Ebola Virus Can Be Effectively Neutralized by Antibody Produced in Natural Human Infection

The activity of antibodies against filoviruses is poorly understood but has important consequences for vaccine design and passive prophylaxis. To investigate this activity, a panel of recombinant human monoclonal antibodies to Ebola virus antigens was isolated from phage display libraries constructed from RNA from donors who recovered from infection in the 1995 Ebola virus outbreak in Kikwit, Democratic Republic of Congo. Antibodies reactive with nucleoprotein (NP), envelope glycoprotein (GP), and secreted envelope glycoprotein (sGP) were characterized by immunofluorescence and radioimmunoprecipitation assays. Four antibodies reacting strongly with sGP and weakly with GP and two antibodies reacting with NP were not neutralizing. An antibody specific for GP neutralized Ebola virus to 50% at 0.4 microgram/ml as the recombinant Fab fragment and to 50% at 0.3 microgram/ml (90% at 2.6 microgram/ml) as the corresponding whole immunoglobulin G1 molecule. The studies indicate that neutralizing antibodies are produced in infection by Ebola virus although probably at a relatively low frequency. The neutralizing antibody may be useful in vaccine design and as a prophylactic agent against Ebola virus infection.     

Defining the N-linked glycosylation site of Hantaan virus envelope glycoproteins essential for cell fusion

The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV.     

Antibodies to rotavirus outer capsid glycoprotein VP7 neutralize infectivity by inhibiting virion decapsidation

The rotavirus capsid is composed of three concentric protein layers. Proteins VP4 and VP7 comprise the outer layer. VP4 forms spikes, is the viral attachment protein, and is cleaved by trypsin into VP8* and VP5*. VP7 is a glycoprotein and the major constituent of the outer protein layer. Both VP4 and VP7 induce neutralizing and protective antibodies. To gain insight into the virus neutralization mechanisms, the effects of neutralizing monoclonal antibodies (MAbs) directed against VP8*, VP5*, and VP7 on the decapsidation process of purified OSU and RRV virions were studied. Changes in virion size were followed in real time by 90 degrees light scattering. The transition from triple-layered particles to double-layered particles induced by controlled low calcium concentrations was completely inhibited by anti-VP7 MAbs but not by anti-VP8* or anti-VP5* MAbs. The inhibitory effect of the MAb directed against VP7 was concentration dependent and was abolished by papain digestion of virus-bound antibody under conditions that generated Fab fragments but not under conditions that generated F(ab')(2) fragments. Electron microscopy showed that RRV virions reacted with an anti-VP7 MAb stayed as triple-layered particles in the presence of excess EDTA. Furthermore, the infectivity of rotavirus neutralized via VP8*, but not that of rotavirus neutralized via VP7, could be recovered by lipofection of neutralized particles into MA-104 cells. These data are consistent with the notion that antibodies directed at VP8* neutralize by inhibiting binding of virus to the cell. They also indicate that antibodies directed at VP7 neutralize by inhibiting virus decapsidation, in a manner that is dependent on the bivalent binding of the antibody.     

Virus-specific phosphoproteins in simian sarcoma virus-transformed primate cells.

Cells transformed by simian sarcoma virus (SSV) express a 115000-dalton protein ( p115 ) that is precipitated by a goat antiserum to disrupted SSV/SSAV-infected and transformed cells but not by antibodies directed against the viral gag protein, p30, or envelope proteins. The protein is detected in productively as well as in nonproductively infected, transformed cells. It is not present in untransformed cells infected with helper virus (SSAV). The protein can be phosphorylated in vivo and in vitro at the tyrosine residue and SSV-transformed cells contain elevated levels of phosphotyrosine.     

Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment.

Foot-and-mouth disease virus structural protein VP1 elicits neutralizing and protective antibody and is probably the viral attachment protein which interacts with cellular receptor sites on cultured cells. To study the relationships between epitopes on the molecule related to neutralization and cell attachment, we tested monoclonal antibodies prepared against type A12 virus, isolated A12 VP1, and a CNBr-generated A12 VP1 fragment for neutralization and effect on viral absorption. The antibodies selected for analysis neutralized viral infectivity with varying efficiencies. One group of antibodies caused a high degree of viral aggregation and inhibited the adsorption of virus to cells by 50 to 70%. A second group of antibodies caused little or no viral aggregation but inhibited the adsorption of virus to cells by 80 to 90%. One antibody, which is specific for the intact virion, caused little viral aggregation and had no effect on the binding of virus to specific cellular receptor sites. Thus, at least three antigenic areas on the surface of foot-and-mouth disease virus which were involved in neutralization were demonstrated. One of the antigenic sites appears to have been responsible for interaction with the cellular receptor sites on the surface of susceptible cells.     

Cell fusion activities of Hantaan virus envelope glycoproteins

Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.     

Neutralizing monoclonal antibodies against three serotypes of porcine rotavirus.

Using three serotypes (four strains) of cultivable porcine rotavirus as immunizing antigens, 10 neutralizing monoclonal antibodies were characterized. One VP4-specific monoclonal antibody directed against porcine rotavirus BEN-144 (serotype G4) neutralized human rotavirus strain ST-3 in addition to the homologous porcine virus. All nine VP7-specific monoclonal antibodies were highly specific for viruses of the same serotype as the immunizing rotavirus strain. One exception was the VP7-specific monoclonal antibody C3/1, which neutralized both serotype G3 and G5 rotaviruses. However, this monoclonal antibody did not neutralize the porcine rotavirus AT/76, also of serotype G3, nor mutants of SA-11 virus (serotype G3) which were selected with monoclonal antibody A10/N3 and are known to have mutations affecting the C antigenic region.     

A simian virus 40-encoded protein of Mr 74,000 daltons is structurally related to the capsid proteins of the virus

We have demonstrated the synthesis of a 74,000-dalton protein (74K protein) in African green monkey kidney cells infected with simian virus (SV)40. The 74K protein was detected late during the lytic cycle. Its synthesis was inhibited by arabinosyl cytosine as was the synthesis of the capsid proteins. Monospecific antibodies raised against VP1 and VP3 precipitated the structural proteins and the 74K protein. The 74K protein was not found in purified virions. Tryptic peptide analysis demonstrated that the 74K protein shares methionine- and serine-containing peptides with VP1 and VP3 and thus is structurally related to the capsid proteins.     

The purified 14-kilodalton envelope protein of vaccinia virus produced in Escherichia coli induces virus immunity in animals.

Vaccinia virus (VV) was successfully used as a live vaccine to eradicate smallpox, but the nature of viral proteins involved in eliciting viral immunity has not yet been identified. A potential candidate is a 14-kDa VV envelope protein that is involved in virus penetration at the level of virus-cell fusion, in cell-cell fusion late in infection, and in virus dissemination. The 14-kDa envelope protein has been produced in Escherichia coli, with properties similar to those of the native protein found in the virus particle and in infected cells (C. Lai, S. Gong, and M. Esteban, J. Biol. Chem. 256:22174-22180, 1990). In this investigation, we showed that mice immunized with purified VV 14-kDa protein synthesized in E. coli in the form of a monomer or a trimer develop high-titer neutralizing antibodies and are protected when challenged with lethal doses of wild-type VV. Our findings demonstrate that it is possible to confer protection against VV through immunization with the 14-kDa envelope protein.     

Monoclonal antibodies against simian virus 40 nuclear large T tumour antigen: epitope mapping, papova virus cross-reaction and cell surface staining.

Thirty six cloned hybridomas have been isolated which produce monoclonal antibodies directed against simian virus 40 (SV40) large T tumour antigen. They have been shown to recognize at least six different epitopes along the T antigen polypeptide according to their reaction with the various truncated forms of T antigen expressed by adenovirus-SV 40 hybrid viruses. Sixteen antibodies cross-react with cells infected by the closely related human BK virus. Only two antibodies, PAb1604 and PAb1614, directed against different epitopes of the SV40 T antigen, cross-react with polyoma large T tumour antigen which has a more limited amino acid sequence homology. This cross-reaction is rarely seen with polyclonal antibodies. Monoclonal antibody PAb1620 gave nuclear immunofluorescence only with murine cells transformed by SV40 and was found to react with a complex of T-antigen and 53 000-dalton host-coded protein. All the monoclonal antibodies react with nuclear T antigen and all but four antibodies stained the surface of SV40-transformed cells. These were four of the five antibodies directed against the central third of the T antigen. Thus the monoclonal antibodies show that cell surface T antigen differs from nuclear T antigen, either in accessibility or structure.     

Blocking immune evasion as a novel approach for prevention and treatment of herpes simplex virus infection

Many microorganisms encode immune evasion molecules to escape host defenses. Herpes simplex virus type 1 glycoprotein gC is an immunoevasin that inhibits complement activation by binding complement C3b. gC is expressed on the virus envelope and infected cell surface, which makes gC potentially accessible to blocking antibodies. Mice passively immunized with gC monoclonal antibodies prior to infection were protected against herpes simplex virus challenge only if the gC antibodies blocked C3b binding. Mice treated 1 or 2 days postinfection with gC monoclonal antibodies that block C3b binding had less severe disease than control mice treated with nonimmune immunoglobulin G (IgG). Mice immunized with gC protein produced antibodies that blocked C3b binding to gC. Immunized mice were significantly protected against challenge by wild-type virus, but not against a gC mutant virus lacking the C3b binding domain, suggesting that protection was mediated by antibodies that target the gC immune evasion domain. IgG and complement from subjects immunized with an experimental herpes simplex virus glycoprotein gD vaccine neutralized far more mutant virus defective in immune evasion than wild-type virus, supporting the importance of immune evasion molecules in reducing vaccine potency. These results suggest that it is possible to block immune evasion domains on herpes simplex virus and that this approach has therapeutic potential and may enhance vaccine efficacy.     

Nuclear localization of poliovirus capsid polypeptide VP1 expressed as a fusion protein with SV40-VP1

The poliovirus cDNA fragment coding for capsid polypeptide VP1 was inserted between the EcoRI and BamHI sites of SV40 DNA, generating a chimaeric gene in which the sequence of the 302 amino acids (aa) of poliovirus capsid polypeptide VP1 was placed downstream from that of the 94 N-terminal aa of SV40 capsid polypeptide VP1. The resulting defective, hybrid virus, SV40-delta 1 polio, was propagated in CV1 cells using an early SV40 mutant, am404, as a helper. Cells doubly infected by SV40-delta 1 polio and am404 expressed a 50-kDal fusion protein which was specifically immunoprecipitated by polyclonal and/or monoclonal antibodies raised against poliovirus capsids or against poliovirus polypeptide VP1. Examination of the infected cells by immunofluorescence after staining with anti-poliovirus VP1 immune sera revealed that the fusion protein was mostly located in the intra- and perinuclear space of the cells, in contrast to the exclusively intracytoplasmic location of genuine poliovirus VP1 polypeptide that was observed in poliovirus-infected cells. This suggests that the N-terminal part of the SV40-VP1 polypeptide could contain an important sequence element acting as a migration signal for the transport of proteins from the cytoplasm to the nucleus.     

牛疱疹病毒Ⅰ型VP22和猪繁殖与呼吸综合征病毒GP5融合基因DNA疫苗的免疫效应

为了提高表达GP5的猪繁殖与呼吸综合征病毒（PRRSV）DNA疫苗的免疫效应,将具有蛋白转导功能的牛疱疹病毒1型（BHV-1）VP22基因插入到经过修饰具有更好免疫原性的PRRSV修饰型ORF5基因（ORF5M）上游,构建VP22和ORF5M融合表达的真核表达质粒pCI-VP22-ORF5M。经间接免疫荧光试验（IFA）和Westernblot检测证实体外表达后,免疫BALB/c小鼠,检测小鼠免疫后的GP5特异性ELISA抗体、抗PRRSV中和抗体和脾淋巴细胞增殖反应,并与非融合的真核表达质粒pCI-ORF5M进行比较。结果显示,融合表达VP22-GP5的DNA疫苗 pCI-VP22ORF5M诱导的体液免疫和细胞免疫反应均明显高于非融合表达的DNA疫苗pCI-ORF5M,表明蛋白转导相关蛋白BHV-1 VP22能显著增强表达GP5的PRRSV DNA 疫苗的免疫效应,有效发挥了基因免疫佐剂效应;这为研制PRRSV高效DNA疫苗奠定了基础,同时也为其它疾病的高效新型疫苗研究提供了思路。     

Identification of a GP64 subdomain involved in receptor binding by budded virions of the baculovirus Autographica californica multicapsid nucleopolyhedrovirus

Enveloped virus entry into host cells is typically initiated by an interaction between a viral envelope glycoprotein and a host cell receptor. For budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, the envelope glycoprotein GP64 is involved in host cell receptor binding, and GP64 is sufficient to mediate low-pH-triggered membrane fusion. To better define the role of GP64 in receptor binding, we generated and characterized a panel of antisera against subdomains of GP64. Eight subdomain-specific antisera were generated, and their reactivities with GP64 proteins and neutralization of virus infectivity and binding were examined. Antibodies directed against the N-terminal region of GP64 (amino acids 21 to 159) showed strong neutralization of infectivity and effectively inhibited binding of (35)S-labeled budded virions to Sf9 cells. In addition, we generated virions displaying truncated GP64 constructs. A construct displaying the N-terminal 274 amino acids (residues 21 to 294) of the ectodomain was sufficient to mediate virion binding. Additional studies of antisera directed against small subdomains revealed that an antiserum against a 40-amino-acid region (residues 121 to 160) neutralized virus infectivity. Site-directed mutagenesis was subsequently used for functional analysis of that region. Recombinant viruses expressing GP64 proteins with single amino acid substitutions within amino acids 120 to 124 and 142 to 148 replicated to high titers, suggesting that those amino acids were not critical for receptor binding or other important GP64 functions. In contrast, GP64 proteins with single amino acid substitutions of residues 153 and 156 were unable to substitute for wild-type GP64 and did not rescue a gp64 knockout virus. Further analysis showed that these substitutions substantially reduced binding of recombinant virus to Sf9 cells. Thus, the amino acid region from positions 21 to 159 was identified as a putative receptor binding domain, and amino acids 153 and 156 appear to be important for receptor binding.     

Characterization of two monoclonal antibodies, 38F10 and 44D11,against the major envelope fusion protein of Helicoverpa armigera nucleopolyhedrovirus

The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor(F_0) and then cleaved into a disulfide-linked F_1 and F_2 subunits during the process of protein maturation and secretion. To facilitate further investigation into the structure and function of F protein during virus infection, monoclonal antibodies(mAbs) against the F_2 subunit of Helicoverpa armigera nucleopolyhedrovirus(HearNPV)(Ha F) were generated. Two kinds of mAbs were obtained according to their different recognition epitopes: one kind of mAbs, as represented by 38F10,recognizes amino acid(aa) 85 to 123 of F_2 and the other kind, represented by 44D11, recognizes aa148 to 173 of F_2. Western blot and immunofluorescence assay confirmed that both of the mAbs recognized the F protein expressed in HearNPV infected cells, however, only 44D11 could neutralize HearNPV infection. The results further showed that 44D11 may not interact with a receptor binding epitope, rather it was demonstrated to inhibit syncytium formation in cells expressing the Ha F protein. The results imply that the monoclonal antibody 44D11 recognizes a region within HaF_2 that may be involved in the F-mediated membrane fusion process.     

Multimeric structure of the membrane erythropoietin receptor of murine erythroleukemia cells (Friend cells). Cross-linking of erythropoietin with the spleen focus-forming virus envelope protein.

In erythroleukemia cells infected with the polycythemia strain of the Friend virus complex, erythropoietin could be cross-linked mainly to a protein of 63 kDa when using disuccinimidyl suberate. In contrast, erythropoietin in other erythroleukemia cells cross-linked to two proteins of 85 and 100 kDa. When native erythropoietin receptor complexes were immunoprecipitated, the 63-kDa erythropoietin-cross-linked protein could be precipitated both by antibodies directed against the intracellular part of the cloned chain of the erythropoietin receptor and by antibodies directed against the envelope proteins of the Friend virus. However, after denaturation of the complexes, the 63-kDa protein was only precipitated by antibodies directed against the envelope proteins of the Friend virus. Enzymatic deglycosylation confirmed that erythropoietin was cross-linked with the envelope protein of the defective virus and bidimensional diagonal gel electrophoresis analyses showed that some of the erythropoietin cross-linked envelope proteins were dimerized by disulfide bonds. Thus, the main erythropoietin-receptor complex in the plasma membrane of these cells consisted of a molecule of the cloned chain of the erythropoietin receptor noncovalently associated with one or two disulfide-bonded molecule(s) of the envelope protein of the defective virus. Moreover, our results also showed that the viral envelope protein associated with the cloned chain of the erythropoietin receptor at a site distinct from the erythropoietin binding site.     

Envelope membranes from mature spinach chloroplasts contain a NADPH:protochlorophyllide reductase on the cytosolic side of the outer membrane

Using fluorescence spectroscopy, we have demonstrated that isolated envelope membranes from mature spinach chloroplasts catalyze the phototransformation of endogenous protochlorophyllide into chlorophyllide in presence of NADPH, but not in presence of NADH. Protochlorophyllide reductase was characterized further using monospecific antibodies (anti-protochlorophyllide reductase) raised against the purified enzyme from oat. In mature spinach chloroplasts, protochlorophyllide reductase is present only in envelope membranes. We have demonstrated that the envelope protochlorophyllide reductase, a 37,000-dalton polypeptide, is only a minor envelope component and is present on the outer surface of the outer envelope membrane. This conclusion is supported by several lines of evidence: (a) the envelope polypeptide that was immunodecorated with anti-protochlorophyllide reductase can be distinguished from the major 37,000-dalton envelope polypeptide E37 (which was identified by monospecific antibodies) only after two-dimensional polyacrylamide gel electrophoresis; (b) the envelope protochlorophyllide reductase was hydrolyzed when isolated intact chloroplasts were incubated in presence of thermolysin; and (c) isolated intact chloroplasts strongly agglutinate when incubated in presence of antibodies raised against protochlorophyllide reductase. These results demonstrate that major differences exist between chloroplasts and etioplasts with respect to protochlorophyllide reductase levels and localization. The presence on the chloroplast envelope membrane of both the substrate (protochlorophyllide) and the enzyme (protochlorophyllide reductase) necessary for chlorophyllide synthesis could have major implications for the understanding of chlorophyll biosynthesis in mature chloroplasts.     

Contribution of ebola virus glycoprotein, nucleoprotein, and VP24 to budding of VP40 virus-like particles

The VP40 matrix protein of Ebola virus buds from cells in the form of virus-like particles (VLPs) and plays a central role in virus assembly and budding. In this study, we utilized a functional budding assay and cotransfection experiments to examine the contributions of the glycoprotein (GP), nucleoprotein (NP), and VP24 of Ebola virus in facilitating release of VP40 VLPs. We demonstrate that VP24 alone does not affect VP40 VLP release, whereas NP and GP enhance release of VP40 VLPs, individually and to a greater degree in concert. We demonstrate further the following: (i). VP40 L domains are not required for GP-mediated enhancement of budding; (ii). the membrane-bound form of GP is necessary for enhancement of VP40 VLP release; (iii). NP appears to physically interact with VP40 as judged by detection of NP in VP40-containing VLPs; and (iv). the C-terminal 50 amino acids of NP may be important for interacting with and enhancing release of VP40 VLPs. These findings provide a more complete understanding of the role of VP40 and additional Ebola virus proteins during budding.     

Hsp70与汉滩病毒S基因融合蛋白的原核表达及免疫小鼠的初步研究

本文构建了hsp70与S基因的原核融合表达载体pGEX-4T-1/hsp70-S,在大肠杆菌中表达,并通过GSTrapFF柱进行了纯化。同时制备了NP和Hsp70两种纯化蛋白。分别用这三种纯化蛋白免疫BALB/c小鼠,结果表明纯化的NP和Hsp70-NP两种蛋白均可同时诱导产生抗汉滩病毒核蛋白(NP)抗体,且后者刺激产生的抗体效价明显高于前者。淋巴细胞增殖实验表明,两组免疫小鼠的脾细胞均能够对体外抗原刺激产生增殖反应,而Hsp70-NP组免疫小鼠脾细胞对NP的增殖指数明显高于NP组免疫组。结果显示,与单独用NP免疫小鼠相比,Hsp70-NP纯化蛋白可以刺激机体产生更强的抗汉滩病毒体液免疫应答和特异性淋巴细胞增殖反应。     

Characterization of an alternate form of Newcastle disease virus fusion protein

The sequence and structure of the Newcastle disease virus (NDV) fusion (F) protein are consistent with its classification as a type 1 glycoprotein. We have previously reported, however, that F protein can be detected in at least two topological forms with respect to membranes in both a cell-free protein synthesizing system containing membranes and infected COS-7 cells (J. Virol. 77:1951-1963, 2003). One form is the classical type 1 glycoprotein, while the other is a polytopic form in which approximately 200 amino acids of the amino-terminal end as well as the cytoplasmic domain (CT) are translocated across membranes. Furthermore, we detected CT sequences on surfaces of F protein-expressing cells, and antibodies specific for these sequences inhibited red blood cell fusion to hemagglutinin-neuraminidase and F protein-expressing cells, suggesting a role for surface-expressed CT sequences in cell-cell fusion. Extending these findings, we have found that the alternate form of the F protein can also be detected in infected and transfected avian cells, the natural host cells of NDV. Furthermore, the alternate form of the F protein was also found in virions released from both infected COS-7 cells and avian cells by Western analysis. Mass spectrometry confirmed its presence in virions released from avian cells. Two different polyclonal antibodies raised against sequences of the CT domain of the F protein slowed plaque formation in both avian and COS-7 cells. Antibody specific for the CT domain also inhibited single-cycle infections, as detected by immunofluorescence of viral proteins in infected cells. The potential roles of this alternate form of the NDV F protein in infection are discussed.