Immunostimulatory properties of a novel adjuvant administered with inactivated influenza virus vaccine

The immunopotentiating activity of a new delivery system was investigated comparatively to Alhydrogel adjuvant, as an antiviral inactivated vaccine after one injection. The efficiency of the new formulation (BioMed) was evaluated with an inactivated porcine strain of influenza (A/Sw/IN/1726/88 H1N1) in the pig model. The first assessment criteria was the follow-up of selected immunological parameters such as, antibody levels, lymphoproliferation, double positive CD4+CD8+ T lymphocytes and cytokine production (IL-2, IL-4, IFN-gamma). The second criteria was the estimate of the protection level of animals exposed to a homologous challenge of 50 PID50 one month after a single immunizing or control injection. In the BioMed group of animals, 4 pigs (out of 6) were free of macroscopic lesion, while lesions could be seen in all individuals of other groups and virus was isolated in only one animal, whereas all other animals of other groups had virus in their lungs. This better protection of BioMed animals seems to be correlated mainly with higher levels of antibodies and to a lesser extent with a slightly better CMI response and probably with the production of memory CD4+CD8+ T cells.     

M148R and M149R are two virulence factors for myxoma virus pathogenesis in the European rabbit

Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). MYXV has a linear double-stranded DNA genome that encodes several factors important for evasion from the host immune system. Among them, four ankyrin (ANK) repeat proteins were identified: M148R, M149R, M150R and M-T5. To date, only M150R and M-T5 were studied and characterized as critical virulence factors. This article presents the first characterization of M148R and M149R. Green Fluorescent Protein (GFP) fusions allowed us to localize them in a viral context. Whereas M149R is only cytoplasmic, interestingly, M148R is in part located in the nucleolus, a unique feature for an ANK repeat poxviral protein. In order to evaluate their implication in viral pathogenicity, targeted M148R, M149R, or both deletions were constructed in the wild type T1 strain of myxoma virus. In vitro infection of rabbit and primate cultured cells as well as primary rabbit cells allowed us to conclude that M148R and M149R are not likely to be implicated in cell tropism or host range functions. However, in vivo experiments revealed that they are virulence factors since after infection of European rabbits with mutant viruses, a delay in the onset of clinical signs, an increase of survival time and a dramatic decrease in mortality rate were observed. Moreover, histological analysis suggests that M148R plays a role in the subversion of host inflammatory response by MYXV.     

猪细小病毒-伪狂犬病病毒二联油乳剂灭活疫苗的研制

猪细小病毒(Porcine paruovirus，PPV)和伪狂犬病病毒(Pseudorabies virus，PRV)均是导致母猪发生繁殖障碍性疾病的主要病原，它们在世界范围内广泛分布，造成了巨大的经济损失，严重制约了养猪业的健康发展，已经成为猪病研究的热点。     

Protection of chickens against infectious bronchitis virus with a multivalent DNA vaccine and boosting with an inactivated vaccine

The protective efficacy of DNA plasmids encoding avian infectious bronchitis virus (IBV) S1, N, or M protein was investigated in chickens. Chickens were inoculated monovalently (with plasmid pVAX1-16S1, pVAX1-16M, or pVAX1-16N alone) or multivalently (combination of the three different plasmids, pVAX1-16S1/M/N). A prime-boost immunization protocol against IBV was developed. Chickens were immunized with the multivalent DNA vaccine twice and then boosted with an inactivated vaccine once. Antibody titers of the chickens immunized with pVAX1-16S1/M/N were much higher than those of the monovalent groups (p < 0.01). A protective rate up to 90% was observed in the pVAX1-16S1/M/N group. The serum antibody titers in the prime-boost birds were significantly higher than those of the multivalent DNA vaccine group (p < 0.01) but not significantly different compared to the inactivated vaccine group at 49 days of age. Additionally, the prime-boost group also showed the highest level of IBV-specific cellular proliferation compared to the monovalent groups (p < 0.01) but no significant difference was found compared to the multivalent DNA vaccine group, and the prime-boost group completely protected from followed viral challenge.     

Construction of a Salmonella Gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens

In order to develop a novel, safe and immunogenic fowl typhoid (FT) vaccine candidate, a Salmonella Gallinarum ghost with controlled expression of the bacteriophage PhiX174 lysis gene E was constructed using pMMP99 plasmid in this study. The formation of the Salmonella Gallinarum ghost with tunnel formation and loss of cytoplasmic contents was observed by scanning electron microscopy and transmission electron microscopy. No viable cells were detectable 24 h after the induction of gene E expression by an increase in temperature from 37 °C to 42 °C. The safety and protective efficacy of the Salmonella Gallinarum ghost vaccine was tested in chickens that were divided into four groups: group A (non-immunized control), group B (orally immunized), group C (subcutaneously immunized) and group D (intramuscularly immunized). The birds were immunized at day 7 of age. None of the immunized animals showed any adverse reactions such as abnormal behavior, mortality, or signs of FT such as anorexia, depression, or diarrhea. These birds were subsequently challenged with a virulent Salmonella Gallinarum strain at 3 weeks post-immunization (wpi). Significant protection against the virulent challenge was observed in all immunized groups based on mortality and post-mortem lesions compared to the non-immunized control group. In addition, immunization with the Salmonella Gallinarum ghosts induced significantly high systemic IgG response in all immunized groups. Among the groups, orally-vaccinated group B showed significantly higher levels of secreted IgA. A potent antigen-specific lymphocyte activation response along with significantly increased percentages of CD4⁺ and CD8⁺ T lymphocytes found in all immunized groups clearly indicate the induction of cellular immune responses. Overall, these findings suggest that the newly constructed Salmonella Gallinarum ghost appears to be a safe, highly immunogenic, and efficient non-living bacterial vaccine candidate that protects against FT.     

Construction of a multiple targeting RNAi plasmid that inhibits target gene expression and FMDV replication in BHK-21 cells and suckling mice

Foot-and-mouth disease (FMD) is a highly contagious disease that afflicts cloven-hoofed animals. The etiological agent of FMD is foot-and-mouth disease virus (FMDV). The VP1 gene of FMDV is essential during the life cycle of the virus and plays a key role in the attachment of the virus to susceptible cells. We constructed a plasmid, pCWN11, that expresses siRNAs multiple-targeting the VP1 genes of FMDV. We evaluated the gene silencing efficiency of the plasmid using an enhanced green fluorescent protein (EGFP) reporter system in BHK-21 cells. The antiviral potential of the plasmid in BHK-21 cells and suckling mice were investigated. The results indicate that cotransfection of pCWN11 with any one of three serotypes VP1-EGFP plasmids resulted in a reduction in the EGFP signal relative to the control. Moreover, the antiviral potential induced by pCWN11 was evident during challenge with one FMDV isolate of either serotype O (HKN/2002) or serotype Asia I (YNBS/58), and the inhibition extended to almost 40 h. Furthermore, subcutaneous injection of pCWN11 in the neck made suckling mice significantly less susceptible to FMDV serotype O and Asia I.     

An inactivated vaccine for the control of bluetongue virus serotype 16 infection in sheep in Italy

Because no suitable products are at the moment available to safely control the spread of BTV-16 in Europe, an inactivated vaccine was produced from the reference field isolate of bluetongue virus serotype 16. One group of six sheep was vaccinated subcutaneously with the inactivated vaccine twice, on days 0 and 28, whereas a second group of eight sheep was inoculated with saline solution and used as mock-vaccinated control animals. Seventy-eight days after the first vaccination, all sheep were inoculated subcutaneously with a suspension containing 10(6.3) TCID(50) of a virulent reference BTV-16 isolate. Apart from a transient inflammatory reaction at the injection site, no adverse effects were reported following vaccination. All vaccinated animals developed high titres (7.3-9.3log(2)(ED50%/50 microl)) of virus-specific neutralising antibodies and were resistant to challenge with BTV-16. Conversely, following challenge, control animals developed hyperthermia and long lasting high-titre viraemia.     

Safety and immunogenicity of a delta inulin-adjuvanted inactivated Japanese encephalitis virus vaccine in pregnant mares and foals

In 2011, following severe flooding in Eastern Australia, an unprecedented epidemic of equine encephalitis occurred in South-Eastern Australia, caused by Murray Valley encephalitis virus (MVEV) and a new variant strain of Kunjin virus, a subtype of West Nile virus (WNV_KUN). This prompted us to assess whether a delta inulin-adjuvanted, inactivated cell culture-derived Japanese encephalitis virus (JEV) vaccine (JE-ADVAX™) could be used in horses, including pregnant mares and foals, to not only induce immunity to JEV, but also elicit cross-protective antibodies against MVEV and WNV_KUN. Foals, 74–152 days old, received two injections of JE-ADVAX™. The vaccine was safe and well-tolerated and induced a strong JEV-neutralizing antibody response in all foals. MVEV and WNV_KUN antibody cross-reactivity was seen in 33% and 42% of the immunized foals, respectively. JE-ADVAX™ was also safe and well-tolerated in pregnant mares and induced high JEV-neutralizing titers. The neutralizing activity was passively transferred to their foals via colostrum. Foals that acquired passive immunity to JEV via maternal antibodies then were immunized with JE-ADVAX™ at 36–83 days of age, showed evidence of maternal antibody interference with low peak antibody titers post-immunization when compared to immunized foals of JEV-naïve dams. Nevertheless, when given a single JE-ADVAX™ booster immunization as yearlings, these animals developed a rapid and robust JEV-neutralizing antibody response, indicating that they were successfully primed to JEV when immunized as foals, despite the presence of maternal antibodies. Overall, JE-ADVAX™ appears safe and well-tolerated in pregnant mares and young foals and induces protective levels of JEV neutralizing antibodies with partial cross-neutralization of MVEV and WNV_KUN.     

NDV、IBDV二联灭活苗对蛋雏鸡免疫程序研究

为探讨鸡新城疫病毒(NDV)、传染性法氏囊病毒(IBDV)二联灭活疫苗在蛋雏鸡养殖中的实际应用效果进行本试验。用鸡新城疫、传染性法氏囊二联灭活疫苗及传染性法氏囊活疫苗以不同免疫程序免疫7~14日龄蛋雏鸡,定期监测NDV、IBDV抗体水平,并在雏鸡60日龄时进行攻毒保护试验。结果表明,各二联苗免疫组NDV抗体水平在免疫后14 d即达到NDV血清学最低有效保护价标准,并维持至雏鸡60日龄。免疫后28 d,灭活苗一次性免疫各组IBDV中和抗体水平显著高于活苗两次免疫组,琼扩抗体水平与活苗两次免疫组无明显差异。雏鸡60日龄时攻毒,各免疫组均100%保护,对照组无保护。以上结果说明该二联灭活疫苗可替代IBDV活毒疫苗用于7~14日龄商品雏鸡免疫,免疫期至少为60d。     

Inhibition of virus replication in the tonsils of pigs previously vaccinated with a Chinese strain vaccine and challenged oronasally with a virulent strain of classical swine fever virus

After an oronasal (O.N.) infection with classical swine fever (C.S.F.) virus, virus multiplication can be detected in the tonsils from Day 2 post infection (p.i.) till death. The course of viral replication during the first 10 days after O.N. challenge exposure of pigs, previously vaccinated with a Chinese strain vaccine in the presence or absence of maternal antibodies, was studied using direct immunofluorescence techniques on cryostat sections and virus isolations. When piglets were challenged O.N. in the presence of maternal antibodies, virus replication in the tonsils still occurred. The multiplication period and the localization of the virus, however, were directly correlated to the maternal antibody levels. The maternal antibody level also seems responsible for the efficacy of the vaccination to prevent challenge virus replication in the tonsils: vaccination in the presence of low maternal antibody titers completely inhibited virus replication; vaccination in the presence of high maternal antibody titers only reduced the multiplication period of the O.N.-administered virulent virus. In both cases, animals were challenged 1 week post vaccination. Vaccination of seronegative animals resulted in an almost complete inhibition of the virus replication in the tonsils during a full fattening period: cryostat sections revealed a limited virus replication in three out of 20 animals. In one of these animals, virus replication was probably so negligible that virus isolation remained negative.     

细胞亲环蛋白A对猪瘟病毒增殖的影响

细胞亲环蛋白A(CyPA)在多种病毒感染中起重要的调控作用。本实验室前期蛋白质组学研究表明,猪瘟病毒(CSFV)感染PK-15后细胞的CyPA明显上调表达。为研究CyPA在CSFV增殖中的作用,本研究首先采用环孢素A(CsA)处理PK-15细胞,特异性地抑制CyPA顺反异构酶活性,观察对CSFV的影响,在CsA处理后24 h、48 h、72 h收集细胞和细胞冻融上清,进行病毒基因组拷贝数和病毒滴度测定。结果表明CsA处理能够抑制CSFV在PK-15细胞中的增殖。同时,采用RNA干扰方法,下调PK-15细胞中CyPA的表达,结果也能够抑制CSFV在细胞中的增殖。本研究结果证明CyPA对CSFV在PK-15细胞中的增殖具有调控作用,对进一步阐明CSFV与宿主细胞蛋白的相互作用具有重要意义。     

Preliminary testing in turkeys of the safety and efficacy of a putative haemorrhagic enteritis virus vaccine

Haemorrhagic enteritis virus (HEV) causes clinical haemorrhagic enteritis in young poults and/or subclinical immunosuppression which is often associated with colibacillosis. This disease is controlled with live vaccines worldwide, however, importation of HEV vaccines or cells that support HEV propagation are not permitted in Australia. A major experiment in isolators was conducted to test the safety and efficacy of a putative HEV vaccine. The study had a factorial design with four factors namely vaccination age (28 and 42 days of age), vaccine dose (0, 10⁵, 10⁶, 10⁷ genomic copies of HEV vaccine), challenge with HEV (yes, no) and vaccination‐challenge interval (7, 21 or 42 days). A total of 315 poults were used providing 6‐8 birds per treatment combination. Turkey growth rate, mortality, pathological findings, anti‐HEV antibodies and viral load were examined. Vaccination lead to significant increases in anti HEV antibody over the following 2‐4 weeks. Overall, vaccination with 10⁶ and 10⁷ was protective against increase in relative splenic weight and splenic viral load in challenged birds. Clinical haemorrhagic enteritis was not induced by any treatment but there was an increased incidence of airsacculitis in groups receiving either HEV vaccine or challenge virus compared to the negative control birds (25.8‐29.3% vs. 9.4%, P < 0.05). Growth rate, mortality and relative bursal weight were unaffected by vaccination. This laboratory level study indicates that the putative vaccine is safe and likely to be efficacious, but may cause elevated levels of airsacculitis. These findings require confirmation in larger scale field trials.     

Combined effects of fasting and diet on interferon production and virus replication in calves infected with a vaccine strain of infectious bovine rhinotracheitis virus

A study was undertaken to investigate the combined effects of fasting and different diets on interferon (IFN) production and virus replication measured in nasal secretions of calves inoculated with a vaccine strain of infectious bovine rhinotracheitis virus. Four groups of calves were inoculated intranasally with infectious bovine rhinotracheitis virus. Two groups were inoculated 24 hours after onset of a 3-day fast; upon refeeding, 1 group was fed a maintenance diet (M diet) of hay, and the other was fed a higher energy diet (HE diet) of hay and concentrate. Nonfasted control groups were fed the M diet or the HE diet. Overall IFN production was highest (P less than 0.01) in nonfasted calves fed the M diet throughout the study and lowest in nonfasted calves fed the HE diet. Fasted calves refed the HE diet produced consistently and significantly more IFN than did nonfasted calves fed this diet. Fasted calves refed the M diet, however, produced significantly less IFN, compared with control calves fed the M diet throughout the study. Overall mean virus excretion was similar in all groups; therefore, the amount of virus replication per se did not account for the differences in IFN production, nor did greater IFN production result in less virus excretion. Serum cortisol concentrations and immune responses were not significantly affected by fasting or diet.     

血红素加氧酶1对猪瘟病毒复制的影响

本实验研究了猪血红素加氧酶1对猪瘟病毒(CSFV)在PK-15细胞中复制的影响,本研究应用siRNA干扰方法抑制PK-15细胞中血红素加氧酶1表达后再接种CSFV,细胞中CSFV基因组增殖与对照组相比显著下降;感染病毒细胞中和培养上清中CSFV滴度也显著降低。本实验首次报道了干扰细胞中HO-1表达对CSFV细胞中增殖的影响,提出了CSFV与血红素加氧酶1新的相互作用关系。     

Comparison of the safety and efficacy of a recombinant feline leukemia virus (FeLV) vaccine delivered transdermally and an inactivated FeLV vaccine delivered subcutaneously

The efficacy of a new recombinant FeLV vaccine (rFeLV), delivered transdermally via a needle-free delivery device was compared to that of an inactivated FeLV vaccine (FeLV-k), administered subcutaneously, with a conventional needle and syringe. Kittens were immunized with either rFeLV (0.25 ml, transdermal) or FeLV-k (1 ml, subcutaneous); or they were sham-vaccinated with physiologic saline (0.25 ml, transdermal). Two vaccinations were administered 21 days apart. Injection sites were monitored for any acute or subacute reactions relative to vaccine administration. Four weeks following the final vaccination, all cats were subject to oro-nasal FeLV challenge. Blood was collected for determination of FeLV antigenemia (p27) at weekly intervals beginning three weeks post-challenge. All of the vaccinated cats from both groups resisted FeLV challenge; and 90% of the control cats developed persistent FeLV antigenemia in response to challenge. No acute or persistent injection site reactions were observed. The rFeLV, delivered transdermally, provides protection against persistent FeLV antigenemia following a robust challenge that is equivalent to that of FeLV-k.     

马传染性贫血病毒疫苗株S2基因不同变异位点逆向突变感染性克隆的体外复制特性分析

为研究EIAV弱毒疫苗株(EIAV_(FCCV15))S2基因发生的稳定性突变对疫苗株特性的作用以及S2基因的功能,以EIAVFDDVl5感染性克隆质粒pFDDV3-8为模板,根据疫苗研制过程中S2基因发生的4个主要稳定性变异位点,构建及拯救出S2基因不同差异位点逆向突变为强毒株相应氨基酸的6株感染性克隆衍生毒株.经实时定量PCR、逆转录酶活性和western blot等检测表明,疫苗株S2基因4个稳定性变异位点全部逆向突变的感染性克隆衍生毒、,pFDDVS2rl-3-4-5在体外培养靶细胞中的复制水平低于亲本疫苗株感染性克隆衍生病毒和其他组合的逆向突变的感染性克隆衍生病毒,提示ELAV疫苗株S2蛋白4个稳定突变位点的综合作用可能是决定疫苗株和强毒株特性差异的因素之一.以上结果为体内感染S2基因逆向突变感染性克隆衍生病毒,进一步揭示S2基因在ELAV弱毒疫苗致弱过程中的作用提供了依据.     

Efficacy of a novel in ovo-attenuated live vaccine and recombinant vaccine against a very virulent infectious bursal disease virus in chickens

Infectious bursal disease (IBD) causes severe economic damage to the poultry industry worldwide. To prevent IBD virus (IBDV) infection, live virus vaccines have been widely used in chickens having wide-ranging levels of maternally derived antibodies. But, the risks of infection with other pathogens because of lesions related to atrophy of the bursa of Fabricius in vaccinated chickens are a concern. To resolve the problems, a recombinant turkey herpesvirus (HVT) vaccine expressing IBDV-VP2 protein (rHVT-IBD) has been developed. However, the induction of neutralizing antibodies by rHVT-IBD against a virulent IBDV might be delayed compared with that by the live IBD vaccine, leading to the high risks of IBDV infection for young chickens. To find the best selection of IBDV vaccine for the onset of immunity, we examine the protective efficacy of a novel in ovo-attenuated live IBDV (IBD-CA) vaccine and the rHVT-IBD vaccine in young chickens challenged with a very virulent IBDV (vvIBDV) strain. We show that the protective efficacy of IBD-CA vaccine was higher than that of the rHVT-IBD vaccine in 14-day-old chickens challenged with the vvIBDV strain, leading to the risk of IBDV infection for young chickens when vaccinated with rHVT-IBD. Our results suggest that farmers should select the best vaccines to maximize vaccine efficacy in consideration of the vaccine characteristics, prevalence levels of IBDV in the areas, and initial MDA levels of the chickens since the attenuated live and recombinant vaccines play a role in the different vaccine efficacies.