum and in splenocyte cultured supernatant, suggesting that rchIL-12 alone or plus MO significantly induced a cell-mediated immune response. Finally, bursal lesion protection from very virulent IBDV (vvIBDV) challenge in chickens receiving rVP2 antigen with rchIL-12 alone or plus MO was much more effective than that with MO alone at two weeks after boosting. Taken together, rchIL-12 alone augmented in vivo the induction of a primary and also a secondary SN antibody production and a cell-mediated immunity against IBDV rVP2 antigen, which conferred the enhancement of bursal lesion protective efficacy from vvIBDV challenge. These data indicated that a potential for chIL-12 as immunoadjuvant for chicken vaccine development such as IBDV rVP2 antigen.     

鸡新城疫重组鸡痘病毒活疫苗的免疫持续期和加强免疫研究

本研究旨在评价表达新城疫病毒（NDV）血凝素-神经氨酸酶（HN）基因的重组鸡痘病毒（rFPV-12LSHN）活疫苗的免疫持续期和加强免疫对疫苗免疫效力的影响。用rFPV-12LSHN活疫苗免疫14日龄SPF鸡,103PFU/羽,7d即可检测到NDV HI抗体应答,对NDV强毒F48E8株攻毒保护率达100%。一次免疫18周后,对NDV强毒攻击依然提供完全保护。鸡痘病毒（FPV）疫苗免疫4周,再接种rFPV-12LSHN活疫苗,攻毒保护率降低至50%。相反,rFPV-12LSHN免疫4周,随后二次免疫可显著提高对NDV的体液免疫应答水平（P〈0.01）,对NDV强毒攻击的保护率仍然为100%。结果表明,表达NDV HN基因的重组鸡痘病毒（rFPV-12LSHN）活疫苗,能够快速建立坚强免疫力,免疫持续期至少可达18周,rFPV-12LSHN的二次免疫可以提高疫苗的免疫力。     

Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay

Eleven monoclonal antibodies (mAbs) which are specific for chicken interleukin-2 (chIL-2) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting and neutralizing assays. These mAbs were used to develop a mAb-based antigen capture ELISA specific for chicken IL-2 detection. Anti-IL-2 mAbs bound specifically to E. coli-derived rchIL-2 in ELISA and identified a 16 kDa IL-2 polypeptide band in Western blot. Several mAbs were shown to neutralize the biological activities of both rchIL-2 and native chicken IL-2 as measured by concanavalin A (ConA)-induced lymphocyte proliferation assay, IL-2 bioassay, and natural killer cell assay. Among the neutralizing mAbs, the mAb chIL-2/11 was most potent in neutralizing IL-2 activity. To develop a sensitive ELISA for the detection of chicken IL-2, an antigen capture ELISA was developed using the mAb chIL-2/16 as the antigen capture antibody and rabbit anti-IL-2 peptide antibody as the detection antibody. Using the mAb-based antigen capture ELISA, significant correlation between the level of IL-2 detected in bioassays and in ELISA was observed. These results showed that the mAb-based antigen capture ELISA is less time-consuming and more reliable compared to a conventional IL-2 bioassay for chicken IL-2. These neutralizing mAbs will facilitate basic immunobiological studies of the role of IL-2 in normal and disease states in chickens.     

Vaccination against Newcastle disease with a recombinant baculovirus hemagglutinin-neuraminidase subunit vaccine

Vaccination of chickens with an oil-emulsion vaccine containing a recombinant baculovirus that expressed the hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV)-induced hemagglutination-inhibition (HI) and virus-neutralizing antibodies against NDV. HI antibody titers obtained in response to vaccination with the live recombinant virus were higher than those obtained when the recombinant was inactivated with beta-propiolactone, and the titers were lower than those obtained in response to the same HN concentrations in live or beta-propiolactone-inactivated NDV strain B1. The serological response to the recombinant baculovirus was differentiated from the response to NDV by an enzyme-linked immunosorbent assay in which purified NDV nucleoprotein was used as antigen. Chickens vaccinated with the live recombinant or with inactivated NDV resisted an oculonasal challenge with the neurotropic velogenic Texas GB strain of NDV, which was lethal in unvaccinated controls. It was concluded that the HN protein of NDV expressed as a subunit by a recombinant baculovirus was protective against Newcastle disease.     

Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2¹³ per 25 µL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4℃. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.     

表达NDV HN基因的重组鸡痘病毒的部分生物学特性研究

为了评价转基因对鸡痘病毒(FPV)生物学特性的影响,通过电镜观察表达新城疫病毒(NDV)血凝素-神经氨酸酶蛋白(HN)基因重组鸡痘病毒(rFPV-12LSHN)感染的鸡胚成纤维细胞(CEF).结果表明:在FPV中插入NDV HN基因后,不改变rFPV的形态、病毒成熟过程;rFPV-12LSHN与FPV在CEF上的产量无...     

Protection of chickens from Newcastle disease and infectious laryngotracheitis with a recombinant fowlpox virus co-expressing the F, HN genes of Newcastle disease virus and gB gene of infectious laryngotracheitis virus

A recombinant fowlpox virus (rFPV) coexpressing the Newcastle disease virus (NDV) fusion and hemagglutinin-neuraminidase genes and infectious laryngothracheitis virus (ILTV) glycoprotein B gene was constructed. This virus was then evaluated for its ability to protect specific-pathogen-free (SPF) chickens against clinical symptoms and death after challenge by virulent NDV and ILTV. SPF chickens were grouped and vaccinated with the rFPV and commercial NDV (La Sota) and ILTV attenuated live vaccine (Nobilis ILT), respectively. After challenge with NDV 10 days postvaccination, 70% of chickens vaccinated with rFPV were protected from death, whereas 100% of the commercial NDV-vaccinated chickens were protected from death. In contrast, 100% of the unvaccinated chickens died after challenge. After challenge with ILTV, both the rFPV and commercial ILTV-vaccinated chickens were completely protected from death and 70% of chickens were protected from respiratory signs. In comparison, 100% of the unvaccinated chickens developed severe respiratory disease and 10% of chickens died. The protective efficacy was also measured by the antibody responses and isolation of challenge viruses. Results showed that this rFPV could be a potential vaccine for preventing NDV and ILTV by a single immunization.     

SIMULATED NATURAL INFECTION OF CHICKENS WITH AUSTRALIAN LENTOGENIC NEWCASTLE DISEASE VIRUS AND SUBSEQUENT CHALLENGE WITH VIRULENT VIRUS

A total of 291 eight-week-old chickens were exposed to chickens infected with either of two Australian lentogenic strains (V4 and AVL NDV-1) of Newcastle disease virus (NDV). At 3 weeks after exposure, all chickens exposed to V4 infected chickens had developed haemagglutination-inhibition (HI) antibody. All chickens exposed to AVL NDV-1 virus infected chickens had developed HI antibody 5 weeks later. This sudden late appearance of HI antibody, to titres higher than those observed with V4 chickens, was explained by V4 virus being introduced to the AVL NDV-1 group of chickens. When groups of these chickens were challenged with Roakin virus (mesogenic NDV) at 3 weeks and Fontana 1083 virus (viscerotropic velogenic NDV) and Texas GB virus (neutrotropic NDV) at 3, 5, 10 and 21 weeks only three chickens developed clinical illness one of which died. These chickens were one AVL NDV-1 chicken contact challenged with Fontana 1083 virus at 3 weeks, one V4 chicken oronasally challenged with Texas GB virus at 5 weeks and one V4 chicken challenged oronasally with Fontana 1083 virus at 10 weeks. Susceptible non-vaccinated chickens died soon after challenge. Challenge by oronasal infection with 10(7.0) ELD50 of virus or contact with susceptible infected chickens enabled virulent virus to be isolated from most chickens and was accompanied by a large anamnestic increase in serum HI antibody.     

The efficacy of recombinant fowlpox vaccine protection against Marek's disease: its dependence on chicken line and B haplotype

Earlier studies have shown that the B haplotype has a significant influence on the protective efficacy of vaccines against Marek's disease (MD) and that the level of protection varies dependent on the serotype of MD virus (MDV) used in the vaccine. To determine if the protective glycoprotein gene gB is a basis for this association, we compared recombinant fowlpox virus (rFPV) containing a single gB gene from three serotypes of MDV. The rFPV were used to vaccinate 15.B congenic lines. Nonvaccinated chickens from all three haplotypes had 84%-97% MD after challenge. The rFPV containing gB1 provides better protection than rFPV containing gB2 or gB3 in all three B genotypes. Moreover, the gB proteins were critical, since the B*21/*21 chickens had better protection than chickens with B*13/*13 or B*5/*5 using rFPV with gB1, gB2, or gB3. A newly described combined rFPV/gB1gEgIUL32 + HVT vaccine was analyzed in chickens of lines 15 x 7 (B*2/*15) and N (B*21/*21) challenged with two vv+ strains of MDV. There were line differences in protection by the vaccines and line N had better protection with the rFPV/gB1gEgIUL32 + HVT vaccines (92%-100%) following either MDV challenge, but protection was significantly lower in 15 X 7 chickens (35%) when compared with the vaccine CVI988/Rispens (94%) and 301B1 + HVT (65%). Another experiment used four lines of chickens receiving the new rFPV + HVT vaccine or CVI988/Rispens and challenge with 648A MDV. The CVI 988/Rispens generally provided better protection in lines P and 15 X 7 and in one replicate with line TK. The combined rFPV/gB1gEgIUL32 + HVT vaccines protected line N chickens (90%) better than did CVI988/Rispens (73%). These data indicate that rFPV + HVT vaccines may provide protection against MD that is equivalent to or superior to CVI988/ Rispens in some chicken strains. It is not clear whether the rFPV/gB1gEgIUL32 + HVT vaccine will offer high levels of protection to commercial strains, but this vaccine, when used in line N chickens, may be a useful model to study interactions between vaccines and chicken genotypes and may thereby improve future MD vaccines.     

Pathogenicity and immunogenicity of different recombinant Newcastle disease virus clone 30 variants after in ovo vaccination

Even though Newcastle disease virus (NDV) live vaccine strains can be applied to 1-day-old chickens, they are pathogenic to chicken embryos when given in ovo 3 days before hatch. Based on the reverse genetics system, we modified recombinant NDV (rNDV) established from lentogenic vaccine strain Clone 30 by introducing specific mutations within the fusion (F) and hemagglutinin-neuraminidase (HN) proteins, which have recently been suggested as being responsible for attenuation of selected vaccine variants (Mast et al. Vaccine 24:1756-1765, 2006) resulting in rNDV49. Another recombinant (rNDVGu) was generated to correct sequence differences between rNDV and vaccine strain NDV Clone 30. Recombinant viruses rNDV, rNDV49, and rNDVGu have reduced virulence compared with NDV Clone 30, represented by lower intracerebral pathogenicity indices and elevated mean death time. After in ovo inoculation, hatchability was comparable for all infected groups. However, only one chicken from the NDV Clone 30 group survived a 21-day observation period; whereas, the survival rate of hatched chicks from groups receiving recombinant NDV was between 40% and 80%, with rNDVGu being the most pathogenic virus. Furthermore, recombinant viruses induced protection against challenge infection with virulent NDV 21 days post hatch. Differences in antibody response of recombinant viruses indicate that immunogenicity is correlated to virulence. In summary, our data show that point mutations can reduce virulence of NDV. However, alteration of specific amino acids in F and HN proteins of rNDV did not lead to further attenuation as indicated by their pathogenicity for chicken after in ovo inoculation.     

非典型鸡新城疫的病因研究初报（一）

应用血清学、生物学方法、从我省产蛋量下降、HI抗体在1：128以上的疑似非典型鸡新城疫（CND）病鸡群中分离到1株病毒，经鉴定和致病力测定，明确是1株ND强毒，毒力与F48E9相近，然后采用该株野毒感染不同抗体水平的3批蛋鸡及肉鸡，结果3批蛋鸡均是低抗体滴度组（1：10－1：40）发生了CND症状，感染后6－7天开始产蛋停止，经15－17天后才恢复产蛋，高抗体滴定组（1：80以上）无明显变化，无抗体对照组出现典型鸡新城疫变化，全部死亡；病毒感染后第6、10天监测HI抗体，结果低滴度组第6天达1：80－1：160，第10天达1：160－1：1280。试验结果表明，我们分离到的NDV，既可引起典型的ND，也可引起非典型的ND；高HI抗体蛋鸡产生非典型鸡新城疫的病因之一，是由于存在低抗体鸡群而发生了CND，其高HI抗体有竞争是由于强毒感染后产生的抗体。     

猪脾转移因子提高LaSota株鸡新城疫弱毒疫苗的免疫保护率

旨在了解猪脾转移因子（TF）对新城疫病毒（NDV）弱毒疫苗LaSota株的免疫增强效果及机制。本研究分别采用不同剂量（10^-2、10^-3、10^-4、10^-5羽份） LaSota疫苗株单独（单独免疫组）、LaSota疫苗株与TF联合（联合免疫组）免疫SPF鸡,14 d后以NDV F₄₈E₉强毒攻毒,同时设立对照组（非免疫攻毒）和空白组（非免疫非攻毒）,并采用ELISA方法与蛋白质芯片技术分别测定外周血IL-4、IFN-γ与IL-12 P40浓度。结果显示,鸡免疫10^-2、10^-3、10^-4、10^-5羽份时疫苗攻毒保护率和半数保护量（PD₅₀）如下：单独免疫组分别为100%、55%、0%、0%和0.000 8羽份,联合免疫组分别为100%、75%、0%、0%和0.000 5羽份,对照组和空白组死亡率分别为100%和0%。免疫10^-3羽份疫苗后,联合免疫组IL-4和IFN-γ含量均高于其他组,并于第7、14天差异极显著（P<0.01）;攻毒后,联合免疫组IL-4、IFN-γ和IL-12 P40含量均高于其他组,IL-4于第1、14天,IFN-γ于第1、3天,IL-12 P40于第1天差异极显著（P<0.01）。综上表明,TF可增强IFN-γ、IL-12介导的细胞免疫和IL-4介导的体液免疫,提高LaSota株弱毒疫苗的免疫保护率,降低疫苗PD₅₀;在抵抗NDV F₄₈E₉强毒攻击时,联合免疫组的免疫保护率明显高于单独免疫组。     

AEROSOL VACCINATION OF CHICKENS WITH THE V4 STRAIN OF NEWCASTLE DISEASE VIRUS

SUMMARY Two-week-old chickens, free of detectable maternal antibody to Newcastle disease virus (NDV), or with low levels of maternal antibody, were vaccinated with the V4 strain of NDV. Haemagglutination inhibition (HI) antibodies were determined at intervals after vaccination. Two hundred chickens were vaccinated by exposure to an aerosol, a dose of 10⁶ 50% embryo infectious doses (EID⁵⁰) being allowed per chicken. Forty unvaccinated chickens were placed in direct contact with vaccinated chickens. Most of the vaccinated chickens and the incontact chickens had developed HI antibodies of titre ≥ 8 within 2 weeks of vaccination. The HI antibodies in many chickens persisted for at least 8 weeks. Control chickens in a shed 15 metres from the shed containing the vaccinated chickens did not develop HI antibodies to NDV. NDV could be isolated from some vaccinated chickens for 15 days after vaccination. An aerosol dose of 10⁵EID₅₀ per chicken failed to induce a serological response in 2 groups of 40 chickens each. HI antibodies were produced in 1 of 2 groups, each of 40 chickens, vaccinated with 10⁶EID₅₀ and in both of 2 groups of 40 chickens each vaccinated with 10⁷EID₅₀. Duplicate groups of 40 chickens were vaccinated with 10⁶EID₅₀ of V4 virus per chicken administered either as an aerosol, a coarse spray or a droplet placed in the conjunctival sac. HI antibodies were produced in all the groups of chickens.     

The effect of dietary lysine deficiency on the immune response to Newcastle disease vaccination in chickens

The effect of lysine deficiency on chicken immune function was evaluated using broiler chickens fed a diet with lysine at 67% of the control diet (1.24% lysine). The evaluation of humoral immune function was conducted by measuring the antibody production to a live Newcastle disease virus (NDV) vaccination using the hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA). The cellular immune function was evaluated through the use of cutaneous basophil hypersensitivity test. The antibody response to NDV vaccination was reduced in broiler chickens fed a lysine-deficient diet when measured by ELISA but not when measured by HI. The cell-mediated immune response was also reduced by lysine deficiency.     

Improved protection from velogenic Newcastle disease virus challenge following multiple immunizations with plasmid DNA encoding for F and HN genes

Specific-pathogen free (SPF) chickens were inoculated with the plasmid constructs encoding the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of Newcastle disease virus (NDV), either individually or in combination and challenged with velogenic NDV. The antibody level against NDV was measured using commercial enzyme linked immunosorbent assay (ELISA). In the first immunization regimen, SPF chickens inoculated twice with NDV-F or NDV-HN constructs elicited antibody responses 1 week after the second injection. However, the levels of the antibody were low and did not confer significant protection from the lethal challenge. In addition, administration of the plasmid constructs with Freund's adjuvant did not improve the level of protection. In the second immunization regimen, chickens inoculated twice with the plasmid constructs emulsified with Freund's adjuvant induced significant antibody titers after the third injection. Three out of nine (33.3%) chickens vaccinated with pEGFP-HN, five of ten (50.0%) chickens vaccinated with pEGFP-F and nine of ten (90.0%) chickens vaccinated with combined pEGFP-F and pEGFP-HN were protected from the challenge. No significant differences in the levels of protection were observed when the chickens were vaccinated with linearized pEGFP-F. The results suggested that more than two injections with both F and HN encoding plasmid DNA were required to induce higher level of antibodies for protection against velogenic NDV in chickens.     

鸵鸟新城疫病毒野毒株的分离及其生物学特性

用鸡胚从疑似鸵鸟新城疫的送检病料中分离到 2株病毒。 2株病毒均能凝集鸡红细胞 ,且能被抗新城疫病毒 ( NDV)阳性血清抑制 ;用抗 NDV单抗 PEG夹心 ELISA测定 2株分离毒均为阳性。对其中 1株作进一步生物学特性鉴定 ,按照国际上规定的 NDV毒力判定标准测定的该毒株最低致死量致死鸡胚的平均死亡时间 ( MDT)为 54h,1日龄雏鸡脑内接种致病指数 ( ICPI)为 1 .88,6周龄雏鸡静脉接种致病指数 ( IVPI)为2 .75。结果表明 ,本分离株为鸵鸟新城疫病毒强毒     

Modulation of systemic and mucosal immunity against an inactivated vaccine of Newcastle disease virus by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interleukin-18 and interferon-α

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to limitations in the efficacy against currently circulating ND viruses, existing vaccination strategies require improvements, and incorporating immunomodulatory cytokines with existing vaccines might be a novel approach. Here, we investigated the systemic and mucosal immunomodulatory properties of oral co-administration of chicken interleukin-18 (chIL-18) and chicken interferon-α (chIFN-α) using attenuated Salmonella enterica serovar Typhimurium on an inactivated ND vaccine. Our results demonstrate that oral administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α provided enhanced systemic and mucosal immune responses, as determined by serum hemagglutination inhibition antibody and NDV Ag-specific IgG as well as NDV Ag-specific IgA in lung and duodenal lavages of chickens immunized with inactivated ND vaccine via the intramuscular or intranasal route. Notably, combined oral administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α significantly enhanced systemic and mucosal immunity in ND-vaccinated chickens, compared to single administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α. In addition, oral co-administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α provided enhanced NDV Ag-specific proliferation of peripheral blood mononuclear cells and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results provide valuable insight into the modulation of systemic and mucosal immunity by incorporation of immunomodulatory chIL-18 and chIFN-α using Salmonella vaccines into existing ND vaccines.