Discriminating antigen and non-antigen using proteome dissimilarity II: viral and fungal antigens

Immunogenicity arises via many synergistic mechanisms, yet the overall dissimilarity of pathogenic proteins versus the host proteome has been proposed as a key arbiter. We have previously explored this concept in relation to Bacterial antigens; here we extend our analysis to antigens of viral and fungal origin. Sets of known viral and fungal antigenic and non-antigenic protein sequences were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we could not determine a threshold able meaningfully to separate non-antigen from antigen. We conclude that viral and fungal antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to mammalian genomes.     

Critical requirement for professional APCs in eliciting T cell responses to novel fragments of histidyl-tRNA synthetase (Jo-1) in Jo-1 antibody-positive polymyositis

Polymyositis (PM) is an autoimmune muscle disease characterized by oligoclonal T cell infiltrates mediating myocytotoxicity. Although antigenic triggers for this process remain undefined, clinically homogeneous subsets of PM patients are characterized by autoantibodies directed against nuclear and cytoplasmic Ags that include histidyl-tRNA synthetase (Jo-1). Available evidence suggests that formation of anti-Jo-1 autoantibodies is Ag-driven and therefore dependent on CD4(+) T cells that may also direct cytolytic CD8(+) T cells involved in myocyte destruction. To assess peripheral blood T cell responses to Jo-1, we first subcloned full-length human Jo-1 as well as novel fragments of Jo-1 into the maltose-binding protein expression vector pMALc2. Expressed proteins were then used in standard proliferation assays with either PBMC or autologous DCs as sources of APCs. Although PBMC-derived APCs and DCs both supported peripheral blood T cell proliferation when primed with full-length human Jo-1, only DCs promoted proliferative responses to a unique amino-terminal fragment of Jo-1. mAb blockade of different HLA Ags revealed that these responses were MHC class II dependent. Therefore, for the first time, these studies demonstrate anti-Jo-1 T cell responses in Jo-1 Ab-positive PM patients as well as in healthy control subjects. More importantly, this work underscores the critical importance of APC type in dictating T cell responses to a novel antigenic fragment of Jo-1.     

真核表达MERS-CoV刺突蛋白亚单位的信号肽序列优化研究

中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)的刺突蛋白(Spike,S)亚单位1(S1)是引起宿主免疫反应和产生中和抗体的主要靶抗原,也是疫苗研发和病原检测的重要靶标,选用适宜的真核表达系统高效表达S1蛋白是进行相关研究的基础。为确定MERS-CoV S1在哺乳动物细胞中高效分泌性表达的信号肽序列,构建了含高斯荧光素酶(Gaussia luciferase,GLuc)、人组织纤溶酶原激活剂(Tissue plasminogen activator,tPA)及小鼠免疫球蛋白G的2a亚型(Mouse immunoglobular G subtype 2a,MIgG2a)7个信号肽(原始序列和改造序列)序列的MERS-CoV S1表达质粒,瞬时转染细胞后,通过Western Blot检测并比较细胞培养上清和裂解液中S1的表达水平及分泌表达效率(条带密度灰度扫描比),并对哺乳动物细胞表达的S1蛋白的纯度与抗原特性进行了分析。结果表明7种信号肽在293T、BHK21和ExpiCHO-S^TM三种细胞系统中介导MERS-CoV S1的高效分泌表达的效率各有不同,其中tPA-1信号肽介导S1抗原在ExpiCHO-S^TM中具有较高的分泌表达效率与产量,纯化的S1蛋白保持了较好的抗原性。本研究为进一步研发基于MERS-CoV S1的亚单位疫苗及免疫学检测试剂奠定了基础。     

Fixation of Thy-1 in nervous tissue for immunohistochemistry: a quantitative assessment of the effect of different fixation conditions upon retention of antigenicity and the cross-linking of Thy-1

It has proved difficult to obtain good immunohistochemical localization of cell surface antigens in nerve for a number of reasons, prominent among which are problems of fixing this class of molecule without destroying their antigenicity. In the course of developing a fixation procedure suitable for one such antigen. Thy-1, we have quantitatively assessed the effect of different fixation parameters upon the retention of Thy-1 antigenicity and upon the extent of cross-linking of the antigen in the tissue. The former was measured using radioimmunoassays adapted for membrane antigens in fixed tissue, the latter by measuring the proportion of antigen rendered insoluble to the detergent, sodium deoxycholate, and by examining the size of the antigen on sodium dodecyl sulfate--polyacrylamide gels. These approaches demonstrated that minor modifications of the standard vascular perfusion fixation of brain, using both glutaraldehyde and paraformaldehyde, were sufficient to fix the Thy-1 molecule, and at the same time substantially spare its antigenicity. In this study we measured Thy-1 using both a conventional rabbit antiserum and a mouse monoclonal antibody to the Thy-1.1 antigenic determinant. The multiple antigenic determinants recognized by the rabbit antibodies were cumulatively more resistant to fixation than the single antigenic determinant recognized by the monoclonal antibody.     

The Antigens Associated with the Cell Walls of Members of the Genus Pseudomonas

Three antigens were associated with the cell walls of pseudomonads. A highly antigenic, strain-specific antigen of high molecular weight and protein or lipoprotein in nature, occurred as an envelope around the cells. It could be washed off the cells closely associated with carbohydrate material but its antigenicity was not dependent on the carbohydrate present. Another antigen, common to all strains tested, was situated below the first antigen. This was less antigenic than the strain-specific antigen and was polysaccharide or lipopolysaccharide in nature. A second common antigen was the mucopeptide of the cell walls. This had an antigenicity similar to that of the second antigen and was dependent on both the carbohydrate and polypeptide components of the macromolecule. There appears to be some correlation between these findings and the structure of cell walls of pseudomonads are shown by electron microscopy.     

Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.

SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.     

Immunocytogenetics. V. A highly conserved NOR antigen (He) is facultatively associated with nucleolar or nucleoplasmic granules

A new protein antigen of the nucleolus organizer region (NOR), designated He, was recognized by human autoantibodies obtained from a patient with Raynaud phenomenon. In mitotic cells of all vertebrate species tested. He serum selectively immunostained the chromosomal NORs. A completely unexpected characteristic of the He antigen was its location during interphase. In mammalian cell substrates, it was concentrated in numerous nucleoplasmic granules, with minor amounts of the antigen uniformly distributed throughout the entire nucleus. In interphase nuclei of lower vertebrate cells, however, the antigen was preferentially located in the nucleolus. The antigenicity of He is not dependent on RNA or DNA; its cytochemical properties operationally classify it as a nonhistone component of the chromosome scaffold. The He antigen was present in the residual nucleolar structures of cells that were not at all active in rRNA synthesis, such as mammalian late spermatids and amphibian erythrocytes.     

Molecular mapping of human band 3 aging antigenic sites and active amino acids using synthetic peptides

An aging antigen, senescent cell antigen appears on old cells and marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes. This antigen is derived from the major anion transport protein, protein band 3, that is involved in respiration and acid base balance. We use synthetic peptides from the transmembrane, anion transport segment of band 3 to walk band 3 to identify potential aging antigenic sites. A competitive inhibition assay with affinity purified IgG autoantibody from senescent red cells was used. Results indicate that: aging antigenic sites reside on human band 3 residues 538–554, 593–601, and 812–830; and that the smallest residues which act as aging antigenic sites are 593–601 and 813–818. The contribution of lysine and/or arginine to antigenicity is examined by synthesizing peptide analogs in which glycines or arginines are substituted for lysines or arginines. Substitution of neutral glycine for the positively charged amino acids arginine or lysine or both arginine and lysine did not result in a significant difference in antigenicity between the analog and the native band 3 peptide. Substitution of the positively charged arginine for the positively charged lysine resulted in a significant reduction in antigenicity. The chicken sequence of band 3 peptides 538–554 and 812–827 differs from that of the human peptides at several sites. Antigenicity of these chicken analogs were tested and compared to the human peptides. The data suggest that the three-dimensional configuration of band 3 segments plays a dominant role in defining the antigenic determinants reactive with senescent cell IgG autoantibodies.     

Antigenic specificity of monoclonal antibodies to human myoglobin

Two monoclonal antibodies directed against different sites of the human myoglobin molecule have been tested for their cross-reactivities against several myoglobins including seven from mammalian species. The relation between their cross-reactivities and their amino acid sequences had led to a possible localization of two antigenic domains in human myoglobin. Each domain includes residues previously considered not to be directly involved in the antigenic structure of myoglobin. Unlike polyclonal serum antibodies, monoclonal hybridoma antibodies directed to a native protein often fail to bind to supposedly antigenic protein fragments. This is explicable in terms of the concept of antigenic domains. Such domains are numerous and overlapping, each comprising a number of contributory amino acid side chains which need not necessarily include continuous sequences of amino acids and which need not exhibit measurable antigenicity in isolation from the rest of the domain.     

Immunochemical characterization of cell wall protein antigen purified from the cell wall autolysate of Clostridium botulinum type A.

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components in immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.     

Molecular mapping of a histocompatibility-restricted immunodominant T cell epitope with synthetic and natural peptides: implications for T cell antigenic structure

We have prepared synthetic and natural peptides that have allowed delineation of a major antigenic site of myoglobin recognized by histocompatibility (I-Ed)-restricted T cell clones. The smallest peptide capable of stimulating T cell proliferation consisted of residues 136-146. Residues Glu 136, Lys 140, and Lys 145 were essential for antigenicity, whereas Lys 133 and Tyr 146 added potency but were not required for antigenicity. The periodicity of these residues suggests that folding the peptide into its native alpha-helical structure may be essential for antigenicity either by forming a hydrophilic binding site for the T cell receptor or by participating in antigen presentation. The same folding could also produce a hydrophobic site on the opposite side of the alpha-helix that could participate in hydrophobic interactions. The extrapolation of these findings to other known peptide antigens suggests that this tendency to form an amphipathic alpha-helix may be a general property of antigenic sites recognized by T cells, perhaps due to a different functional role of each type of site in eliciting T cell responses.     

Immunochemical Characterization of Cell Wall Protein Antigen Purified from the Cell Wall Autolysate of Clostridium botulinum Type A

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components on immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.     

Loss of T cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1(27-35) tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1(27-35) antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide·MHC complex. These results help explain how the "anchor-fixing" strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.     

Differentiation of human monocytic cell lines confers susceptibility to Bacillus anthracis lethal toxin

Anthrax lethal toxin (LT) is comprised of protective antigen and lethal factor. Lethal factor enters mammalian cells in a protective antigen-dependent process and cleaves mitogen-activated protein kinase kinases. Although LT has no observable effect on many cell types, it causes necrosis in macrophages derived from certain mouse strains and apoptosis in activated mouse macrophages. In this study, we observed that LT treatment of three different human monocytic cell lines U-937, HL-60 and THP-1 did not induce cell death. Cells did become susceptible to the toxin, however, after differentiation into a macrophage-like state. Treatment with LT resulted in decreased phosphorylation of p38, ERK1/2 and JNK in both undifferentiated and differentiated HL-60 cells, suggesting that the change in susceptibility does not result from differences in toxin delivery or substrate cleavage. Death of differentiated HL-60 cells was accompanied by chromosome condensation and DNA fragmentation, but was not inhibited by the pan-caspase inhibitor Z-VAD-FMK. In addition, we observed that the macrophage differentiation process could be inhibited by LT. Our results indicate that LT-mediated death of mouse and human macrophages may occur through distinct processes and that the differentiation state of human cells can determine susceptibility or resistance to LT.     

单抗LC-1与针对的人肺腺癌SPC-A-1细胞肿瘤相关抗原复合物的内化研究

我们运用抗人肺癌单抗LC-1结合胶体金免疫电镜技术研究了人肺腺癌SPC-A-1细胞表面抗原抗体复合物被内吞的全过程,发现该细胞表面抗原抗体复合物是通过受体介导内吞途径被内化,经多泡体富集后至溶酶体消化降解。此外我们还用流式细胞仪分析了内吞前后该细胞表面抗原量的变化和短期内的恢复情况。LC-1在诱发该细胞表面抗原内化的同时还诱发了它对该核糖体的自噬。     

A mouse plasma cell surface antigenic determinant (PLKB) recognized by xenoantiserum. Its relationship to the PC.1 antigenic determinant.

The specificities of the xenoantisera made against mouse myeloma cells have been compared to those recognized by alloantiserum by studying patterns of cytotoxicity on both normal and malignant plasma cells. Goat antiserum obtained by immunization with Balb/c mouse myeloma ADJ-PC-22A cells and purified by in vivo absorption could detect cell surface antigenic determinants present on plasma cells and on cells of liver, kidney, and brain (PLKB antigen), as we had previously reported for a similarly prepared rabbit antiserum. In spite of an apparent similarity between the tissue representation of the PLKB determinant and that of PC.1 antigenic determinants which were detected by DBA/2 anti-ADJ-PC-22A cell alloantiserum, the PLKB antigenic determinant is not identical with the PC.1 antigenic determinant, since the former is found on the tissues of PC.1-negative as well as PC.1-positive strains of mice. However, it was deduced that the PLKB antigenic determinant and the PC.1 antigenic determinant reside in close proximity on the cell surface or maybe even on the same molecule, since Fab fragments of antiserum against either PLKB or PC.1 blocked the cytotoxicity against both antigens. On the other hand, these Fab fragments did not inhibit the cytotoxicity of anti-H-2 antiserum, indicating that neither PLKB nor PC.1 antigenic determinants are in close proximity to H-2 antigens. Association of PLKB and PC.1 determinants was further supported by the finding that the loss of the PLKB determinant in a variant of myeloma MOPC-70A corresponds to the loss of PC.1 determinant on the same cells.     

Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas

The murine monoclonal antibody (MAb) designated DF3 has defined a high m.w. antigen detectable in human breast carcinomas and in human milk. DF3 antigen is detectable on apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells. DF3 antigen expression has been shown to correlate with the degree of human breast tumor differentiation, and the detection of a cross-reactive species in human milk has suggested that DF3 antigen might be useful as a biochemical marker of differentiated mammary epithelial cells. To further characterize DF3 antigen, we have developed an approach to purify the cross-reactive species by using gel filtration and antibody affinity chromatography. The affinity column-purified DF3 antigen was absorbed by wheat germ agglutinin and peanut agglutinin, but not by concanavalin A or lentil lectin. In contrast, wheat germ agglutinin inhibited MAb DF3 reactivity with the purified antigen, whereas there was little, if any, inhibition when using peanut agglutinin. These findings are thus consistent with the involvement of terminal N-acetyl-D-neuraminic acid and/or N-acetylglucosamine residues in the antigenic site. DF3 antigenicity was also sensitive to neuraminidase, but not chondroitinase ABC, chondroitinase AC, chondroitin-4-sulfatase, or hyaluronidase. Furthermore, DF3 antigen was sensitive to Pronase, subtilisin BPN', and alpha-chymotrypsin. The presence of O-glycosidic linkages between carbohydrate and protein in the DF3 antigenic site was further supported by the presence of NaBH4-sensitive sites. Together, these results suggest that sialyl oligosaccharides present on a peptide backbone are required for maintaining DF3 antigenicity. Similar findings have been demonstrated for DF3 antigen purified from both human milk and breast cancer effusions. However, the DF3 antigen in human milk consisted of a single high m.w. species, whereas the tumor-associated antigen consisted of two distinct glycoproteins with m.w. of 330,000 and 450,000. These findings may be relevant to the recent demonstration that distinct high m.w. DF3 antigens are elevated in the circulation of patients with breast carcinoma.     

Target specificity of an autoreactive pathogenic human γδ-T cell receptor in myositis

In polymyositis and inclusion body myositis, muscle fibers are surrounded and invaded by CD8-positive cytotoxic T cells expressing the αβ-T cell receptor (αβ-TCR) for antigen. In a rare variant of myositis, muscle fibers are similarly attacked by CD8-negative T cells expressing the γδ-TCR (γδ-T cell-mediated myositis). We investigated the antigen specificity of a human γδ-TCR previously identified in an autoimmune tissue lesion of γδ-T cell-mediated myositis. We show that this Vγ1.3Vδ2-TCR, termed M88, recognizes various proteins from different species. Several of these proteins belong to the translational apparatus, including some bacterial and human aminoacyl-tRNA synthetases (AA-RS). Specifically, M88 recognizes histidyl-tRNA synthetase, an antigen known to be also targeted by autoantibodies called anti-Jo-1. The M88 target epitope is strictly conformational, independent of post-translational modification, and exposed on the surface of the respective antigenic protein. Extensive mutagenesis of the translation initiation factor-1 from Escherichia coli (EcIF1), which served as a paradigm antigen with known structure, showed that a short α-helical loop around amino acids 39 to 42 of EcIF1 is a major part of the M88 epitope. Mutagenesis of M88 showed that the complementarity determining regions 3 of both γδ-TCR chains contribute to antigen recognition. M88 is the only known example of a molecularly characterized γδ-TCR expressed by autoaggressive T cells in tissue. The observation that AA-RS are targeted by a γδ-T cell and by autoantibodies reveals an unexpected link between T cell and antibody responses in autoimmune myositis.     

A New Monoclonal Antibody that Recognizes 180 kDa Polypeptide Expressed on Early Mouse Embryos and Mouse Embryonal Carcinoma Cells

A hybridoma clone 1F7 producing a monoclonal antibody against OTF9-63 mouse embryonal carcinoma cell line was isolated with the aid of the intrasplenic primary immunization technique. This antibody was capable of recognizing embryonal carcinoma cell lines originated from certain mouse strains, including 129/Sv, but not those which were established from other strains as well as human and other murine cell lines except FM3A, a mouse mammary carcinoma cell line. Indirect immunofluorescence study revealed that 1F7 antigen was expressed on early mouse embryos in a stage specific manner. In order to characterize the 1F7 antigenic molecules, we analyzed both glycoshingolipids and glycoproteins recovered from OTF9-63 by means of immunostaining after thin layer chromatography and SDS-polyacrylamide gel electrophoresis followed by Western blotting, respectively. It was concluded that 1F7 antigenic determinants were carried by 180 kDa polypeptides. One of the most interesting characteristics of 1F7 antigen is complete failure to express on the embryonic ectoderm of 5.5d and 6.5d embryos, one of cell types most closely related to embryonal carcinoma cells. 1F7 antibody may help analyse the process of teratocarcinogenesis in 129/Sv mice.