Protection against lethal challenge by Ebola virus-like particles produced in insect cells

Ebola virus-like particles (VLPs) were produced in insect cells using a recombinant baculovirus expression system and their efficacy for protection against Ebola virus infection was investigated. Two immunizations with 50 μg Ebola VLPs (high dose) induced a high level of antibodies against Ebola GP that exhibited strong neutralizing activity against GP-mediated virus infection and conferred complete protection of vaccinated mice against lethal challenge by a high dose of mouse-adapted Ebola virus. In contrast, two immunizations with 10 μg Ebola VLPs (low dose) induced 5-fold lower levels of antibodies against GP and these mice were not protected against lethal Ebola virus challenge, similar to control mice that were immunized with 50 μg SIV Gag VLPs. However, the antibody responses against GP were boosted significantly after a third immunization with 10 μg Ebola VLPs to similar levels as those induced by two immunizations with 50 μg Ebola VLPs, and vaccinated mice were also effectively protected against lethal Ebola virus challenge. Furthermore, serum viremia levels in protected mice were either below the level of detection or significantly lower compared to the viremia levels in control mice. These results show that effective protection can be achieved by immunization with Ebola VLPs produced in insect cells, which give high production yields, and lend further support to their development as an effective vaccine strategy against Ebola virus.     

狂犬病病毒糖蛋白和核蛋白基因的克隆及其重组蛋白在昆虫细胞中的表达

目的从国内狂犬病疫苗ａＧ株中克隆狂犬病病毒糖蛋白（ＧＰ）基因和核蛋白（ＮＰ）基因，应用杆状病毒－昆虫细胞表达系统使其在昆虫细胞中表达。方法从狂犬病病毒感染细胞上清液中提取病毒ＲＮＡ，应用ＲＴ－ＰＣＲ方法扩增ＧＰ基因和ＮＰ基因。扩增的基因与转移质粒连接并转化大肠杆菌，得到重组转移质粒。将其与野生杆状病毒（ＡｃＭＮＰＶ）线性ＤＮＡ共转染Ｓｆ９昆虫细胞，通过有限稀释法筛选含有ＧＰ基因和ＮＰ基因重组病毒。初步检测了重组蛋白的抗原性。结果用ＲＴ－ＰＣＲ方法扩增得到ＧＰ基因和ＮＰ基因，通过重组转移后重组病毒感染的细胞经免疫印染实验表明可分别表达ＧＰ和ＮＰ重组蛋白。重组蛋白的分子量分别为５８×１０３和５３×１０３。用重组病毒感染的细胞免疫小鼠后可诱导动物产生特异性抗体。结论可以应用杆状病毒－昆虫细胞表达系统表达狂犬病病毒ＧＰ和ＮＰ重组蛋白，为开发基因工程化狂犬病疫苗提供有意义的资料     

麻疹病毒血凝素和融合蛋白基因的克隆与表达及其重组蛋白的免疫学评价

采用逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）的方法从麻疹病毒Ｅｄｍｏｎｓｔｏｎ株基因组中扩增出血凝素Ｈ基因和融合蛋白Ｆ基因，并利用转移质粒将这两个基因分别重组到杆状病毒多角体蛋白启动子（ＰＨ）控制之下，获得重组病毒ｖＢＭＶＨ和ｖＢＭＶＦ。重组杆状病毒感染Ｓｆ９昆虫细胞，表达的重组蛋白分别具有血凝、血溶活性。红细胞吸附抑制试验、免疫印迹、酶联免疫试验结果显示重组蛋白在生物学、生化学特性与天然蛋白类似，并能被天然麻疹免疫血清（人、鼠、兔）所识别。动物实验表明，重组蛋白具有诱生中和抗体的能力。重组血凝素免疫血清还具有血凝抑制抗体活性。这些结果说明杆状病毒－昆虫细胞体系表达的麻疹病毒血凝素和融合蛋白具有较好的生物学活性及免疫原性和抗原性。     

Serological cross-reactivities between antibodies to simian virus 40, BK virus,and JC virus assessed by virus-like-particle-based enzyme immunoassays

Enzyme immunoassays (EIAs) for detection of serum antibodies to simian virus 40 (SV40), BK virus (BKV), and JC virus (JCV) were developed by using virus-like-particles (VLPs) produced in insect cells from recombinant baculoviruses expressing the VP1 protein of the respective virus. Rhesus macaque sera with neutralizing antibodies to SV40 showed a high level of reactivity in the SV40 VLP-based EIA, and these sera also showed lower levels of reactivity in the BKV and JCV VLP-based EIAs. Rhesus macaque sera negative for neutralizing antibodies to SV40 were negative in all three EIAs. Competitive binding assays showed that SV40 VLPs inhibited BKV reactivity. In rhesus macaque sera, high optical density (OD) values for antibodies to SV40 VLPs were correlated with high OD values for antibodies to BKV but not with high OD values for antibodies to JCV VLPs. Human sera with neutralizing antibodies to SV40 were more reactive to SV40 VLPs than human sera without neutralizing antibodies to SV40. The greater SV40 reactivities of human sera were correlated with greater reactivities to BKV VLPs but not JCV VLPs. These data suggest that cross-reactivity with BKV antibodies may account for part of the low-level SV40 reactivity seen in human sera. With their greater versatility and their suitability for large-scale testing, the VLP-based EIAs for SV40, BKV, and JCV are likely to contribute to a better understanding of the biology of these viruses.     

Serological Cross-Reactivities between Antibodies to Simian Virus 40, BK Virus, and JC Virus Assessed by Virus-Like-Particle-Based Enzyme Immunoassays

Enzyme immunoassays (EIAs) for detection of serum antibodies to simian virus 40 (SV40), BK virus (BKV), and JC virus (JCV) were developed by using virus-like-particles (VLPs) produced in insect cells from recombinant baculoviruses expressing the VP1 protein of the respective virus. Rhesus macaque sera with neutralizing antibodies to SV40 showed a high level of reactivity in the SV40 VLP-based EIA, and these sera also showed lower levels of reactivity in the BKV and JCV VLP-based EIAs. Rhesus macaque sera negative for neutralizing antibodies to SV40 were negative in all three EIAs. Competitive binding assays showed that SV40 VLPs inhibited BKV reactivity. In rhesus macaque sera, high optical density (OD) values for antibodies to SV40 VLPs were correlated with high OD values for antibodies to BKV but not with high OD values for antibodies to JCV VLPs. Human sera with neutralizing antibodies to SV40 were more reactive to SV40 VLPs than human sera without neutralizing antibodies to SV40. The greater SV40 reactivities of human sera were correlated with greater reactivities to BKV VLPs but not JCV VLPs. These data suggest that cross-reactivity with BKV antibodies may account for part of the low-level SV40 reactivity seen in human sera. With their greater versatility and their suitability for large-scale testing, the VLP-based EIAs for SV40, BKV, and JCV are likely to contribute to a better understanding of the biology of these viruses.     

Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

Infectious hemorrhagic fevers caused by the Marburg and Ebola filoviruses result in human mortality rates of up to 90%, and there are no effective vaccines or therapeutics available for clinical use. The highly infectious and lethal nature of these viruses highlights the need for reliable and sensitive diagnostic methods. We assembled a protein microarray displaying nucleoprotein (NP), virion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses for use as a surveillance and diagnostic platform. Using the microarrays, we examined serum antibody responses of rhesus macaques vaccinated with trivalent (GP, NP, and VP40) virus-like particles (VLP) prior to infection with the Marburg virus (MARV) (i.e., Marburg marburgvirus) or the Zaire virus (ZEBOV) (i.e., Zaire ebolavirus). The microarray-based assay detected a significant increase in antigen-specific IgG resulting from immunization, while a greater level of antibody responses resulted from challenge of the vaccinated animals with ZEBOV or MARV. Further, while antibody cross-reactivities were observed among NPs and VP40s of Ebola viruses, antibody recognition of GPs was very specific. The performance of mucin-like domain fragments of GP (GP mucin) expressed in Escherichia coli was compared to that of GP ectodomains produced in eukaryotic cells. Based on results with ZEBOV and MARV proteins, antibody recognition of GP mucins that were deficient in posttranslational modifications was comparable to that of the eukaryotic cell-expressed GP ectodomains in assay performance. We conclude that the described protein microarray may translate into a sensitive assay for diagnosis and serological surveillance of infections caused by multiple species of filoviruses.     

Expression and self-assembly in baculovirus of porcine enteric calicivirus capsids into virus-like particles and their use in an enzyme-linked immunosorbent assay for antibody detection in swine

Porcine enteric calicivirus (PEC) causes diarrhea and intestinal lesions in pigs. PEC strain Cowden grows to low to moderate titers in cell culture but only with the addition of intestinal contents from uninfected gnotobiotic pigs (W. T. Flynn and L. J. Saif, J. Clin. Microbiol. 26:206--212, 1988; A. V. Parwani, W. T. Flynn, K. L. Gadfield, and L. J. Saif, Arch. Virol. 120:115--122, 1991). Cloning and sequence analysis of the PEC Cowden full-length genome revealed that it is most closely related genetically to the human Sapporo-like viruses. In this study, the complete PEC capsid gene was subcloned into the plasmid pBlueBac4.5 and the recombinant baculoviruses were identified by plaque assay and PCR. The PEC capsid protein was expressed in insect (Sf9) cells inoculated with the recombinant baculoviruses, and the recombinant capsid proteins self- assembled into virus-like particles (VLPs) that were released into the cell supernatant and purified by CsCl gradient centrifugation. The PEC VLPs had the same molecular mass (58 kDa) as the native virus capsid and reacted with pig hyperimmune and convalescent-phase sera to PEC Cowden in enzyme-linked immunosorbent assay (ELISA) and Western blotting. The PEC capsid VLPs were morphologically and antigenically similar to the native virus by immune electron microscopy. High titers (1:102,400 to 204,800) of PEC-specific antibodies were induced in guinea pigs inoculated with PEC VLPs, suggesting that the VLPs could be useful for future candidate PEC vaccines. A fixed-cell ELISA and VLP ELISA were developed to detect PEC serum antibodies in pigs. For the fixed-cell ELISA, Sf9 cells were infected with recombinant baculoviruses expressing PEC capsids, followed by cell fixation with formalin. For the VLP ELISA, the VLPs were used for the coating antigen. Our data indicate that both tests were rapid, specific, and reproducible and might be used for large-scale serological investigations of PEC antibodies in swine.     

Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D

Summary The DNA sequence encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) was inserted into a baculovirus transfer vector under control of the polyhedrin gene promoter of the baculovirusAutographa california nuclear polyhedrosis virus (AcNPV). After co-transfection ofSpodoptera frugiperda (Sf9) insect cells with wild-type AcNPV DNA and the recombinant transfer vector DNA, polyhedrin-negative recombinants that expressed high levels of HSV-1 gD were isolated using immunoaffinity selection with antibody coated magnetic particles followed by plaque purification. These recombinant baculoviruses expressed a protein that was slightly smaller than virion HSV-1 gD made in Vero cells. This recombinant protein was expressed at high levels. The expressed protein was glycosylated, was found on the membrane of Sf9 cells, and reacted with gD specific antibodies. Antibodies raised in mice to the recombinant gD neutralized HSV-1 as measured by plaque reduction assays. Mice inoculated with the recombinant baculovirus were completely protected from lethal challenge with HSV-1.     

The induction and characterization of monoclonal antibodies specific to GP of Ebola virus

The Ebola virus is highly infectious and characterized by hemorrhagic fever, headache, and so on with a high mortality rate. Currently, there are neither therapeutic drugs or vaccines against the Ebola virus nor fast diagnostic methods for the detection of Ebola virus infection. This study reported the induction and isolation of two monoclonal antibodies that specifically recognized the glycoprotein (GP) and secreted glycoprotein (sGP) of the Ebola virus. Plasmids encoding either GP or sGP were constructed and immunized BALB/c mice, accordingly purified sGP was boosted. The antisera were analyzed for binding activity against sGP protein in enzyme-linked immunosorbent assay (ELISA) and neutralization activity in a pseudotyped virus neutralization assay. A number of reactive clones were isolated and two monoclonal antibodies T231 and T242 were identified to react with both GP and sGP. Western blot and ELISA assays showed that the monoclonal antibodies could react with GP and sGP, respectively. Moreover, they could recognize Ebola pseudovirus by cellular immunochemistry assay. We labeled the monoclonal antibody T231 with biotin and analyzed the competitiveness of the two antibodies by the ELISA test. The results showed that the binding epitopes of the two monoclonal antibodies to sGP were partially overlapped. In summary, two GP-specific mAbs were identified, which will be used to detect the Ebola virus or investigate GP.     

森林脑炎病毒prM-E蛋白在昆虫细胞中的表达及免疫活性测定

目的为了表达森林脑炎病毒prME蛋白,为森林脑炎快速诊断试剂的研制奠定基础。方法经过RTPCR扩增、重组转移载体构建、细菌内转座和昆虫细胞转染,以杆状病毒昆虫细胞表达系统成功地表达了森林脑炎病毒MDJ01株prME蛋白。结果从感染细胞上清中电镜观察到重组蛋白形成的球型颗粒,说明重组病毒感染细胞后产生病毒样表达颗粒(viruslikeparticlesVLPs),并且分泌至细胞外。免疫印迹试验和间接免疫荧光试验表明,表达的重组蛋白能够与抗森林脑炎病毒抗体特异结合,具有良好的抗原性。ELISA和间接免疫荧光染色证实,重组prME蛋白可以作为抗原用于检测患者血清特异性抗体。结论在昆虫细胞中表达的prME具有良好的抗原性,本研究为森林脑炎快速特异诊断试剂研制奠定了基础。     

Baculovirus-produced influenza virus-like particles in mammalian cells protect mice from lethal influenza challenge

Influenza virus-like particles (VLPs) are effective vaccines against influenza infection, which can be produced either in insect cells by recombinant baculovirus (BV) infection or in mammalian cells by DNA plasmid transfection. However, VLPs produced from baculovirus/insect cells are difficult to purify due to baculovirus contamination; VLPs produced by plasmid transfection are limited by scale-up capability. In this study, a BacMam BV, in which three CMV-promoters drive the hemagglutinin, neuraminidase, and matrix of influenza virus was constructed. This baculovirus can deliver these genes into mammalian cells/hosts and subsequently influenza VLPs can be produced and secreted from transduced cells. Transduction conditions were optimized and influenza VLPs were purified from transduced 293T cells. Mice were vaccinated with BV transduction-produced VLPs, plasmid transfection-produced VLPs, and BacMam BV. Two vaccinations of each vaccine induced high hemagglutination-inhibition (HAI) titers and prevented influenza virus infection. In contrast, following a single vaccination, all mice vaccinated with each vaccine had significantly lower lung viral titers compared to unvaccinated mice. Remarkably, mice vaccinated with a single dose of BV transduction-produced VLPs survived challenge, whereas mice vaccinated with one dose of BacMam BV- or plasmid transfection-produced VLPs had 60-80% survival. This finding is particularly significant for producing easily purified VLPs. The BacMam system is an alternative strategy for VLP production, which is easy to scale up and purify. Besides, BacMam BV can be used as a gene delivery vector to produce VLPs in vivo, to stimulate immune responses.     

人乳头瘤病毒6b L1/16E7嵌合蛋白的基因克隆和表达

目的 研究HPV6bL1/16E7嵌合蛋白的基因克隆及其在昆虫细胞的表达,为防治尖锐湿疣和宫颈癌的基因工程疫苗研究作准备。方法 用PCR扩增出HPV6bL1/16E7嵌合蛋白基因,将其克隆到杆状病毒转移载体pVL1393,制备重组杆状病毒并感染昆虫细胞表达HPV6bL1/16E7嵌合蛋白。结果 HPV6bL1/16E7嵌合蛋白基因在昆虫细胞中得到了表达,并可自组装形成病毒样颗粒。结果 昆虫细胞表达的HPV6bL1/16E7嵌合病毒样颗粒可进一步用于HPV感染的免疫机理及基因工程疫苗研究。     

柯萨奇病毒A组16型病毒样颗粒免疫原性的初步研究

目的 研究柯萨奇病毒A组16型（Coxsackievirus A16,CA16）病毒样颗粒（Virus-like particles,VLPs）的免疫原性.方法 利用杆状病毒-昆虫细胞表达系统共表达3CD及P1蛋白,制备CA16病毒样颗粒,通过SDS-PAGE及透射电镜等方法对VLPs进行鉴定,以氢氧化铝佐剂免疫ICR小鼠,并对乳鼠经颅腔攻毒.对病毒样颗粒疫苗的免疫原性及保护效果进行评价.结果 将重组CA163CD及P1蛋白的杆状病毒转染SF9细胞,可以产生类似CA16病毒颗粒的大小为27 ～ 30 nm的VLPs.小鼠免疫试验结果显示CA16 VLPs可以刺激产生较高水平的抗CA16病毒的特异性IgG抗体及中和抗体,乳鼠攻毒试验结果显示,母传抗体保护率高达90％.结论 CA16 VLPs可以刺激小鼠产生较高水平的体液免疫反应,并且母传抗体可以保护乳鼠抵御经颅腔的病毒攻击.     

Immune responses in mice vaccinated with virus-like particles composed of the GP5 and M proteins of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) induces reproductive failure in sows and respiratory problems in pigs of all ages. Live attenuated and inactivated vaccines are used on swine farms to control PRRSV. However, their protective efficacy against field strains of PRRSV remains questionable. New vaccines have been developed to improve the efficacy of these traditional vaccines. In this study, virus-like particles (VLPs) composed of the GP5 and M proteins of PRRSV were developed, and the capacity of the VLPs to elicit antigen-specific immunity was evaluated. Serum antibody titers and production of cytokines were measured in BALB/C mice immunized intramuscularly three times with different doses (0.5, 1.0, 2.0, and 4.0 μg) of the VLP vaccine. A commercial vaccine consisting of inactivated PRRSV and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. IgG titers to GP5 were significantly higher in all groups of mice vaccinated with the VLPs than in control mice. Neutralizing antibodies were only detected in mice vaccinated with 2.0 and 4.0 μg of the VLPs. Cytokine levels were determined in cell culture supernatants after in vitro stimulation of splenocytes with the VLPs for 3 days. Mice immunized with 4.0 μg of the VLPs produced a significantly higher amount of interferon-gamma (IFN-γ) than mice immunized with the commercial inactivated PRRSV vaccine and PBS. In contrast, immunization with the commercial vaccine induced higher production of IL-4 and IL-10 in mice than mice vaccinated with VLPs. These data together demonstrate the capacity of VLPs to induce both neutralizing antibodies and IFN-γ in immunized mice. The VLP vaccine developed in this study could serve as a platform for the generation of improved VLP vaccines to control PRRSV.     

Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination

Beak and feather disease virus (BFDV) is a common avian circovirus infection of wild Psittaciformes and is a recognised threat to endangered psittacine species. Currently, there is a requirement to develop BFDV antigen for diagnostic purposes and since efforts to propagate BFDV in vitro have so far been unsuccessful the entire coding region of BFDV ORF C1 was expressed in Sf9 insect cells using a baculovirus expression system. The entire coding region of BFDV ORF C1, the presumptive capsid, was expressed in Sf9 insect cells using baculovirus expression system. Electron microscopic examination of negatively stained material demonstrated that the recombinant protein self-assembled to produce virus-like particles (VLPs) thus confirming that ORF C1 is likely to be the sole determinant for capsid construction in vivo. BFDV VLPs also possessed haemagglutinating activity which provides further evidence that self-assembled BFDV VLPs retain receptor mediated biological activity and that the determinants for BFDV haemagglutination activity rely solely on the capsid protein. The recombinant protein reacted with anti-BFDV sera from naturally immune parrots and cockatoo and from chickens experimentally inoculated with native BFDV in both Western blots and haemagglutination inhibition (HI) assay. BFDV VLPs were also a suitable replacement antigen for serological detection of BFDV antibody by HI.     

登革2型病毒ZS01/01株病毒样颗粒免疫原性研究

目的 研究登革2型病毒（Dengue virus type 2,DENV-2）病毒样颗粒（virus-Like particles,VLPs）的免疫原性.方法 利用已构建的DENV-2 ZS01/01株病毒样颗粒的表达质粒转染293T细胞,对分泌型VLPs进行大量培养并通过蔗糖密度梯度离心法对其进行纯化.纯化的VLPs经Western Blot及透射电镜观察等方法鉴定后免疫BALB/c小鼠.利用ELISA及中和试验等方法对体液免疫反应进行检测,ELISPOT法测定细胞免疫水平.结果 登革2型病毒样颗粒表达质粒转染哺乳动物细胞所得上清经蔗糖密度梯度离心后,电镜下可观察到类似于天然登革病毒的大小在45～55nm之间的病毒样颗粒.体液及细胞免疫检测结果显示登革2型VLPs可以刺激小鼠产生较高水平的登革E蛋白特异性抗体及一定水平的中和抗体,免疫小鼠脾淋巴细胞经体外刺激后IFN-γ水平显著升高.结论 登革2型病毒病毒样颗粒免疫BALB/c小鼠后可引起一定水平的细胞免疫及体液免疫反应,该研究结果为四价登革病毒样颗粒疫苗的研制奠定了基础.     

Surface display of IgG Fc on baculovirus vectors enhances binding to antigen-presenting cells and cell lines expressing Fc receptors

Recombinant baculoviruses (recBV) were constructed with dual cassettes for constitutive expression of human IgG Fc following infection of insect cells and the structural proteins of hepatitis C virus (core, E1 and E2) following transduction of mammalian cells. The IgG Fc was expressed in insect cells as a fusion protein with the signal sequence and transmembrane region of either the native baculovirus envelope protein gp64 or the human transferrin receptor as a type I or type II integral membrane protein, respectively. The IgG Fc fusion proteins formed functional homodimers on the surface of recBV-infected insect cells and were incorporated into the envelope of recBV particles during egress from the infected cell. Both pseudotyped recBV bound specifically to recombinant soluble FcγRIIα receptor and to cell lines and antigen-presenting cells expressing Fc receptors (FcRs). These novel baculoviral vectors, which target cells of the immune system that express FcRs, have potential applications for vaccination or gene therapy.     

Empty virus-like particle-based enzyme-linked immunosorbent assay for antibodies to hepatitis E virus

Hepatitis E, an enterically transmitted non-A, non-B hepatitis, is a serious viral infection that occasionally causes large epidemics in developing countries. In developed countries, the disease only appears sporadically due to the transmission routes, and it is considered to be less important. The hepatitis E virus (HEV) cannot grow in cultured cells and no reliable assay system has ever been developed. In addition, the present diagnostic are not perfect, and actual rates of HEV infection may be underestimated. Highly purified empty virus-like particles (VLPs) of HEV have been produced by the use of a recombinant baculovirus vector in insect cells. Using these VLPs as an antigen, an enzyme-linked immunosorbent assay (ELISA) for antibodies to HEV was developed. A panel of 164 sera that were randomized and coded, and sera collected periodically from three patients with hepatitis E were used for the evaluation. The sensitivity of the assay was shown to be equal to or better than that obtained in previous research that used the same serum panel. The ELISA demonstrated that the serum IgM level of the patients was highest at the onset of the clinical illness and then rapidly decreased. In contrast, a high level of circulating IgG antibody titers lasted for more than 4 years. In Japan, a non-endemic country, the prevalence of the IgG class antibody to HEV in healthy individuals was found to range from 1.9% to 14.1%, depending on the geographical area. Only one out of 900 (0.1%) serum samples was IgM-positive. The IgM class antibody to HEV was detected in 10.8% of non-A, non-B, and non-C acute hepatitis patients in northeast China, whereas none of the patients in Korea had the IgM antibody. The ELISA utilizing the VLPs is sensitive and specific in its detection of the IgM and IgG antibodies to HEV. The ELISA is therefore useful for diagnosing HEV infection and for seroepidemiological study of hepatitis E.