The Immune Response to Artificial Proteins Containing Biologically Active Fragments of Human IFN-α2 and Insulin

A study was made of the humoral immune response of BALB/c mice to various doses of artificial proteins that contained biologically active fragments of human interferon 2 (IFN-2) and insulin. The insulin fragment had no effect on the response to any protein construct. The IFN-2 fragment increased the titer of antibodies against the construct. Mapping of continuous B epitopes with immune sera revealed several antigenic determinants, the C end of the IFN-2 fragment with the adjacent de novo protein region being immunodominant. The more effective binding of serum antibodies with the constructs containing the IFN-2 fragment was attributed to antibody interaction with the fragment and to better recognition of the entire protein construct by the immune system.     

基因免疫制备西尼罗病毒NS5蛋白特异性单抗

研究了NS5蛋白在西尼罗病毒的特异性检测方面的应用及NS5在黄病毒复制中的作用机理。采用RT．PCR方法扩增了西尼罗病毒株的NS5基因片段,将其克隆至真核表达载体pVAX1,构建真核表达质粒。以重组质粒免疫BALB／c小鼠后取脾脏进行杂交瘤细胞融合,建立能稳定分泌西尼罗NS5单克隆抗体的杂交瘤细胞株。构建了真核表达质粒pVAX1-WNV—NS5,免疫动物后获得了28289等4株稳定分泌特异性抗体的杂交瘤细胞株,均为IgM型。真核表达质粒免疫后成功地诱导了针对NS5蛋白的体液免疫应答,单抗特异性分析显示4株单抗与其他黄病毒存在一定交叉反应。     

Neutralizing interferon alpha as a therapeutic approach to autoimmune diseases

Therapeutic antibodies directed against tumor necrosis factor alpha (TNF-alpha) for the treatment of rheumatoid arthritis, and against the human EGF receptor-2 (HER2) receptor for the treatment of breast cancer have provided significant clinical benefit for the patients. The success of these antibodies has also provided strong support for the possibility that increased activity of cytokines or growth factors is causally implicated in a variety of human diseases. Interferon alpha (IFN-alpha) is induced by viruses (linked by epidemiological studies to autoimmune diseases), has significant direct effects on both epithelial cells and the immune system, and then can be further induced by the autoantibodies and apoptotic cells generated by the actions of IFN-alpha. The direct and deleterious impact on target tissues, the ability to induce an autoimmune response, and the potential for a self-sustaining cycle of induction and damage suggests that IFN-alpha could be a pivotal factor in the development of autoimmune diseases. This review will evaluate the rationale for, possible approaches to, and safety concerns associated with, targeting interferon alpha (IFN-alpha) as a therapeutic strategy for the treatment of autoimmune diseases. While the approach may be applicable to several autoimmune diseases, there will be an emphasis on systemic lupus erythematosus and insulin dependent diabetes mellitus.     

Elicitation of immunity in mice after immunization with the S2 subunit of the severe acute respiratory syndrome coronavirus

The S2 domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein is responsible for fusion between virus and target cell membranes, and is expected to be immungenic. In this study, we investigated the immune responses against the S2 subunit in BALB/c mice, which were vaccinated either with plasmid DNA encoding the S2 domain (residues 681-1120), the recombinant S2 fragment (residues 681-980) in incomplete Freund's adjuvant, or with inactivated SARS-CoV. The increased number of specific cytotoxic cells (CTLs) and the high titer of specific antibody showed stimulation of both arms of the immune system in these groups. The shift in cytokines suggested that Th1-polarized immune response was induced by plasmid pCoVS2, meanwhile the Th2-dominant response was induced by recombinant S2 fragment and inactivated vaccine. However, the titer of neutralizing antibodies was only detectable in mice immunized with inactivated virus, but not with pCoVS2 plasmid. Taken together, the S2 domain could induce specific cellular immune response and a high level of total IgG but little neutralizing antibodies against infection by SARSCoV.     

M135R is a novel cell surface virulence factor of myxoma virus

Myxoma virus (MV) encodes a cell surface protein (M135R) that is predicted to mimic the host alpha/beta interferon receptor (IFN-alpha/beta-R) and thus prevent IFN-alpha/beta from triggering a host antiviral response. This prediction is based on sequence similarity to B18R, the viral IFN-alpha/beta-R from vaccinia virus (VV), which has been demonstrated to bind and inhibit type I interferons. However, M135R is only half the size of VV B18R. All other poxvirus-encoded IFN-alpha/beta-R homologs align only to the amino-terminal half of M135R. Peptide antibodies raised against M135R were used for immunoblotting and immunofluorescence and indicate that M135R is expressed as an early gene and that the product is a cell surface N-linked glycoprotein that is not secreted. In contrast to the predicted properties of M135R as an inhibitor of type I interferon, all binding and inhibition assays designed to demonstrate whether M135R can interact with IFN-alpha/beta have been negative. However, pathogenesis studies with a targeted M135-knockout MV construct (vMyx135KO) indicate that the deletion of M135R severely attenuates MV pathogenesis in the European rabbit. We propose that M135R is an important immunomodulatory virulence factor for myxomatosis but that the target immune ligand is not from the predicted type I interferon family and remains to be identified.     

Regulation of indoleamine 2,3-dioxygenase gene expression in human fibroblasts by interferon-gamma. Upstream control region discriminates between interferon-gamma and interferon-alpha

The interferon (IFN)-gamma-mediated induction of indoleamine 2,3-dioxygenase (IDO) enzyme, which converts tryptophan into N-formylkynurenine, has been implicated in the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, and in the antiproliferative effect of IFN-gamma on tumor cells. The IDO activity is induced strongly in many cell types by IFN-gamma but rather poorly by IFN-alpha or -beta. A genomic DNA clone containing part of the transcribed region of the IDO gene and approximately 13 kilobases (kb) of the 5'-upstream DNA sequence was isolated and analyzed. An approximately 1.4-kb fragment of this clone, containing 329 nucleotides of the transcribed sequence and approximately 1.1 kb of the 5'-upstream sequence, when ligated to chloramphenicol acetyltransferase (CAT) structural gene made its expression inducible by IFN-gamma, but this construct responded poorly, if at all, to IFN-alpha 2. Deletion constructs derived from this plasmid narrowed down the IFN-gamma-responsive region to a 151-nucleotide segment (-495/-344) which also contained a 14-nucleotide sequence (GGTTTCAGTTTTCC) highly homologous to the IFN(alpha)-stimulated response element (ISRE) that has been found so far in all cellular genes inducible with IFN-alpha or -beta. Expression of CAT activity was stimulated by IFN-gamma more effectively than by IFN-alpha 2 when a 155-nucleotide fragment (-495/-340) containing the 151-nucleotide segment required for IFN-gamma response was inserted before herpes simplex virus thymidine kinase promoter linked to CAT structural gene. The results indicate that despite the presence of an ISRE, the control region of the IDO gene can distinguish between IFN-gamma and IFN-alpha. This may account for the differential activation of IDO gene expression by IFN-gamma as against IFN-alpha or -beta in intact cells, and suggests that the response of ISRE to IFN-alpha or -beta may be governed by other features in the upstream control region of this gene.     

Herpes simplex virus type 2 virion host shutoff protein regulates alpha/beta interferon but not adaptive immune responses during primary infection in vivo

The herpes simplex virus (HSV) virion host shutoff (vhs) protein, the product of the UL41 (vhs) gene, is an important determinant of HSV virulence. vhs has been implicated in HSV interference with host antiviral immune responses, down-regulating expression of major histocompatibility complex molecules to help HSV evade host adaptive immunity. The severe attenuation of vhs-deficient viruses in vivo could reflect their inability to escape immune detection. To test this hypothesis, BALB/c or congenic SCID mice were infected intravaginally (i.vag.) with the HSV type 2 (HSV-2) vhs null mutant 333d41 or the vhs rescue virus 333d41(R). vhs-deficient virus remained severely attenuated in SCID mice compared with rescue virus, indicating that vhs regulation of adaptive immune responses does not influence HSV pathogenesis during acute infection. Innate antiviral effectors remain intact in SCID mice; prominent among these is alpha/beta interferon (IFN-alpha/beta). The attenuation of HSV-2 vhs mutants could reflect their failure to suppress IFN-alpha/beta-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) mice were infected i.vag. with wild-type virus, vhs null mutants 333-vhsB or 333d41, or the vhs rescue virus 333d41(R). Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type or vhs rescue viruses, they were restored to nearly wild-type levels of replication in IFN-alpha/betaR(-/-) mice over the first 2 days postinfection. Only wild-type and vhs rescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-alpha/betaR(-/-) mice restored the virulence of vhs-deficient viruses. vhs-deficient viruses replicated as vigorously as wild-type and rescue viruses in the nervous systems of IFN-alpha/betaR(-/-) mice. Restoration was specific for the vhs mutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-alpha/betaR(-/-) mice. Furthermore, the defect in the IFN-alpha/beta response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-alpha/beta-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-alpha/beta response in vivo, because an HSV-1 vhs null mutant did not recover replication and virulence after i.vag. inoculation into IFN-alpha/betaR(-/-) mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-alpha/beta-mediated antiviral response.     

The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments

Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice.     

Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags”

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.     

Constitutive expression of alpha interferon by skin dendritic cells confers resistance to infection by foot-and-mouth disease virus

The role of dendritic cells (DC) in the initiation of immune responses against foot-and-mouth disease virus (FMDV) is poorly understood. We analyzed the innate response of freshly isolated swine skin DC to the virus and show a rapid induction of beta interferon (IFN-beta) mRNA but not IFN-alpha mRNA. However, these DC secreted both IFN-alpha and IFN-beta proteins in response to live virus but not killed virus. Furthermore, the surface expression of swine major histocompatibility complex class II (SLA II) or CD80/CD86 molecules and antigen processing functions were not affected by FMDV exposure. Given the demonstrated sensitivity of FMDV to IFN-alpha/beta, there was no productive or nonproductive infection of these cells. Finally, freshly isolated skin DC constitutively expressed intracellular IFN-alpha protein in the absence of stimulation, with no detectable secretion of the cytokine until virus exposure. In situ analysis of these DC showed that these cells express and store IFN-alpha in uninfected animals. This is the first demonstration of the constitutive expression of IFN-alpha in resident, tissue-derived DC and indicates that skin DC can play an important role in the innate immune response of swine to viral infections.     

Immunoregulation of the anti-bovine serum albumin response by polyclonal and monoclonal antibodies

Monoclonal or polyclonal antibodies directed toward determinants on limited structures of bovine serum albumin (BSA) (P505-582) were shown to regulate the entire anti-bovine serum albumin (BSA) immune response when passively administered to mice 24 hr prior to immunization. Regulation was observed as suppression of the humoral IgG immune response toward all BSA determinants except those on fragment P505-582. By Day 21 suppression of humoral response was most pronounced toward determinants present on the carboxy terminal end of the molecule (N 307-582). These observations demonstrate that monoclonal antibodies directed against a single determinant on a protein molecule have the capacity to regulate the immune response to a multiplicity of determinants present on the same protein. The data lend support to concepts of antibody-induced regulation by induction of suppressor cells or idiotype recognition.     

Human receptor for measles virus (CD46) enhances nitric oxide production and restricts virus replication in mouse macrophages by modulating production of alpha/beta interferon

Complement regulatory protein CD46 is a human cell receptor for measles virus (MV). In this study, we investigated why mouse macrophages expressing human CD46 restricted MV replication and produced higher levels of nitric oxide (NO) in response to MV and gamma interferon (IFN-gamma). Treatment of MV-infected CD46-expressing mouse macrophages with antibodies against IFN-alpha/beta blocked NO production. Antibodies against IFN-alpha/beta also inhibited the augmenting effect of MV on IFN-gamma-induced NO production in CD46-expressing mouse macrophages. These antibodies did not affect NO production induced by IFN-gamma alone. These data suggest that MV enhances NO production in CD46-expressing mouse macrophages through action of IFN-alpha/beta. Mouse macrophages expressing a human CD46 mutant lacking the cytoplasmic domains were highly susceptible to MV. These cells produced much lower levels of NO and IFN-alpha/beta upon infection by MV, suggesting the CD46 cytoplasmic domains enhanced IFN-alpha/beta production. When mouse macrophages expressing tailless human CD46 were exposed to culture medium from MV-infected mouse macrophages expressing intact human CD46, viral protein synthesis and development of cytopathic effects were suppressed. Pretreating the added culture medium with antibodies against IFN-alpha/beta abrogated these antiviral effects. Taken together, these findings suggest that expression of human CD46 in mouse macrophages enhances production of IFN-alpha/beta in response to MV infection, and IFN-alpha/beta synergizes with IFN-gamma to enhance NO production and restrict viral protein synthesis and virus replication. This novel function of human CD46 in mouse macrophages requires the CD46 cytoplasmic domains.     

Idiotypic determinants on human anti-insulin antibodies are cyclically expressed

Insulin-treated diabetics develop a heterogeneous antibody response to the protein hormone. To understand the repertoire of this diverse immune response, we developed monoclonal antibodies (Mab) that recognize idiotypic determinants on anti-insulin antibodies. Two Mab that recognize distinct idiotopes on anti-insulin antibodies were studied in detail. These idiotopes were present on IgG, but not IgM, anti-insulin antibodies from the proband and from four of 11 insulin-treated diabetics. The determinants recognized were distinct from conventional allotype (Gm) markers and were found in both Type I and Type II diabetics. The partial inhibition of anti-idiotope binding by beef insulin suggests that idiotopes related to the binding site for beef insulin may be recognized. When the presence of idiotope-positive molecules was determined sequentially during insulin therapy, in one patient, idiotope expression was found to be cyclical. In contrast, the total amount of anti-insulin antibody was constant, whereas idiotope expression varied. These data suggest that common V region determinants are employed in the anti-insulin response, and their variable expression may reflect dynamic regulation during chronic insulin therapy.     

Interferon regulatory factor 7 is negatively regulated by the Epstein-Barr virus immediate-early gene, BZLF-1

Virus infection stimulates potent antiviral responses; specifically, Epstein-Barr virus (EBV) infection induces and activates interferon regulatory factor 7 (IRF-7), which is essential for production of alpha/beta interferons (IFN-alpha/beta) and upregulates expression of Tap-2. Here we present evidence that during cytolytic viral replication the immediate-early EBV protein BZLF-1 counteracts effects of IRF-7 that are central to host antiviral responses. We initiated these studies by examining IRF-7 protein expression in vivo in lesions of hairy leukoplakia (HLP) in which there is abundant EBV replication but the expected inflammatory infiltrate is absent. This absence might predict that factors involved in the antiviral response are absent or inactive. First, we detected significant levels of IRF-7 in the nucleus, as well as in the cytoplasm, of cells in HLP lesions. IRF-7 activity in cell lines during cytolytic viral replication was examined by assay of the IRF-7-responsive promoters, IFN-alpha4, IFN-beta, and Tap-2, as well as of an IFN-stimulated response element (ISRE)-containing reporter construct. These reporter constructs showed consistent reduction of activity during lytic replication. Both endogenous and transiently expressed IRF-7 and EBV BZLF-1 proteins physically associate in cell culture, although BZLF-1 had no effect on the nuclear localization of IRF-7. However, IRF-7-dependent activity of the IFN-alpha4, IFN-beta, and Tap-2 promoters, as well as an ISRE promoter construct, was inhibited by BZLF-1. This inhibition occurred in the absence of other EBV proteins and was independent of IFN signaling. Expression of BZLF-1 also inhibited activation of IRF-7 by double-stranded RNA, as well as the activity of a constitutively active mutant form of IRF-7. Negative regulation of IRF-7 by BZLF-1 required the activation domain but not the DNA-binding domain of BZLF-1. Thus, EBV may subvert cellular antiviral responses and immune detection by blocking the activation of IFN-alpha4, IFN-beta, and Tap-2 by IRF-7 through the medium of BZLF-1 as a negative regulator.     

CD69 is expressed on Daudi cells in response to interferon-alpha.

An approach to obtain monoclonal antibodies directed against cell surface proteins induced by interferon has been developed in order to characterize such proteins and determine their role. Hybridomas obtained by fusion of murine myeloma cells and spleen cells of mice immunized with interferon-alpha-treated Daudi cells were screened for the production of antibodies reacting differentially with interferon-alpha-treated and untreated Daudi cells. One such hybridoma, 2D5, produced an antibody reacting with a 28/32 kDa homodimeric protein (p28/32) expressed at the surface of Daudi cells in response to IFN-alpha treatment. IFN-alpha treatment also increased the basal level of p28/32 detected on peripheral blood leukocytes (PBL). 2D5 Antibody was used to probe the expression of p28/32 on different cells and in response to various inducers. It appears that 2D5 reacted in fact with CD69, a marker of leukocyte activation and that, following IFN-alpha treatment, CD69 was not induced on all cultured cell lines tested. Interestingly, IFN-gamma was also able to induce CD69 expression on a restricted number of cell lines but the induction pattern only partially overlapped that of IFN-alpha. As expected, activation of cells with phorbol myristate acetate (PMA) resulted in a notable increase in the level of CD69 on all cell lines considered except for the epithelial and fibroblastic types.     

Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags”

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system''s ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.