Expression of gag precursor protein and secretion of virus-like gag particles of HIV-2 from recombinant baculovirus-infected insect cells

A recombinant baculovirus carrying the gag gene but lacking the protease coding sequences of human immunodeficiency virus type 2 (HIV-2) has been constructed. When this recombinant baculovirus is used to infect insect cells, a high level of gag precursor protein, gag pr41, is expressed. Electron microscopy showed that the majority of gag pr41 was budding through the plasma membrane and being released into the culture medium in spherical virus-like particles with a diameter of approximately 100 nm. Metabolic labeling demonstrates that gag pr41 is myristylated. Our results demonstrated that HIV-2 gag pr41 can be assembled into virus-like particles in the absence of other HIV proteins. Rabbits immunized with purified gag pr41 particles produced high-titer antibody and Western blot analysis showed that anti-gag pr41 rabbit sera recognize p17, p24, and p55 gag proteins of HIV-1. These results show that gag pr41 particles are highly immunogenic and that gag proteins of HIV-1 and HIV-2 have similar antigenic epitopes.     

Hydrophilicity dependent budding and secretion of chimeric HIV Gag-V3 virus-like particles

Virus-like particles (VLPs) of numerous viruses have been considered as possible candidates for vaccine development. We have constructed HIV chimeric genes by coupling the gag gene of HIV-2 with the V3 domain of the gp120 gene of either HIV-1 or HIV-2 and expressed the chimeric genes in SF21 cells using the recombinant baculovirus expression system. Although the level of expression of the chimeric HIV-2 gag gene with the V3 domain of either HIV-1 gp120 (gagC-1V3) or HIV-2 gp120 (gagC-2V3) was high, the VLP assembly and extracellular release of GagC-1V3 was very poor. In contrast, GagC-2V3 chimeric proteins formed VLPs and released efficiently. We have constructed substitution mutants to investigate the effects of the hydrophobic region of the V3 domain of HIV-1 Gp120 (1V3) in VLP assembly and release. The substitution mutant analyses revealed that in replacing the hydrophobic region of the 1V3 in GagC-1V3 with the hydrophilic sequence of the V3 domain of HIV-2 Gp120 (2V3) enhanced the extracellular VLP. We demonstrate here that disruption of the hydrophobic character of the C-terminus of the chimeric protein improves assembly and release of the VLPs. Our results suggest that the poor GagC-1V3 VLP release was attributed to the hydrophobic region in the V3 sequence of the chimeric protein, and that not only the N-terminal myristylation and positively charged domain of the Gag protein functioned as a targeting signal to direct membrane binding, but also that the C-terminal hydrophobic region affected release of chimeric VLPs.     

Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles

Technical difficulties have severely limited the yield of methods for the generation of human antiviral monoclonal antibodies (Mabs) in the past. We describe here a novel method for the efficient development of human Mabs against viruses. Rotavirus (RV) is a major cause of gastroenteritis in infants and adults worldwide. We generated fluorescent virus-like particles (VLPs) to identify and physically sort single RV-specific B cells from healthy adult blood donors, or RV-infected infants or adults. We expanded the sorted single B cells in culture, tested for RV-specific antibody secretion, and cloned and sequenced the antibody heavy and light chain variable region (VH and VL) genes. The percentage of wells that produced antibodies after sorting and expanding RV-specific adult B cell clones was high at 23%. The overall efficiency of RV-specific antibody gene recovery after the isolation, confirmation, and cloning of RV-specific VH segments was 1.3% of sorted cells in adults. RV-specific variable gene segments also were obtained from acutely infected infants, although infant B cells did not proliferate and differentiate in culture as well as adult B cells. We expressed recombinant Fabs incorporating the VH and VL genes from RV-specific B cell clones using a new modified bacterial Fab expression vector that we describe. Finally, we demonstrated binding of purified Fabs to RV proteins by immunofluorescence and ELISA. This method for the generation of recombinant human Mabs to RV from single antigen-specific B cell clones selected with fluorescent VLPs could be used to generate human Mabs to many other viruses whose proteins can self-assemble into VLPs.     

重组人乳头瘤病毒6型病毒样颗粒的免疫原性分析

目的　研究重组人乳头瘤病毒 6型 (humanpapillomavirustype 6 ,HPV 6 )病毒样颗粒(virus likeparticle ,VLP)的免疫原性。方法　重组杆状病毒在昆虫细胞中表达制备的HPV 6L1VLP(L1 VLP)和HPV 6L1+L2VLP(L1+2 VLP)经鉴定后 ,用于免疫BALB/c小鼠 ,对诱导的体液免疫和细胞免疫反应进行了检测。结果　电镜观察显示L1 VLP和L1+2 VLP二者形态上无明显差异 ,为圆形颗粒 ,直径约 5 0nm ,SDS PAGE和Westernblot分析表明 ,L1+2 VLP中L1和L2蛋白摩尔比例为 4∶1。用ELISA法测定免疫小鼠血清抗体滴度 ,加佐剂L1 VLP免疫组和加佐剂L1+2 VLP免疫组血清针对HPV 6L1VLP的滴度在 1∶10 0 0 0以上 ,高于未加佐剂组免疫血清滴度 (1∶2 0 0 0 ) ,L1+2 VLP免疫诱导出了特异于L2抗原的抗体。血清抗体主要识别HPV 6构象依赖性抗原表位 ,与HPV 11抗原显示出一定的交叉反应 ,而与HPV 16无明显交叉反应。免疫小鼠脾淋巴细胞体外经HPV 6L1VLP再激活后出现了特异性增殖反应 ,3H TdR掺入值与未免疫组之间差异有显著性 (P <0 .0 1) ,L1 VLP和L1+2 VLP两组间差异无显著性 (P >0 .0 5 ) ,L1 VLP和L1+2 VLP免疫组刺激指数 (SI)分别为 6 .4和 6 .2 ,阴性对照组SI为 1.1。HPV 6L1VLP再刺激特异地诱导免疫组脾淋巴细胞IL 2和IL 10分     

Ebola and Marburg virus-like particles activate human myeloid dendritic cells

The filoviruses, Ebola (EBOV) and Marburg (MARV), are potential global health threats, which cause deadly hemorrhagic fevers. Although both EBOV and MARV logarithmically replicate in dendritic cells (DCs), these viruses do not elicit DC cytokine secretion and fail to activate and mature infected DCs. Here, we employed virus-like particles (VLPs) of EBOV and MARV to investigate whether these genome-free particles maintain similar immune evasive properties as authentic filoviruses. Confocal microscopy indicated that human myeloid-derived DCs readily took up VLPs. However, unlike EBOV and MARV, VLPs induced maturation of DCs including upregulation of costimulatory molecules (CD40, CD80, CD86), major histocompatibility complex (MHC) class I and II surface antigens, and the late DC maturation marker CD83. The chemokine receptors CCR5 and CCR7 were also modulated on VLP-stimulated DCs, indicating that DC could migrate following VLP exposure. Furthermore, VLPs also elicited DC secretion of the pro-inflammatory cytokines TNF-alpha, IL-8, IL-6, and MIP-1alpha. Most significantly, in stark contrast to DC treated with intact EBOV or MARV, DC stimulated with EBOV or MARV VLPs showed enhanced ability to support human T-cell proliferation in an allogenic mixed lymphocyte response (MLR). Thus, our findings suggest that unlike EBOV and MARV, VLPs are effective stimulators of DCs and have potential in enhancing innate and adaptive immune responses.     

Physical interaction of human papillomavirus virus-like particles with immune cells

Human papillomavirus virus-like particles (HPV VLP) and chimeric VLP are immunogens that are able to elicit potent anti-viral/tumor B and T cell responses. To investigate the immunogenicity of VLP, we determined which cells of the immune system are able to bind HPV-16 VLP. VLP were found to bind very well to human and mouse immune cells that expressed markers of antigen-presenting cells (APC) such as MHC class II, CD80 and CD86, including dendritic cells, macrophages and B cells. mAb blocking studies identified Fc gamma RIII (CD16) as one of the molecules to which the VLP can bind both on immune cells and foreskin epithelium. However, transfection of a CD16(-) cell line with CD16 did not confer binding of VLP. Splenocytes from Fc gamma RIII knockout mice showed a 33% decrease in VLP binding overall and specifically to subsets of APC. These combined data support a role for CD16 as an accessory molecule in an HPV VLP-receptor complex, possibly contributing to the immunogenicity of HPV VLP.     

Characterization of self-assembled virus-like particles of human polyomavirus BK generated by recombinant baculoviruses

The major structural protein of the human polyomavirus BK (BKV), VP1, was expressed by using recombinant baculoviruses. A large amount of protein with a molecular mass of about 42 kDa was synthesized and identified by Western blotting. The protein was detected exclusively in the nuclei by immunofluorescent analysis and it was released into culture medium. The expressed BKV VP1 protein was self-assembled into virus-like particles (BK-VLPs) with two different sizes (50 and 26 nm in diameter), which migrated into four different bands in CsCl gradient with buoyant densities of 1.29, 1.30, 1.33, and 1.35 g/cm(3). The immunological studies on the BK-VLPs suggested that they have similar antigenicity with those of authentic BKV particles. Cryoelectron microscopy and 3D image analysis further revealed that the larger BK-VLPs were composed of 72 capsomers which all were pentamers arranged in a T = 7 surface lattice. This system provides useful information for detailed studies of viral morphogenesis and the structural basis for the antigenicity of BKV.     

Interaction of papillomavirus virus-like particles with human myeloid antigen-presenting cells

Papillomavirus-like particles (VLPs) are potent inducers of humoral and cellular immune responses, making them attractive candidates for noninfectious viral subunit vaccines. To further our understanding of how VLPs activate the immune system, we have investigated their interaction with human myeloid antigen-presenting cells. We found that VLPs bound, with increasing density, to the cell surface of human monocytes, macrophages, and monocyte-derived dendritic cells (DCs). Interestingly, there was a negative correlation between binding intensity and CD83 expression in DCs, suggesting that the main receptor for binding of VLPs may be downregulated during maturation. Exposure to VLPs resulted in acute phenotypic activation of monocytes and DCs. Furthermore, VLPs rapidly induced production of inflammatory cytokines in monocytes, macrophages, and DCs, as assessed by intracellular cytokine staining. For each cell type, the patterns of interleukin-1beta, interleukin-12, tumor necrosis factor-alpha, and interleukin-6 production were distinct from the pattern induced by lipopolysaccharide (LPS), a bacterial activator of myeloid antigen-presenting cells. Our results indicate that VLPs target multiple cells of the immune system, which helps to account for VLPs being so effective in priming humoral and cellular immune responses even in the absence of adjuvant.     

HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles

Recombinant human immunodeficiency virus type-1 (HIV-1)-like gag-env particles produced in mammalian cells were inoculated into two New Zealand white rabbits. In parallel, two control rabbits were inoculated with the homologous HIV-1 virions inactivated by ultra violet light (uv) and psoralen treatments. The humoral and cellular immune responses to HIV-1 were evaluated for both groups of animals. Recombinant particles elicited humoral immunity that was specific for all the viral structural proteins. The antibodies recognized both denatured and nondenatured proteins. Moreover, the sera neutralized the in vitro infectivity of the homologous virus in CEM cells. Importantly, the recombinant particles also generated a T helper response by priming with the HIV proteins. Similar results were observed with inactivated virus immunization. Therefore, our results suggest that the recombinant HIV-like particles elicit functional humoral immunity as well as cellular immunity and represent a novel vaccine candidate for AIDS.     

Expression and characterization of human group C rotavirus virus-like particles in insect cells

Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpC RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans.     

Expression of human immunodeficiency virus type 1 Gag protein precursor and envelope proteins from a vesicular stomatitis virus recombinant: high-level production of virus-like particles containing HIV envelope

Recombinant vesicular stomatitis viruses have been developed as high-level expression vectors which serve as effective vaccine vectors in animals (Roberts et al., 1998, J. Virol. 72, 4704-4711; Roberts et al., 1999, J. Virol. 73, 3723-3732). Here we show that two genes can be expressed simultaneously from a single, live-attenuated VSV recombinant. The genes used encode the Pr55(gag) protein precursor of HIV-1 (1.7-kb gene) and an HIV-1 envelope (Env) protein (2.4 kb gene). Our results show that VSV can accommodate up to a 40% increase in genome size with only a threefold reduction in virus titer. Recombinants expressing the Pr55(gag) protein precursor with or without Env protein produced abundant HIV virus-like particles (VLPs) in addition to bullet-shaped VSV particles. HIV Env protein expressed from a VSV recombinant also expressing Gag was specifically incorporated into the HIV VLPs but not into the VSV particles. In contrast, VSV G protein was found in both VSV particles and in HIV VLPs. Such VSV/HIV recombinants producing HIV VLPs with Env protein could be an effective source of HIV-like particles inducing both cellular and antibody-mediated immunity to HIV-1.     

Immune response modulation by recombinant peptides expressed in virus-like particles

Aspergillus fumigatus, a ubiquitous fungus, is implicated in the pathogenesis of a number of clinically different allergic diseases in man, including allergic bronchopulmonary aspergillosis. Peptide-based immunotherapy may offer an alternative treatment strategy for the management of allergic disease. The objective of this study was to alter the allergen-specific immune response using dominant T cell epitopes of a major A. fumigatus allergen, Asp f2, expressed in yeast as virus-like particles (VLP). The T cell epitopes of Asp f2, recognized in mice with an H-2d background, were determined by producing T-cell hybridomas. Two dominant T cell epitopes, aa60--71 and aa235--249, were identified and expressed in a yeast VLP system. To induce tolerance VLP-peptides were injected subcutaneously into mice previously immunized with recombinant Asp f2. The T cell immune response was abrogated totally in 3 weeks following a single injection of VLP but was restored 2 months later following intranasal antigen exposure. T-cell depletion resulted in the reduction of 20-30% of all antigen-specific immunoglobulin classes. Thus, recombinant peptides expressed in the VLP system can be used successfully in the modulation of Asp f2-induced immune response in mice, although a single administration is not sufficient to maintain a state of tolerance for a long period of time.     

SARS-CoV N基因重组腺病毒对树突状细胞前炎症细胞因子mRNA表达和分泌的影响

目的 探讨严重急性呼吸综合征相关的冠状病毒(SARS-CoV)感染后炎症细胞因子的mRNA表达和分泌水平的动态变化,在蛋白质水平上阐明SARS-CoV的致病机理.方法 本研究在建立树突状细胞(dendritic cells,DC)培养方法的前提下,利用SARS-CoV的N基因重组腺病毒(rAd-N)和对照的腺病毒(rAd-LacZ)来感染成熟的DC,用RT-PCR和ELISA法检测DC对IL-1β、IL-6、IL-8和TNF-a的mRNA表达和分泌水平的变化.结果 IL-6和TNF-a mRNA的表达和分泌水平在前24 h之内是逐渐升高的,与对照组(rAd-LacZ感染)相比,差异有统计学意义(P<0.05或P<0.01).结论 SARS-CoY的N蛋白与SARS的急性期DC分泌过量的前炎症细胞因子有关.     

Impaired interleukin 2 production by spleen cells from mice infected with human adenovirus.

Spleen cells from mice infected with human adenovirus type 6 (Ad6) showed defective interleukin 2 (IL2) production 3-5 days after the infection. The response of spleen cells to exogenous IL 2 was also deficient. The impaired capacity of concanavalin A--(Con A)-activated spleen cells from Ad6--infected mice to utilize IL 2 seemed to be related to the depressed capacity of the infected splenocytes to express IL 2 receptors. The immunologic dysfunction following infection with Ad6 may be a consequence of deficiencies in both the elaboration of and response to IL 2.     

Microcarrier culture of COS-1 cells producing Japanese encephalitis and dengue virus serotype 4 recombinant virus-like particles

Two stably transfected COS-1 cell lines that secrete recombinant Japanese encephalitis and dengue virus serotype 4 virus-like particles (VLPs) have been adapted to grow on Cytodex 3 microcarriers in an orbital shaker flask platform. The VLPs are used as antigens in diagnostic enzyme-linked immunosorbent assays to detect anti-arboviral IgM and IgG antibodies in human serum samples. Converting from a stationary flask batch system to a microcarrier fed-batch system has led to increases in antigen concentration while decreasing costs by reducing the amount of cell culture medium, disposables, and labor. The cell culture longevity was increased by 48 days in this optimized system, which may be due to a continual supply of nutrients resulting in prolonged survival of the cells on the microcarrier surface. An initial trial using a serum-free medium with this cell line was promising and may lead to reductions in cost, while reducing the variability between batches introduced by fetal bovine serum.     

Protein composition of adenovirus nucleoprotein complexes extracted from infected cells

A viral nucleoprotein complex was extracted from the nuclei of human cells 20 hr after infection with adenovirus type 2 or several of its temperature-sensitive mutants. In its sedimentation property, density in CsCl, and digestion pattern with micrococcal nuclease, the complex resembled viral cores. The polypeptides V, PVII, IIK, and 36K were found associated with this complex which is formed prior to or in the absence of virus assembly. The results suggest that this nucleoprotein complex is a direct precursor to virus assembly.     

Antitumor activity of TRAIL recombinant adenovirus in human malignant glioma cells

Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. One of disadvantages to previous in vivo protocols was the need for large quantities of TRAIL recombinant protein to suppress tumor growth. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene (Ad.hTRAIL) and transferred them into malignant glioma cells in vitro and tumors in vivo, as an alternative to recombinant soluble TRAIL protein. The results show that TRAIL-sensitive glioma cells infected Ad.hTRAIL undergo apoptosis through the production and expression of TRAIL protein. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of cleavage of poly (ADP-ribose) polymerase. Furthermore, in vivo administration of Ad.hTRAIL at the site of tumor implantation suppressed the outgrowth of human glioma xenografts in SCID mice. These results further define Ad.hTRAIL as an anti-tumor therapeutic and demonstrate its potential use as an alternative approach to treatment for malignant glioma.     

Expression of sapovirus virus-like particles in mammalian cells

Summary. Sapovirus (SaV) is an etiological agent of acute gastroenteritis in human and swine. SaV can be divided into five genogroups, GI to GV. Virus-like particles (VLPs) morphologically similar to native SaV have been expressed for GI, GII, GIII and GV strains in insect cells, although only low expression levels were observed for GII strains. In this study, we report the successful expression of SaV GII VLPs using cultured mammalian COS-7 and 293T cells. Our results demonstrated that this mammalian expression system was able to express and form SaV VLPs.