重组杆状病毒表达的EIAV Env蛋白与含有env基因重组痘苗病毒的联合免疫

目的：构建新型的马传染性贫血病毒(EIAV)的候选疫苗：方法：利用BAC—To—BAC杆状病毒表达系统，将中国马传贫驴白细胞弱毒疫苗(EIAV DLV)及其亲本株(EIAV LN)env基因导入到杆状病毒基因组中。转染昆虫细胞后，得到的重组病毒用SDS—PAGE和Western blot检测表达产物。以本实验室构建的含有EIAV env基因的重组痘苗病毒，单独或与重组杆状病毒表达的EIAV Env蛋白联合免疫小鼠。结果：构建的重组杆状病毒能正确表达全长Env蛋白。与单独免疫组相比，联合免疫组免疫应答显著增强，其中中和抗体的滴度提高5～9倍。结论：含有EIAV env基因的重组痘苗病毒与Env蛋白抗原联合免疫，能够诱导高滴度的中和抗体。     

Synthesis and immunogenicity of hepatitis B virus envelope antigen expressed by recombinant vaccinia virus

Summary Five different recombinant vaccinia viruses expressing the envelope antigen of hepatitis B virus (HBsAg) under the control of the P_7·5 promoter were constructed. Cell cultures infected with some of the recombinant viruses synthesized both middle (M) and major surface (S) protein of HBsAg. It was shown that the length of the nontranslated sequence preceding preS2-ATG influenced the extracellular or intracellular HBV antigen distribution and the preS2:S antigen ratio. Some recombinants synthesized an M protein that was enlarged by additional 35 amino acids of preS1 domain and was entirely retained within the infected cells. Antibody responses to the S and preS2 antigens in mice revealed significant differences in the immunogenicity of individual recombinants.     

Characterization of rabies virus glycoprotein expressed by recombinant baculovirus.

A cDNA of the glycoprotein (G protein) gene of rabies virus Nishigahara strain was cloned and inserted into a baculovirus genome under the control of the polyhedrin promoter. Infection of Spodoptera frugiperda cells with this recombinant virus produced a large quantity of new protein instead of the parental polyhedrin protein. By immunofluorescent and immunoblotting analyses, the recombinant protein was antigenically similar to the authentic G protein. Its molecular mass estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, however, was slightly smaller than that of the authentic one, and this observation was suggested to be due to the difference in glycosylation level between the two G proteins. The recombinant G protein expressed on the cell surface of the insect cells showed a fusion activity at low pH. The fusion activity was inhibited by antiserum against either whole virions or G protein of rabies virus.     

Characterization of Japanese encephalitis virus envelope protein expressed by recombinant baculoviruses

Recombinant baculoviruses containing the coding sequences of the viral structural proteins, i.e., the capsid (C) protein, the precursor to premembrane (preM) protein, and the envelope (E) protein, as well as a nonstructural protein, NS1, of Japanese encephalitis virus (JEV) were constructed. Infection of Spodoptera frugiperda cells with these recombinant viruses produced PreM and E proteins. The E proteins synthesized by the recombinants were shown to be glycosylated and similar in size to the authentic E protein. The E protein was found on the surface of infected cells. The antigenic properties of recombinant E proteins were evaluated using a panel of monoclonal antibodies produced against JEV E protein. It was demonstrated that all of the epitopes detectable on the authentic JEV E protein were present on the recombinant E protein expressed by a recombinant baculovirus containing the coding sequence for a part of C, PreM, E, and a part of NS1 proteins. However, for E protein expressed by a recombinant baculovirus having the coding sequence of only a part of PreM, but all of E and a part of NS1, one of the flavivirus cross-reactive epitopes was not detected. Mice immunized with cells infected with the recombinant baculoviruses developed neutralization antibodies.     

Human antibody response to Toscana virus glycoproteins expressed by recombinant baculovirus

The arthropod-borne Toscana virus has been associated with acute neurological disease in humans. In this study, the viral envelope glycoproteins were expressed in soluble form in a baculovirus system. The recombinant sGN and sGC proteins were used as viral antigens in a Western blot assay to analyze the specific immune response in sera from patients with recognized virus-associated aseptic meningitis. The anti-glycoprotein and the anti-nucleoprotein N IgG responses were compared by an immunoassay based on the recombinant proteins. In this system, all the sera showed a high reactivity to the N protein, but they differed in the response to the glycoproteins.     

Intracellular processing of the porcine coronavirus transmissible gastroenteritis virus spike protein expressed by recombinant vaccinia virus.

The Spike (S) protein from a virulent British field isolate of porcine transmissible gastroenteritis virus (TGEV) FS772/70 was constructed from cDNA and inserted into the vaccinia virus (VV) thymidine kinase gene locus under the control of the VV early/late gene P7.5k promoter. Recombinant S protein was synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive glycoprotein with high mannose simple oligosaccharides (gp 190) that underwent post-translational modification to an Endo H-resistant glycoprotein with complex oligosaccharides (gp210). Immunofluorescence analysis demonstrated that the majority of recombinant S protein was retained at the Golgi but some S protein was expressed on the plasma membrane. Monoclonal antibodies (mAbs) raised against native S protein reacted with this recombinant S protein; also, mice infected with the recombinant vaccinia virus (rVV) expressing the S protein induced TGEV neutralizing antibodies. A truncated S protein (S delta) was also expressed in rVV-infected cells by introducing a deletion into the S protein cDNA that removed 292 amino acids from the C-terminus. The S delta protein (gp 170) was shown to be antigenically similar to TGEV S protein by immunofluorescence and immunoprecipitation tests but was retained in the endoplasmic reticulum and not expressed on the cell surface.     

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response.     

MHV S peplomer protein expressed by a recombinant vaccinia virus vector exhibits IgG Fc-receptor activity.

We have previously shown that cells infected with mouse hepatitis virus (MHV) bind rabbit, mouse, and rat IgG by the Fc portion of the IgG molecule. This Fc-binding activity appeared to be mediated by the MHV S protein. S protein could also be precipitated from MHV-infected cells by a monoclonal antibody directed against the murine Fc gamma receptor (Fc gamma R). To prove definitively that the S protein mediates Fc-binding activity, we have expressed the MHV S protein utilizing recombinant vaccinia viruses. The anti-Fc gamma R monoclonal antibody, 2.4G2, precipitated recombinant S protein in cells of murine, human, and rabbit origin. Since the anti-Fc receptor monoclonal antibody does not react with human and rabbit Fc receptors these results demonstrate that the epitope recognized by this antibody is carried on the MHV S protein and is not murine in origin. Examination of various MHV isolates and escape mutants failed to identify the precise sequences in S responsible for the molecular mimicry of the murine Fc gamma R. These data are consistent with the hypothesis that a previously identified region of similarity between the S protein and the Fc gamma R mediates this activity. The Fc binding activity of S was expressed on the cell surface, since MHV-JHM-infected cells, but not uninfected cells, formed rosettes with anti-sheep red blood cell (SRBC) antibody-coated SRBC. The anti-Fc gamma R monoclonal antibody neutralized MHV-JHM and inhibited syncytium formation induced by the MHV S protein.     

The transmembrane domain of vesicular stomatitis virus glycoprotein suffices to anchor HIV-1 envelope gp120 expressed by a recombinant vaccinia virus

The shedding of HIV-1 glycoprotein gp120 results from the proteolytic cleavage of its precursor gp160 into gp120 and an anchoring gp41, to which gp120 is non-covalently attached. This report is directed toward the anchorage of gp120 expressed by three recombinant vaccinia virus (rvv): vPE16 expresses wild-type gp160; vvE13 the gp120-vesicular stomatitis virus G transmembrane and cytoplasmic tail (VSVGTMCT) fusion protein; and vvE14 the gp120 with 52 amino acids (aa) from the vector. In order to convert gp120 into an integral membrane protein, a gp120- VSVGTMCT chimerical gene fragment has been constructed and expressed in mammalian cells. This gp120 fusion protein expressed by an rvv, vvE13, has been shown to elicit better immunogenicity and protection against a gp160-expressing tumor cell line than the full-length envelope (env) glycoprotein gp160 in mice. The results show that gp120-VSVGTMCT expressed by vvE13 is not shed because it is membrane associated. The hydrophobic fragment of vesicular stomatitis virus G (VSVG) furnishes an anchor of the chimeric protein just as it does in the native VSVG.     

Immunogenic and protective properties of rabies virus glycoprotein expressed by baculovirus vectors

The gene encoding the glycoprotein of rabies virus (G protein, CVS strain) has been cloned and inserted into the baculovirus transfer vector pAcYM1 derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). The gene was placed under the control of the AcNPV polyhedrin promoter and expressed to high levels by the derived recombinant virus using a Spodoptera frugiperda cell line. It has been established that the antigenic characteristics of the protein were conserved by comparison with those of the native glycoprotein of rabies virions. The immunogenicity of the expressed product was also demonstrated. Intraperitoneal or intramuscular injection of G antigen conferred protection to mice and was associated with the induction of high titers of neutralizing antibodies. The availability of large quantities of antigenically and immunogenically reactive rabies G protein may make feasible crystallographic studies and the safe preparation of a low cost subunit vaccine for the disease.     

Expression of bovine leukaemia virus envelope gene by recombinant vaccinia viruses

Recombinant vaccinia viruses (VV) containing the envelope gene of bovine leukaemia virus (BLV) were constructed. Three virus constructs were designed: VV-BLV1 which contained the open reading frame for envelope glycoprotein gp51 alone, under control of VVP7.5 promoter; VV-BLV2 and VV-BLV3 contained the entire gene (gp51 + gp30) coding sequence downstream of VP7.5 and the fowlpox virus early/late promoter (PFE/L) respectively. All three VV recombinants expressed envelope glycoproteins as determined by the agar gel diffusion assay. By immunofluorescence techniques it was shown that while VV-BLV2 and VV-BLV3 expressed envelope glycoprotein on the surface of virus-infected cells, VV-BLV1 failed to do so. Rabbits inoculated with VV-BLV1 failed to show an anti envelope glycoprotein antibody response, however, significant levels of antibodies against envelope glycoprotein were detected in sera from rabbits inoculated with VV-BLV2 and VV-BLV3.     

重组杆状病毒表达HEV ORF2抗原片段

目的：用重组杆状病毒表达戊型肝炎病毒（hepatitis E virus,HEV)ORF2基因片段,并对其免疫学特性进行研究。方法：与中间转染载体pVL1393相连构建重组质粒,在Lipofectin介导下将其与杆状病毒线性DNA（BaculoGold^TM)共转染Sf9昆虫细胞得到重组病毒,用免疫荧光,Western blot等方法对表达产物进行免疫学特性初步研究,并进行动物免疫试验。结果：含ORF2结构基因片段的重组杆状病毒在Sf9昆虫细胞中获得了高效表达,经免疫荧光,Western blot,动物免疫试验证实该重组蛋白为HEV特异性蛋白,可刺激机体产生相应抗体。结论：重组杆状病毒能有效表达HEV抗原基因,所表达的ORF2重组蛋白具有HEV抗体识别的抗原表位,具有较好的抗原性和免疫原性。     

Synthesis, oligomerization, and biological activity of the human immunodeficiency virus type 2 envelope glycoprotein expressed by a recombinant vaccinia virus

The full-length envelope gene from an infectious human immunodeficiency virus type 2 (HIV-2) molecular clone was expressed in CD4+ and CD4- cells by a recombinant vaccinia virus vector. Pulse-chase experiments indicated that gp160 was processed into gp120 and gp41 subunits. Although large amounts of gp120 were shed into the medium, the recombinant vaccinia virus-infected cells fused with uninfected CD4+ cells. The receptor binding of HIV-2 gp120 was further analyzed using a panel composed of nine soluble CD4 mutants containing insertions of 2 amino acids within the first and second immunoglobulin-like domains. Of three mutations previously shown to interfere with HIV-1 gp120 binding, two also interfered with binding of the HIV-2 glycoprotein indicating use of the same binding site. Chemical crosslinking, sucrose gradient sedimentation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were employed to study the oligomerization of the envelope protein. The data indicated that gp160 assembles posttranslationally into dimers and higher oligomers that are probably tetramers.     

Expression of bovine immunodeficiency-like virus envelope glycoproteins by a recombinant baculovirus in insect cells.

The bovine immunodeficiency-like virus (BIV) env open reading frame (ORF) contains both sequences encoding env and sequences for exon 1 of the putative rev gene. Recombinant baculoviruses incorporating BIV env ORF sequences were constructed to characterize the expression, processing, and immunogenicity of products of the BIV env ORF in insect cells and to develop reagents to study native BIV Env glycoproteins. A recombinant baculovirus containing the entire env ORF synthesized a nonglycosylated, 20-kDa, BIV-specific protein, apparently unrelated to native BIV Env proteins. In contrast, a recombinant baculovirus containing a truncated env ORF in which the coding sequences for rev exon 1 were deleted synthesized three size classes of glycosylated proteins in insect cells related to the BIV Env precursor (gp145), surface (gp100), and transmembrane (gp45) glycoproteins observed in BIV-infected mammalian cells. Oligomers of recombinant BIV Env proteins also formed in these baculovirus-infected insect cells. Immunofluorescence staining of intact insect cells infected by the baculovirus expressing BIV Env with BIV-specific serum demonstrated that the recombinant Env glycoproteins were expressed on the cell surface. Antisera raised to recombinant Env glycoproteins immunoprecipitated native gp145, gp100, and gp45 in BIV-infected bovine cells similar to sera from animals naturally or experimentally infected with BIV.     

Comparison of protective immunity elicited by recombinant vaccinia viruses that synthesize E or NS1 of Japanese encephalitis virus.

Immunization with recombinant vaccinia viruses that specified the synthesis of Japanese encephalitis virus (JEV) glycoproteins protected mice from a lethal intraperitoneal challenge with JEV. Recombinants which coexpressed the genes for the structural glycoproteins, prM and E, elicited high levels of neutralizing (NEUT) and hemagglutination inhibiting (HAI) antibodies in mice and protected mice from a lethal challenge by JEV. Recombinants expressing only the gene for the nonstructural glycoprotein, NS1, induced antibodies to NS1 but provided low levels of protection from a similar challenge dose of JEV. Antibodies to the NS3 protein in postchallenge sera, representing the degree of infection with challenge virus, were inversely correlated to NEUT and HAI titers and levels of protection. These results indicate that although vaccinia recombinants expressing NS1 can provide some protection from lethal JEV infection, recombinants expressing prM and E elicited higher levels of protective immunity.     

Immunogenicity of the Plasmodium falciparum glutamate-rich protein expressed by vaccinia virus.

The glurp gene of Plasmodium falciparum F32 has been inserted into a vaccinia virus, and the recombinant virus was designated VVG4. Expression of glurp in VVG4-infected Vero cells was analyzed by immunoprecipitation and revealed a primary GLURP product of approximately 220,000 Da; GLURP was detected both intracellularly and in culture supernatants. To study the immunogenicity of vaccinia virus-expressed GLURP, mice were immunized with VVG4 and serum samples were analyzed for antibody reactivity with three polypeptides, covering almost the entire GLURP molecule; these three polypeptides were produced in recombinant form in Escherichia coli. The immune response was primarily directed against a carboxy-terminal repeat region. The mouse anti-GLURP serum recognized authentic GLURP by immunoprecipitation analysis from P. falciparum grown in vitro. These results demonstrate that vaccinia virus-expressed glurp product can induce a humoral immune response against GLURP derived from blood-stage parasites.     

Expression of envelope glycoprotein (E) of Japanese encephalitis virus by recombinant vaccinia virus

Vaccinia virus recombinants inserted with cDNA clones of Japanese encephalitis (JE) virus envelope glycoprotein (E) gene were constructed. The E gene product was detected in the recombinant virus-infected BHK21 cells by immunofluorescence (IF) and Western blotting. The intensity of IF observed was higher by the recombinant of the TK promoter--P7.5 promoter--inserted cDNA construct than by the P7.5 promoter--TK promoter--inserted cDNA construct. The E gene product was hardly detected by the recombinant carrying the TK promoter only upstream to the inserted cDNA, although the glycoprotein E mRNA had been transcribed.     

Feline leukemia virus envelope protein expression encoded by a recombinant vaccinia virus: apparent lack of immunogenicity in vaccinated animals

We have constructed a recombinant vaccinia virus encoding the expression of the feline leukemia virus (FeLV) envelope gene of the Gardner-Arnstein strain of FeLV subgroup B. Human cells infected with the recombinant virus (vFeLVenv) express and process the FeLV envelope protein similarly to cells infected with authentic FeLV. The mature gp 70 protein is transported to and accumulates on the surface of vFeLVenv-infected cells. Vaccinia virus replication and FeLV gp70 accumulation was also observed in cells of feline and murine origin, albeit at levels somewhat reduced from those in human cells. Toward the goal of developing a recombinant vaccinia virus as a live vaccine for feline leukemia disease in cats, immunogenicity studies were performed in cats and mice. These experiments yielded surprising results: although animals mounted a typical virus-neutralizing antibody response to the vaccinia virus vector, we were unable to detect antibodies against FeLV gp70 in any of the vaccinated animals. A subsequent 'booster' immunization with killed FeLV was unable to elicit evidence of immunologic 'priming' by the recombinant virus. We are presently unable to explain the apparent lack of immunogenicity. These results may point to complexities involved in the development of vaccines to protect against retrovirus infection.     

Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry

We have tested Green Fluorescent Protein (GFP) expressed by a vaccinia virus recombinant as a marker for viral infection. Virus recombinants expressing either wild-type GFP, or a Ser₆₅ to Thr mutated version (GFP-S65T) were used to infect cultured cells, and the appearance of fluorescence was followed during infection by flow cytometry. Although both versions were detectable in infected cells, GFP-S65T gave up to 26-fold brighter fluorescence than wild-type GFP when excited by an argon laser beam (488 nm). In addition, GFP-S65T fluorescence appeared earlier, and infected cells could be detected above background as soon as 1 h after infection. We have used this construct to infect porcine peripheral blood lymphocytes, and show its usefulness to study virus tropism when used in combination with cell-type specific markers. Thus, GFP provides a direct, fast and convenient way to monitor infection by flow cytometry.     

Characterization of the antibody response to three different versions of the HTLV-I envelope protein expressed by recombinant vaccinia viruses: induction of neutralizing antibody.

Recombinant vaccinia viruses (RVV) designated RVV E1, RVV E2, and RVV E3, were constructed to express three different versions of the human T cell leukemia virus type I (HTLV-I) envelope proteins to determine which configuration elicits an optimal antibody response. RVV E1 expressed the native HTLV-I envelope proteins gp46 (surface protein) and gp21 (transmembrane protein), while RVV E2 expressed the envelope precursor with the proteolytic cleavage site deleted. The RVV E3 construct expressed only the external surface glycoprotein, gp46. Radioimmunoprecipitation and FACS analysis confirmed that the appropriate envelope proteins were expressed by RVV E1-, E2-, and E3-infected cells. Immunization studies were carried out using Balb/c, A/J, and C57BL/6 strains of mice. Balb/c mice responded poorly to immunization with all of the three RVV constructs. C57BL/6 mice produced neutralizing antibodies in response to immunization with all three constructs, whereas A/J mice developed neutralizing antibodies only when immunized with the RVV E1s construct. The results indicate that the humoral immune responses depend on the form of HTLV-I envelope proteins expressed by each RVV.