Soluble Antigens of Vaccinia-infected Mammalian Cells: III. Relation of “Early” and “Late” Proteins to Virus Structure

The structural proteins of vaccinia virus can be divided into two classes on the basis of their times of synthesis in the infected cell. The production of one of these classes of proteins begins prior to the onset of viral deoxyribonucleic acid (DNA) replication. These are referred to as "early" proteins. Synthesis of the second class of structural proteins follows the onset of viral DNA replication; hence, the term "late" proteins for this class. We are able, by immunological procedures, to identify three "early" virus-structural proteins. These materials, when incorporated into virions, appear to be associated with the "core" of the virion and do not elicit production of virus-neutralizing antibody. It would seem, therefore, that those virus-structural proteins synthesized early in the course of infection act as internal components of the virion. The "late" proteins may be subdivided into two groups on the basis of certain physical properties and molecular weight differences. The first of these groups, comprised of at least two proteins, corresponds to the classical LS antigens and elicits production of neutralizing antibodies. These proteins, when incorporated into virions, are found only in the outer ("coat") fraction of the virion. The second group of "late" antigens, also comprised of two proteins, termed the G antigens, do not elicit synthesis of neutralizing antibody. One of these proteins is associated with the virus "core"; the other is found in the "coat" fraction of the virion and appears to occupy an intermediary, subsurface position. Procedures suitable for the isolation of the G antigens are described, in addition to the partial characterization of these antigens.     

Affinity separation using an Fv antibody fragment-"smart" polymer conjugate

Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled. Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior. Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates. In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody. For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL). The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer. After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80%. We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution. It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine. The conjugate can then be recycled for second use. In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody. Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody and tailoring of the conjugate.     

Antinuclear Antibodies and Nuclear Antigens

The lupus erythematosus (LE) cell factor is one of a variety of heterogeneous antibodies directed against many nuclear antigens. These antinuclear antibodies are found in a wide variety of clinical disorders. As detected by immunofluorescence techniques, they are so frequently found in association with systemic lupus erythematosus that their absence essentially excludes that diagnosis.It is suggested that in certain situations other than systemic lupus, antinuclear antibodies may be the result of some inflammatory and destructive processes. Antinuclear antibodies may be detected in certain lower animals frequently in association with disease. In lower animals, immunization with nuclear antigens appropriately complexed^* to protein carriers consistently results in the induction of antinuclear antibodies. In both man and animals, antinuclear antibodies are often detected without associated disease. Furthermore, antinuclear antibodies are not injurious to intact tissue culture cells. Nonetheless, antigen-antibody complexes consisting of nuclear antigens and antinuclear antibodies may contribute to the propagation of certain diseases, particularly lupus nephritis.The use of antinuclear antibodies as immunochemical tools holds great promise for the better understanding of such nuclear antigens as are found in viruses and in the nuclei of certain malignant cells.     

Role of Non-Surface Antigens in Controlling Paramecium Surface Antigen Synthesis*

SYNOPSIS. Paramecium aurelia exposed to antisera prepared against cells of a different surface antigenic type are often induced to transform to a new serotype. One possible explanation is that paramecia that are so affected have antigens related to the ciliary antigens, but not accessible to immobilizing antibodies. It is these secondary antigens that are bound by the antibodies, thereby forcing the cells to alter their pattern of antigen synthesis. Examination of affected paramecia has disclosed that secondary antigens are often present but the specificity of these antigens cannot account for the activity of the antisera. It is therefore proposed that antibodies directed against substances other than the immobilization antigens may induce transformation. Two kinds of antiserum, neither of which contains immobilizing antibodies of any sort, are able to markedly alter the expression of the serotypes. One was obtained by immunizing rabbits with culture fluid in which paramecia had been growing. The 2nd was made by injecting rabbits with normal sera from other rabbits.     

Immunochemical characterization of the outer membrane complex of Serratia marcescens and identification of the antigens accessible to antibodies on the cell surface

Serratia marcescens New CDC O14:H12 contains major outer membrane proteins of 43.5 kDal, 42 kDal (the porins) and 38 kDal (the OmpA protein) which can be separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Immunoblotting of whole cell or outer membrane preparations using antiserum raised against the whole cells revealed similar complex patterns of antigens. The OmpA protein was the major immunogen, although six other outer membrane proteins were also detected; the porins reacted only weakly with antibodies in this system. Immunoabsorption of antisera with whole cells showed that only the O antigenic chains of lipopolysaccharide and the H (flagella) antigens were accessible to antibody on the cell surface. Failure to detect the OmpA protein and other envelope antigens in this way suggests that their antigenic sites are not able to react with antibodies and are possibly masked by the O antigen.     

Antibodies to tumor necrosis factor: a component of B cell immune responses with a role in tumor/host interaction

Infiltrating B lymphocytes are found within tumors, where their role and the antigens they recognize are poorly defined. After in vitro expansion of these cells, we were able to detect the production of antibodies to tumor necrosis factor (TNF) in 13 of 17 human tumors studied. These antibodies were detected by both enzyme-linked immunosorbent assay and by neutralization. Anti-TNF antibodies were not produced by resting peripheral blood B cells of normal subjects. However, anti-TNF antibodies were produced by B cells obtained from healthy individuals, after either in vivo or in vitro antigenic stimulation. This suggests that anti-TNF antibody production may constitute part of the overall B cell response to antigens. The intratumoral production of anti-TNF antibody may play a role in tumor/host interactions.This work was supported by grants from FAPESP (90/1844-4) and from the Share Foundation and the Concern Foundation     

Physical characterization of the "immunosignaturing effect"

Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer's disease display a characteristic and reproducible "immunosignature" on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.     

Development of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscle

Each vertebrate skeletal muscle fiber is ensheathed by a basal lamina (BL) which passes through the synaptic cleft of the neuromuscular junction. In the adult, the synaptic portion of the BL is both functionally and chemically specialized. We have used an immunofluorescence method to compare the development of synaptic and extrasynaptic portions of BL in embryonic rat intercostal muscles. Immunohistochemical staining of adult muscle fibers with monoclonal and serum antibodies defines "synaptic" antigens (including acetylcholinesterase) that are concentrated in synaptic BL, "extrasynaptic" antigens that are concentrated in extrasynaptic regions, and "shared" antigens (including collagen IV, fibronectin, laminin, and a heparan sulfate proteoglycan) that are present in both synaptic and extrasynaptic BL ( Sanes and Chiu , 1983). Synapses appear on newly formed myotubes on embryonic Day 14 (E14; birth is on E22 ). Patches of BL that contain shared and extrasynaptic antigens are present on myotube surfaces by E15, and BL forms a continuous sheath by E17. Shared antigens are present at but not confined to synaptic areas by E15. Two synaptic antigens appear in synaptic areas a day later, and are not detectable extrasynaptically . At least one extrasynaptic antigen is present at immature synapses, and lost or masked by E19 . Thus synaptic BL is not assembled as a unit; rather, components are added, lost, or modified as synaptogenesis proceeds.     

Masking of protein antigen by modification of amino groups with carbobenzoxychloride (benzyl chloroformate) and demasking by treatment with nonspecific protease.

Cryostat sections of various substrates were treated with carbobenzoxychloride in acetone to modify antigens. By applying specific fluorescent antibodies, it could be shown that the antigenic determinants of rabbit gamma-globulin and bovine insulin were totally masked. The antigenicity of ACTH was markedly reduced, whereas the polysaccharide antigens of Salmonella typhimurium were only partially masked. After masking, antigenicity could be restored by treatment with nonspecific protease. The reversible protection of amino groups by carbobenzoxychloride may be a way to preserve protein antigens during embedding in plastics, as such materials also bind to amino groups, blocking the antigenicity of proteins.     

Discovery of "masked" ribonucleases in highly purified preparations of RNA

It was found that RNA preparations isolated with the help of various inhibitors of RNAses from different eukariotic tissues, followed by thorough deproteinization contain particular ribonucleases ("masked" RNAses). These RNAses were supposed to be connected with RNA molecules and are not active. They may be activated by changes of RNA molecule conformation. Apparently the "marked" RNAses can take part in (a) processing of large precursor RNA molecules; (b) regulation of gene expression by means of specially cut RNA fragments.     

Development of a "reverse capture" autoantibody microarray for studies of antigen-autoantibody profiling

Diagnosing cancers based on serum profiling is a particularly attractive concept. However, the technical challenges to analysis of the serum proteome arise from the dynamic range of protein amounts. Cancer sera contain antibodies that react with a unique group of autologous cellular antigens, which affords a dramatic amplification of signal in the form of antibodies relative to the amount of the corresponding antigens. The serum autoantibody repertoire from cancer patients might, therefore, be exploited for antigen-antibody profiling. To date, studies of antigen-antibody reactivity using microarrays have relied on recombinant proteins or synthetic peptides as arrayed features. However, recombinant proteins and/or synthetic peptides may fail to accurately detect autoantibody binding due to the lack of proper PTMs. Here we describe the development and use of a "reverse capture" autoantibody microarray. Our "reverse capture" autoantibody microarray is based on the dual-antibody sandwich immunoassay platform of ELISA, which allows the antigens to be immobilized in their native configuration. As "proof-of-principle", we demonstrate its use for antigen-autoantibody profiling with sera from patients with prostate cancer and benign prostate hyperplasia.     

Immuno-electron microscopic localization of lipopolysaccharide antigens on ultrathin sections of Salmonella typhimurium.

Lipopolysaccharide antigens were demonstrated on ultrathin sections of styrene-embedded Salmonella typhimurium by direct postembedding staining with ferritin-labeled antibodies. The antigenicity, partially masked in the embedding process, could be satisfactorily recovered by treatment of ultrathin sections with nonspecific protease. As judged from the reaction site of the ferritin-labeled antibodies, the lipopolysaccharides were localized in two zones. The broader zone of densely distributed ferritin molecules was superimposed over the whole outer cellwall, and a smaller zone revealing antigenicity was found over the cell membrane, which strongly supports the concept that the latter is the site of synthesis of lipopolysaccharides. The well-defined labeled areas between these two antigenic zones may be the routes whereby the synthesized polysaccharide molecules reach the cell wall.     

Haptenic oligosaccharides in antigenic variants of mycobacterial C-mycosides antagonize lipid receptor activity for mycobacteriophage D4 by masking a methylated rhamnose.

The simple apolar C-mycosides, i.e., structurally well-defined hydrophobic glycopeptidolipids of several Mycobacterium species (see diagram below), were earlier shown to behave as receptors for adsorption of mycobacteriophage D4. This phage is usually virulent for Mycobacterium smegmatis. More complex, polar C-mycosides with additional carbohydrate substituents attached solely to the deoxytalose have recently been described. They are the highly specific serotyping antigens discovered by W. B. Schaefer--lipids which characterize members of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex. Both kinds are depicted in the structure below: (Formula: see text) where X equals H (for simple, apolar C-mycosides) and X equals small oligosaccharides (for antigenic forms; more complex, polar C-mycosides). The present investigations showed that the purified polar antigenic lipids exhibit considerably less adsorptive activity for D4 than do the apolar C-mycosides. Thus, the haptenic oligosaccharides are believed to shield the site in the molecule that the phage recognizes, and the blocking is reinforced by the specific antibodies that the antigens elicit. Although the MAIS serovars usually also produce the phage-reactive apolar C-mycosides, they are not permissive hosts for D4, nor do whole cells adsorb the phage. We suggest that in these species the apolar forms are probably "covered" at the cell surface by the antigenic lipids. Therefore, these antigenic mycosides may play a putative role in virulence of the MAIS members by protecting these mycobacteria from their own potential pathogen. The results of chemical transformations at specific sites of the mycoside core coupled with studies of simple synthetic lipid glycosides indicated that the principal phage receptor activity resides in the terminal methylated rhamnose (see diagram). It is this sugar which is evidently masked by the (seemingly remote) haptenic oligosaccharides.     

Diagnostic Mr 31,000 Schistosoma mansoni proteins: requirement of infection, but not immunization, and use of the "miniblot" technique for the production of monoclonal antibodies

Antibodies directed against diagnostic Mr 31,000 polypeptide(s) of adult Schistosoma mansoni were already formed in mice during prepatency. In contrast, repeated immunization of mice with homogenates of adult schistosomes failed to elicit antibodies detectable in immunoblots in the Mr 31,000 region. Therefore, spleen cells of infected mice were used to produce hybridoma lines. The "miniblot technique" was developed in order to detect in hybridoma supernatants antibodies against schistosome Mr 31,000 components. Electrophoretically separated total S. mansoni proteins were transferred onto nitrocellulose, and the position of the Mr 31,000 components was determined with polyclonal antisera and immunoblotting. Pieces of about 3 square mm of nitrocellulose bearing the diagnostic proteins were incubated with about 100 microliter of hybridoma supernatant in microtitre plates and subsequently probed with peroxidase-conjugated antibody to mouse IgG. This screening technique identified hybridomas secreting antibody to the relevant S. mansoni antigens. It is applicable to other defined parasite antigens, which are, however, not available in biochemically purified form. The monoclonal antibodies produced against the proteins with diagnostic potential reacted with antigens localized in the schistosome gut.     

Cell-surface associated proteins of corneal fibroblasts: dissection with monoclonal antibodies

It is now generally accepted that the cell surface is involved in the interaction of the cells with the extracellular matrix. To identify and characterize cell-surface-associated components of corneal fibroblasts, several monoclonal antibodies were developed. Hybridomas were developed by fusing mouse myeloma cells SP2/OAg14 with spleen cells from mice immunized with membrane fractions of corneal fibroblasts grown in culture. Twenty-five hybridomas secreting monoclonal antibodies to cell-surface components were selected by an enzyme-linked immunosorbent assay using corneal fibroblasts grown in microtiter plates as the substrate. Immunohistochemical staining demonstrated that the antigenic determinants recognized by these antibodies were not present on corneal epithelial cells, but were present on skin fibroblasts. The antigenic determinants recognized by two of these antibodies, designated 10D2 and 716, were matrix components of the corneal stroma. Immunochemical characterization of the antigens was carried out by indirect precipitation of the radioactively labeled cellular proteins with the monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the precipitates. Four antibodies were able to precipitate antigens from cell extract in detectable amounts. Antibodies designated 5E2, 9G2, and 10D2 recognized antigens consisting of polypeptides of approximate molecular weights 105K and 110K, while antibody 716 recognized an antigen of 100K molecular weight. However, based on the tissue distribution and cell-surface distribution, these antibodies reacted with different antigenic determinants. The antigen recognized by 716 was also secreted by cells in culture but consisted of 220K and 200K polypeptide chains. It was tentatively identified as cellular fibronectin, based on the reaction of this antigen with polyclonal antibodies to plasma fibronectin.     

Purification of cytoplasmic antigens from the mycelial phase of Candida albicans: Possible advantages of its use in Candida serology

The serology of candidiasis is complicated by the use of poorly defined antigens. Total extracts of the yeast phase have been commonly used as cytoplasmic antigen, without regard to the significant amounts of carbohydrate that may contaminate such preparations. This is particularly true in the case of commercially available antigens that have been used as cytoplasmic antigens but actually are richer in carbohydrate than in protein. Affinity chromatography in concanavalin A — Sepharose provides a simple procedure to separate carbohydrates, mainly mannan, from protein antigens in whole Candida extracts. By using mannan-poor antigens, the specificity of serological reactions can be increased considerably, since both the positive reactions seen in asymptomatic donors and the cross-reactions seen in patients infected with other fungi are due to anti-mannan antibodies. In contrast, both anti-mannan and anti-cytoplasmic antigen antibodies can be detected in patients suspected of systemic candidiasis. On the other hand, absolute specificity may never be achieved for systemic candidiasis. We have found antibodies against cytoplasmic antigen in a patient allergic to C. albicans, in whom the microorganism was isolated from fecal material. It appears that, under favorable conditions, mucosal sensitization may also trigger a systemic reaction directed against both mannan and cytoplasmic antigens.Publication no. 341 from The Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina.     

Masking the cryptodeterminant on the 54-kilodalton mouse sperm surface antigen.

Previously, we reported that the epitope of a 54-kDa sperm surface sialoglycoprotein on the flagellum is masked by sialic acid residues. The epitope is referred to as a hidden determinant or cryptodeterminant. This paper reports the manner in which the epitope is masked as evaluated qualitatively and quantitatively by means of SDS-PAGE/immunoblots (Western blot) and ELISA. Immunoblotting with four specific monoclonal antibodies to the 54-kDa sialoglycoprotein--T21 (IgM), MC71 (IgG1), MC81 (IgM), and MC91 (IgM)--demonstrated that not only IgM but also IgG antibody MC71 and the Fab fragment MC71 are masked. Quantitative evaluation with ELISA to compare the antibody titration curves of the masked and unmasked antigens on sialidase-treated and untreated sperm, respectively, indicated that sialidase caused the antibody-binding ability of the epitopes to increase to a different level for each antibody. There were 32-256-, 8-16-, 16-, and 2-4-fold increases in binding to T21, MC71, MC81, and MC91 antibodies, respectively. These results suggest that the antigen-masking through the cryptodeterminant does not depend upon the subtype or the molecular mass of the antibody, but upon the biochemical nature of the epitope region that is closely related to the sialic acid. The mechanism and physiological roles of the antigen-masking are discussed.     

肠毒素大肠杆菌定居因子CS3和肠毒素融合抗原基因在减毒鼠伤寒沙门氏菌中的共表达

不耐热肠毒素（LT）和耐热肠毒素（ST）是产肠毒素大肠杆菌的主要致病因素,CS3为该菌的优势定居因子,是定居因子CFA/Ⅱ菌毛抗原的共有抗原组分。采用基因操作技术将编码CS3和融合肠毒素蛋白基因转化到减毒鼠伤寒沙门氏菌疫苗侯选株X4072中进行表达。用重组菌株口服免疫小鼠后,免疫动物能产生抗CS3、LT和ST的血清抗体。特别有意义的是,所产生的抗ST抗体能中和天然ST的生物活性。这一结果为研究载体疫苗防治肠毒素大肠杆菌腹泻疾病奠定了基础。     

Genetic regulation of the antibody response to H-2Db alloantigens in mice. IV. Antigens that activate helper T cells for IgG, coded for by the non-H-2 genome

When B10.A(5R) mice are immunized with congenic C57BL/10 cells only 2-ME-sensitive antibodies (IgM type) are found directed against H-2Db. To obtain 2-ME-resistant antibodies (IgG type) 5R mice must be immunized with noncongenic cells (e.g., A.BY); in this case non-H-2 cell surface antigens will activate helper T cells to induce anti-Db IgG antibody production by B cells. An attempt was made to define helper antigens that activate helper T cells. Neither N-2 antigens of seven H-2Db recombinant strains nor a limited set of non-H-2 cell surface antigens were able to serve as helper antigens. By using individual backcross mice as antigen, one helper antigen was found on the background of strain A under the conditions used, whereas other backgrounds may carry more than one antigen. The helper antigen is dominantly expressed in F1 mice and has to be presented on the same cell as H-2Db to induce the switch from IgM to IgG.     

Autoantibodies and immunologic theories]

A hypothesis is put forward to the effect that immunological phenomena represent a particular case of the transport of metabolites, rather than obligatory "defense mechanism". This hypothesis excludes the necessity in additional postulates (forbidden clones, somatic mutations, cells-repressors etc.) to account for the basic immunological phenomena, such as recognition, appearance of autoantibodies and tolerance. It suffices to assume that: 1) autolytic enzymes destroy "their" antigens but cannot destroy completely "foreign" antigens; 2) as a result of decomposition of antigens by enzymes, "tolerogens" may appear which block the receptors in immunocompetent cells thus preventing the appearance of antibodies; 3) cells capable to synthesize autoantibodies exist in the normal organism but not activated due to the absence of "their" antigens. When such antigens appear, they initiate the synthesis of antibodies. The hypothesis advanced may appear too simple as compared with the existing theories, but experiments have to confirm it.