Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV), an alpha-herpesvirus, has been developed as a live viral vector for animal vaccines. However, the PRV recombinant virus TK⁻/gE⁻/GP5⁺ expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV), based on the PRV genetically depleted vaccine strain TK⁻/gE⁻/LacZ⁺, scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV. To develop a booster-specific immune response of such PRV recombinants, the ORF5m gene (the modified ORF5 gene having better immune responses) was substituted for the ORF5 gene and introduced into PRV TK⁻/gE⁻/LacZ⁺, resulting in a PRV recombinant named TK⁻/gE⁻/GP5m⁺, which expressed the modified GP5m protein. The recombinant virus was confirmed using PCR, Southern blotting and Western blotting. TK⁻/gE⁻/GP5m⁺ and TK⁻/gE⁻/GP5⁺ expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses. The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16) and cell immune responses induced by TK⁻/gE⁻/GP5m⁺ against PRRSV were higher than that induced by TK⁻/gE⁻/GP5⁺. Thus, the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV. Translated from Journal of Biotechnology, 2005, 21(6): 858–864 [译自: 生物工程学报]     

Construction and immune efficacy of recombinant pseudorabies virus expressing PrM-E proteins of Japanese encephalitis virus genotype І

Background

Japanese encephalitis (JE) is an arboviral disease with high case fatality rates and neurologic or psychiatric sequelae among survivors in Asia, western Pacific countries and northern Australia. Japanese encephalitis virus (JEV) is the cause of JE and the emergence of genotype ? (GI) JEV has displaced genotype III (GIII) as the dominant strains circulating in some Asian regions. The currently available JE vaccines are safe and effective in preventing this disease, but they are developed based on the GIII JEV strains.

Methods

The recombinant virus PRV TK^?/gE^?/PrM-E⁺ which expressed the premembrane (prM) and envelope (E) proteins of JEV SX09S-01 strain (genotype I, GI) was constructed by homologous recombination between the genome of PRV TK^?/gE^?/LacZ⁺ digested with EcoRI and plasmid pIE-CAG-PrM-E-BGH. Expression of JEV PrM and E proteins was analyzed by Western blot analysis. Immune efficacy of PRV TK^?/gE^?/PrM-E⁺ was further evaluated in mouse model.

Results

A recombinant pseudorabies virus (PRV TK^?/gE^?/PrM-E⁺) was successfully constructed. Mice experiments showed that PRV TK^?/gE^?/PrM-E⁺ could induce a high level of ELISA antibodies against PRV and JEV, as well as high titer of PRV neutralizing antibodies. After challenge with 1?×?10⁷ PFU virulent JEV SX09S-01 strain, the time of death was delayed and the survival rate was improved in PRV TK^?/gE^?/PrM-E⁺ vaccinated mice.

Conclusions

PRV TK^?/gE^?/PrM-E⁺ is a potential vaccine candidate against PRV and JEV GI infection in the future.

     

Construction of a recombinant BHV-1 expressing the VP1 gene of foot and mouth disease virus and its immunogenicity in a rabbit model

Foot-and-mouth disease (FMD) and infectious bovine rhinotracheitis (IBR) are two important infectious diseases of cattle. Using bovine herpesvirus type 1 (BHV-1) as a gene delivery vector for development of live-viral vaccines has gained widespread interest. In this study, a recombinant BHV-1 was constructed by inserting the synthetic FMDV (O/China/99) VP1 gene in the the gE locus of BHV-1 genome under the control of immediately early gene promoter of human cytomegalovirus (phIE CMV) and bovine growth hormone polyadenylation (BGH polyA) signal. After homologous recombination and plaque purification, a recombinant virus named BHV-1/gE⁻/VP1 was acquired and identified. The immunogenicity was confirmed in a rabbit model by virus neutralization test and enzyme-linked immunosorbent assay (ELISA). The result indicated that the BHV-1/gE⁻/VP1 has the potential for being developed as a bivalent vaccine for FMD and IBR.     

A Novel Mucosal Vaccine Against Foot-and-Mouth Disease Virus Induces Protection in Mice and Swine

Epitopes of a foot-and-mouth disease virus (FMDV) capsid protein VP1 complex and a chimera of 6×His-tagged cholera toxin B subunit (hCTB) were expressed in Hansenula polymorpha and used together as a mucosal vaccine. Antibody and cytokine responses to VP1–hCTB vaccine and protection against FMDV were evaluated by ELISA and a virus challenge test in mice, respectively. VP1–hCTB directly enhanced the expression of interleukin-5 (IL-5) both in serum and supernatants of cultured spleen cells. After challenging suckling mice with 10⁵ FMDV (=50% lethal dosage per mouse) a greater protection was seen after intraperitoneal and intranasal vaccinations than after oral vaccination. In swine immunized with VP1–hCTB, immune responses were achieved after three administrations, and the vaccine protected swine (80%) when challenged with 10^6.5 FMDV (=50% infectious dosage per swine). These results demonstrated the possibility of using CTB as a mucosal adjuvant to elicit protective immune responses against FMDV. Houhui Song, Zhiliang Wang and Dongxia Zheng contributed equally to this work.     

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

Background

Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene.

Methods

A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein.

Results

Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts.

Conclusions

In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

     

共表达PCV2 ORF2和猪IL-18基因的重组猪伪狂犬病毒对小鼠的免疫原性

【目的】研制猪伪狂犬病毒(PRV)和猪圆环病毒2型(PCV2)的二联活疫苗,并用猪IL-18作为免疫佐剂。【方法】将猪IL-18基因插入到质粒p GO中,获得的重组转移质粒p GO18与猪PRV弱毒HB98株DNA共转染ST细胞,并进行空斑筛选和纯化;RT-PCR和Western blot分别从转录和蛋白水平鉴定其表达情况。将重组病毒PGO18和PGO、PRV弱毒株HB98、PCV2灭活商品苗和1640细胞培养基分别免疫6周龄雌性昆明小鼠,4周后二次免疫,二免后4周用PCV2 DF强毒和PRV Min/A强毒接种小鼠。通过ELISA、血清中和试验和流式细胞术及攻毒保护试验评价重组病毒的免疫原性。【结果】获得了重组病毒PGO18,并且可在ST细胞内表达;PGO18可诱导小鼠机体产生PCV2的ELISA和PRV的中和抗体水平,刺激CD3+、CD4+、CD8+T细胞亚群的增殖,且能有效抵抗PCV2和PRV强毒攻击。【结论】IL-18基因可增强重组病毒的免疫效果,使重组病毒具有良好的免疫原性,有望成为防治PCV2和PRV的候选疫苗株。     

口蹄疫病毒结构蛋白VP1容忍外源标签的能力

【目的】利用口蹄疫病毒的反向遗传操作技术,构建含不同外源标签口蹄疫病毒的全长克隆,鉴定口蹄疫病毒结构蛋白VP1容忍不同外源标签的能力。【方法】通过融合PCR技术,在FMDV O/HN/93全长感染性克隆的VP1 G-H环分别引入V5、TC12、KT3、3FLAG外源标签,构建全长质粒。全长质粒经Not I线化后转染表达T7 RNA聚合酶的稳定细胞,拯救重组病毒。RT-PCR、序列测定、间接免疫荧光鉴定病毒,噬斑和一步生长曲线分析重组病毒的生物学特性。【结果】成功拯救到表达V5或KT3表位标签的重组病毒,未能拯救到表达TC12或3×FLAG的重组病毒。V5和KT3表位标签的插入均影响了口蹄疫病毒的复制能力。【结论】重组口蹄疫病毒的成功拯救为未来标记疫苗以及口蹄疫病毒作为表达载体等的研究奠定了基础。     

猪伪狂犬病病毒流行株HLJ-01的分离鉴定及致病性分析

【背景】自2011年以来,猪伪狂犬病毒(pseudorabies virus,PRV)发生变异,经典的疫苗株已不能完全抵抗PRV变异株的感染,国内多个猪场出现伪狂犬病的暴发,PRV变异毒株开始在我国大规模流行。【目的】通过分离PRV流行变异毒株,并对其进行遗传进化和致病性分析,为PRV流行病学调查及疫苗研制提供实验数据。【方法】采集黑龙江某猪场感染PRV的脑组织病料,根据GenBank PRV gE和gB保守序列设计引物,进行PCR鉴定。通过对gE和gC基因进行序列测定和遗传进化分析。利用BHK-21细胞分离病毒,采用噬斑纯化方法对病毒进行纯化。通过电镜、间接免疫荧光对病毒进行鉴定,测定病毒生长曲线并进行致病性研究。【结果】经PCR和测序鉴定分离株为PRV流行株,将其命名为HLJ-01。遗传进化分析结果显示,该分离毒株与我国近几年分离的流行变异株位于同一分支;氨基酸序列分析结果显示,gE和gC存在国内流行变异株的特征序列,表明该分离毒株为流行变异株。生长曲线显示,分离株HLJ-01在感染48h时滴度最高(10^8.5TCID₅₀/mL)。电镜观察结果显示,病毒颗粒直径约150nm,呈球形,有囊膜,囊膜外有放射状纤突,呈现典型PRV病毒特征。动物感染实验结果显示,10^7.0TCID₅₀剂量感染组死亡率为100%;10^6.0TCID₅₀剂量感染组死亡率为80%;10^5.0TCID₅₀剂量感染组死亡率为60%。仔猪在接种病毒后均出现PRV感染的典型症状和病理变化,证实分离毒株对仔猪有较强致病力。【结论】分离获得一株猪伪狂犬病毒,经鉴定该分离株为流行变异株,而且具有较强的致病力,这为PRV流行病学分析及疫苗候选株的筛选奠定了基础。     

Virus-like particles of recombinant PCV2b carrying FMDV-VP1 epitopes induce both anti-PCV and anti-FMDV antibody responses

Mixed infection of porcine circovirus type 2 (PCV2) and foot-and-mouth disease virus (FMDV) is devastating to swine populations. To develop an effective vaccine that can protect the pigs from the infection of PCV2 and FMDV, we used the neutralizing B cell epitope region (aa 135–160) of FMDV to replace the regions aa 123–151 and aa 169–194 of the PCV2b Cap protein to generate a recombinant protein designated as Capfb. The Capfb protein was expressed in Escherichia coli system and the purified Capfb protein assembled into virus-like particles (VLPs) through dialysis. The ability of the Capfb protein to induce effective immune response against FMDV and PCV2b was tested in mice and guinea pigs. The results showed that the Capfb-VLPs could elicit anti-PCV2b and anti-FMDV antibody response in mice and guinea pigs without inducing antibodies against decoy epitope. Moreover, the Capfb-VLPs could enhance the percentage and activation of B cells in lymph nodes when the mice were stimulated with inactivated FMDV or PCV2b. These data suggested that the Capfb-VLPs could be an efficacious candidate antigen for developing a novel PCV2b-FMDV bivalent vaccine.

     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK-/gE-/GP5+expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV),based on the PRV genetically depleted vaccine strain TK-/gE-/LacZ+,scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene (the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK-/gE-/LacZ+,resulting in a PRV recombinant named TK-/gE-/GPSm+,which expressed the modified GPSm protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK-/gE-/GPSm+and TK-/gE-/GP5+expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK-/gE-/GPSm+against PRRSV were higher than that induced by TK-/gE-/GP5+.Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.     

口蹄疫O型重组病毒的构建及其免疫原性分析

【目的】利用反向遗传操作技术,构建含O型口蹄疫病毒(food-and-mouth disease virus, FMDV) 3个拓扑型免疫优势结构蛋白基因的重组FMDV,评估其作为猪O型口蹄疫(food-and-mouth disease, FMD)疫苗候选株的潜力。【方法】通过基因合成,在FMD疫苗株O/HN/CHA/93 (古典中国拓扑型)的基因中嵌合流行株O/NXYCh/CHA/2018 (东南亚拓扑型) VP1结构蛋白的重组病毒骨架上,用O/TUR/5/2009疫苗株(中东-南亚拓扑型) VP1蛋白的G-H环基因替换其对等基因,构建含O型3个拓扑型FMDV结构蛋白基因的重组全长质粒,Not I线性化后转染表达T7 RNA聚合酶的BSR/T7细胞,拯救重组病毒。通过RT-PCR、序列测定、间接免疫荧光鉴定重组病毒;噬斑试验和一步生长曲线分析重组病毒的生物学特性。重组病毒制备疫苗免疫猪,用病毒中和试验分析其对当前流行的O型3个拓扑型FMDV的交叉反应性。【结果】成功拯救到含O型3个拓扑型FMDV结构蛋白基因的重组病毒,重组病毒与亲本病毒具有相似的生物学特性。亲本病毒和重组病毒制备的疫苗免疫猪,均能够对中东-南亚型(Middle East-South Asia, ME-SA)拓扑型和东南亚型(South-East Asia, SEA)拓扑型病毒株产生保护性平均中和抗体(>1.65log₁₀);均不能对古典中国型(Cathay)拓扑型流行株产生保护性平均中和抗体(<1.65log₁₀),但与亲本病毒相比,O/TUR/5/2009疫苗株G-H环基因的替换显著提高了对ME-SA和SEA拓扑型病毒株的交叉反应性(p<0.05)。【结论】本研究对未来FMD疫苗的设计具有重要的指导意义。     

Enhancing effects of the chemical adjuvant levamisole on the DNA vaccine pVIR‐P12A‐IL18‐3C

DNA‐based vaccination is an attractive alternative for overcoming the disadvantages of inactivated virus vaccines; however, DNA vaccines alone often generate only weak immune responses. In this study, the efficacy of LMS as a chemical adjuvant on a DNA vaccine (pVIR‐P12A‐IL18‐3C) encoding the P1‐2A and 3C genes of the FMDV and swine IL‐18, which provides protection against FMDV challenge, was tested. All test pigs were administered booster vaccinations 28 days after the initial inoculation, and were challenged with 1000 ID₅₀ FMDV O/NY00 20 days after the booster vaccination. Positive and negative control groups were inoculated with inactivated virus vaccine and PBS respectively. The DNA vaccine plus LMS induced greater humoral and cell‐mediated responses than the DNA vaccine alone, as evidenced by higher concentrations of neutralizing and specific anti‐FMDV antibodies, and by higher concentrations of T‐lymphocyte proliferation and IFN‐γ production, respectively. FMDV challenge revealed that the DNA vaccine plus LMS provided higher protection than the DNA vaccine alone. This study demonstrates that LMS may be useful as an adjuvant for improving the protective efficiency of DNA vaccination against FMDV in pigs.     

Aujeszky’s disease virus production in disposable bioreactor

A novel, disposable-bag bioreactor system that uses wave action for mixing and transferring oxygen was evaluated for BHK 21 C13 cell line growth and Aujeszky’s disease virus (ADV) production. Growth kinetics of BHK 21 C13 cells in the wave bioreactor during 3-day period were determined. At the end of the 3-day culture period and cell density of 1.82 × 10⁶ cells ml^-1, the reactor was inoculated with 9 ml of gE^- Bartha K-61 strain ADV suspension (10^5.9 TCID₅₀) with multiplicity of infection (MOI) of 0.01. After a 144 h incubation period, 400 ml of ADV harvest was obtained with titre of 10^7.0 TCID₅₀ ml⁻¹, which corresponds to 40,000 doses of vaccine against AD. In conclusion, the results obtained with the wave bioreactor using BHK 21 C13 cells showed that this system can be considered as suitable for ADV or BHK 21 C13 cell biomass production.     

表达猪瘟病毒E2蛋白的BacMam病毒的构建及其免疫原性分析

含具有哺乳动物细胞活性的启动子的重组杆状病毒(BacMam病毒)可有效转导多种哺乳动物细胞,并被广泛用于开发新型非复制型载体疫苗．将水泡性口炎病毒G蛋白(VSV-G)基因插入多角体启动子下游,得到经修饰的杆状病毒转移载体,将对虾白斑综合症病毒(WSSV)ie1启动子控制下的猪瘟病毒E2基因表达盒插入此载体中,构建了BacMam病毒BacMam/G-ie1-E2,以其感染Sf9细胞和转导HeLa细胞,通过间接免疫荧光试验和Western blot分析检测E蛋白的表达,同时用BacMam病毒直接免疫小鼠,用检测猪瘟病毒抗体的间接ELISA方法检测免疫小鼠血清抗体,用基于CFSE和WST-8的淋巴细胞增殖试验评价其细胞免疫应答．结果显示,BacMam/G-ie1-E2能同时在昆虫细胞和哺乳动物细胞中高效表达E2蛋白,免疫小鼠能诱导产生针对猪瘟病毒的特异性抗体,免疫小鼠脾细胞经猪瘟病毒刺激后能诱导特异性的淋巴细胞增殖．这表明,由BacMam病毒介导的基因转移有望用于开发针对猪瘟病毒的非复制型载体疫苗．     

猪口蹄疫病毒多抗原表位重组腺病毒的构建与鉴定

本研究设计构建了含有猪O型口蹄疫病毒VP1（21—60）-（141-160）-（200—213）位氨基酸的基因的重组腺病毒质粒pAd-VP,经PacI酶切后转染HEK-293A细胞,3次噬斑纯化获得了重组腺病毒rAd—VP。该重组腺病毒于HEK-293A细胞连续传代至20代效价稳定,TCID50为10^-10／mL。RT—PCR检测证明目的基因在mRNA水平上可有效表达;应用O型口蹄疫病毒标准阳性血清进行间接荧光抗体试验,在rAdVP感染的HEK-293A细胞的胞质可见清晰荧光。证明该重组腺病毒对VP1（21-60）-（141—160）-（200—213）位氨基酸的基因进行了成功的表达,从而为FMDV多抗原表位腺病毒活载体疫苗的研究奠定了基础。     

重组鸡痘病毒和DNA疫苗对口蹄疫病毒的豚鼠免疫保护

将构建的携带FMDV衣壳蛋白P1-2A和蛋白酶3C编码基因的重组鸡痘病毒活载体疫苗vUTAL3CP1以及编码FMDVP1-2A基因和猪IL-18基因的重组DNA疫苗pVIRIL18P1,分别以单独和混合的方式给豚鼠进行2次免疫,然后测定FMDV特异性结合抗体、中和抗体和T淋巴细胞增殖反应,并用250ID50的FMDV进行攻击,观察其保护效果。结果表明这2种基因工程疫苗均能诱导豚鼠产生特异性的体液免疫及细胞免疫应答。其中以vUTAL3CP1两次免疫组的效果最好,其诱导的抗体水平已接近于常规灭活疫苗,而细胞免疫水平则比后者高得多。攻击保护结果表明该组完全保护率可达3/4,而另外两组也具有一定保护效果。上述研究结果为进一步进行大动物免疫攻毒试验,并最终筛选出最佳疫苗和免疫程序奠定了基础。     

Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera

To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).     

猪源胰酶中特异病毒和非特异病毒安全检测简

目的：对猪源胰酶样品进行病毒检测,以评价其病毒安全性。方法：非特异性病毒检测采用致细胞病变、血凝吸附试验和形态学检测方法;特异性病毒检测包括猪繁殖与呼吸障碍综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪圆环病毒（PCV）、猪细小病毒（PPV）、猪口蹄疫病毒（FMDV）和猪伪狂犬病病毒（PRV）特异性核酸检测,以及CSFV、PPV、PRV特定性抗原蛋白直接免疫荧光检测。结果：受检样品非特异性病毒检测中,未见可观察到的细胞病变产生和病毒粒子,对豚鼠、鸡和人的0型红细胞无凝集现象。特异性病毒检测中,PRRSV、CSFV、PCV、PPV、FMDV和PRV核酸检测均为阴性,CSFV、PPV、PRV免疫荧光检测均为阴性。结论：猪源胰酶样品经非特异性病毒检测和特异性病毒检测均无可检出的病毒存在。