Heterogenetic antigens of gram-positive bacteria

Chorpenning, Frank W. (The Ohio State University, Columbus), and Matthew C. Dodd. Heterogenetic antigens of gram-positive bacteria. J. Bacteriol. 91:1440-1445. 1966.-Soluble antigens obtained by various methods from gram-positive bacteria were used to modify erythrocytes whose hemagglutinating reactions with immune rabbit sera and normal human sera were then studied. Antigens from all gram-positive organisms studied except corynbacteria altered red cells, causing them to react with specific bacterial antisera and with normal human sera; however, cross-absorption and inhibition tests indicated that at least three different specificites were involved. One of these antigens seemed to be similar to Rantz's streptococcal NSS, which is shared with Staphylococcus aureus and Bacillus spp., and is therefore heterogenetic. Another was found in streptococci but was apparently not present in S. aureus and Bacillus spp. A third antigen, also heterogenetic, appeared to be shared by several species of Bacillus and by S. aureus, but not by streptococci or any gram-negative bacteria. The third antigen was heat-stable at pH 8.0, and appeared to be essentially polysaccharide in nature. Normal human sera varied in their content of antibodies which reacted with erythrocytes modified by extracts from gram-positive bacteria. Whereas some sera reacted very broadly with red cells modified by extracts of practically any gram-positive organism, other sera agglutinated only cells which had been modified by streptococcal antigen.     

The use of bacteria as probes for lectins in preparations of solubilized human tonsil cell membranes

In earlier work we have shown that some bacteria bind naturally to lymphocyte subpopulations and that this binding may be due to lectin-carbohydrate interactions. Here we determined the possibility of using bacteria to probe for these lectins in solubilized tonsil cell membrane preparations. Since lectins are capable of agglutination, we determined the ability of human tonsil cell membrane extract (TCME) to agglutinate bacteria. We used Escherichia coli strain YS57 which does not bind to human lymphocytes and a mutant strain derived from it, E. coli UI 2023, which binds to about 50 percent of human lymphocytes. The UI 2023 was agglutinated while the YS57 was not; this agglutination was not due to antibodies or DNA. When E. coli UI 2023 was treated with periodate, it lost its ability to be agglutinated. The agglutination of E. coli UI 2023 was not blocked by any of the monosaccharides and disaccharides used but was blocked by the E. coli LPS, more specifically, by its carbohydrate moiety. Also, the E. coli UI 2023 absorbed the agglutinating factor while its parental strain, YS57, did not. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of TCME after absorption with bacteria showed that a band around 67kD was absent in the TCME absorbed by E. coli prevented the absorption by E. coli UI 2023 whereas Na2IO4-treated LPS did not. In addition, tonsil cell membrane was radioiodinated before obtaining the TCME; sodium dodecylsulfate-polyacrylamide gel electrophoresis of the radioiodinated TCME recovered after elution from E. coli UI 2023, but not from E. coli YS57, showed again a band around 67 kD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of tumour antigens by serological analysis of cDNA expression cloning

The identification of antigens that distinguish normal cells from cancer cells is an important challenge in the field of tumour immunology and immunotherapy. The immunoscreening of cDNA expression libraries constructed from human tumour tissues with antibodies in sera from cancer patents (SEREX: serological identification of antigens by recombinant expression cloning) provides a powerful approach to identify immunogenic tumour antigens. To date, over 2,000 tumour antigens have been identified from a variety of malignancies using SEREX. These antigens can be classified into several categories, of which the cancer/testis (CT) antigens appear to be the most attractive candidates for vaccine development. The SEREX-defined tumour antigens facilitate the identification of epitopes (antigenic peptides) recognised by antigen-specific cytotoxic T lymphocytes (CTLs) and provide a basis for peptide vaccine and gene therapy in a wide variety of human cancers. Moreover, some of these antigens seem to play a functional role in the pathogenesis of cancer.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Heart-reactive antibodies in rabbit anti-Streptococcus mutans sera fail to cross-react with Streptococcus mutans

Immunization of rabbits with Streptococcus mutans antigens results in the production of serum antibodies that bind in vitro to human, rabbit, and monkey cardiac muscle. Antibodies to heart, however, have also been reported to occur at lower titers in the sera of unimmunized rabbits. In this study, the specificities of heart-reactive antibodies (HRA) in sera of unimmunized and S. mutans-immunized rabbits were compared using indirect immunofluorescence, Western blot, and Bio-Dot immunoassays. Both groups of sera gave striational indirect immunofluorescence-staining patterns on thin sections of native human and monkey cardiac muscle. Western blot analyses revealed that antibodies in normal sera bound 9 to 20 components of human, rabbit, and monkey heart. The major bands had Mr of 205,000, 160,000, 135,000, and 70,000. Several of the normal sera did not have antibody activity to S. mutans antigens, indicating that these HRA do not cross-react with these bacteria. Although immunization of rabbits with S. mutans caused increased titers of HRA (two to three doubling dilutions), Western blot assays using anti-S. mutans sera showed banding patterns qualitatively similar to those of normal sera on heart extracts. Antibodies to skeletal muscle myosin were detected in both serum groups. Of eighteen normal rabbit sera sixteen had antimyosin titers of 10 to 40, whereas all eighteen anti-S. mutans sera had titers of 10 to 160. Affinity-purified antimyosin antibodies isolated from anti-S. mutans serum did not bind to S. mutans components. Conversely, affinity-purified antibodies to S. mutans antigens did not bind to myosin or to other cardiac muscle components. Among these were antibodies to the 185-kDa cell wall protein (also known as B, I/II, IF, Spa A, and P1) previously believed to possess antigenic mimicry. HRA were removed from anti-S. mutans sera by absorption with S. mutans but this effect was not specific, because a non-cross-reactive internal standard antibody was also absorbed to the same extent. Because previous evidence for antigenic mimicry between S. mutans and cardiac muscle was based on serum cross-absorption experiments, this immunologic relationship is not substantiated. These results indicated that naturally occurring antibodies to cardiac muscle components are present in the sera of unimmunized rabbits and that immunization with S. mutans does not stimulate production of new heart-reactive antibody, but rather serves to boost antibody production by preexisting clones of self-reactive B-lymphocytes.     

Sera from patients with multiple sclerosis react with human cell T lymphotropic virus-I gag proteins but not env proteins--Western blotting analysis

To study the possible involvement of human T cell lymphotropic virus type I (HTLV-I)-related agent in Japanese multiple sclerosis (MS), we performed a Western blotting analysis, using purified viral antigens, on sera from 46 patients with MS, nine patients with other neurologic diseases, and 11 healthy controls. Of 46 MS patients, 11 (24%) had antibodies reactive with antigens corresponding to the group-specific antigen (gag) proteins (p15, p19, and p24), although the prevalence was lower than that reported in a recent study using an enzyme-linked immunosorbent assay (ELISA). Despite the lower frequency of immunoreactivity, Western blotting technique had merits of identification of multiple antigens and higher specificity for detection of antibodies than ELISA. Those sero-positive patients consisted of four cases with IgG antibodies reactive mainly to the gag p24 and/or p15, four with IgM antibodies mainly to the gag p24 and/or p19, and three with both IgG and IgM antibodies. These immunostaining patterns of MS sera were clearly distinguishable from those of adult T cell leukemia patients who had antibodies to the envelope (env) proteins and its precursors in addition to the gag proteins. The antibody in MS sera was generally of low titer and reactive at a high serum concentration (1/10 dilution). None of the sera from patients with other neurologic diseases and healthy controls had the viral antibodies. These findings indicate that at least one quarter of Japanese MS patients have antibody responses to a hitherto unidentified agent related to HTLV-I, which possibly plays a part, primarily or secondarily, in the pathogenesis of those patients.     

Enzymatic large-scale synthesis of MUC6-Tn glycoconjugates for antitumor vaccination

In cancer, mucins are aberrantly O-glycosylated, and consequently, they express tumor-associated antigens such as the Tn determinant (alpha-GalNAc-O-Ser/Thr). As compared with normal tissues, they also exhibit a different pattern of expression. In particular, MUC6, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. Recently, we have shown that the MCF7 breast cancer cell line expresses MUC6-Tn glycoproteins in vivo. Cancer-associated mucins show antigenic differences from normal mucins, and as such, they may be used as potential targets for immunotherapy. To develop anticancer vaccines based on the Tn antigen, we prepared several MUC6-Tn glycoconjugates. To this end, we performed the GalNAc enzymatic transfer to two recombinant MUC6 proteins expressed in Escherichia coli, using UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), which catalyze in vivo the Tn antigen synthesis. We used either a mixture of ppGalNAc-Ts from MCF7 breast cancer cell extracts or a recombinant ppGalNAc-T1. In both cases, we achieved the synthesis of MUC6-Tn glycoconjugates at a semi-preparative scale (mg amounts). These glycoproteins displayed a high level of Tn antigens, although the overall density depends on both enzyme source and protein acceptor. These MUC6-Tn glycoconjugates were recognized by two anti-Tn monoclonal antibodies that are specific to human cancer cells. Moreover, the MUC6-Tn glycoconjugate glycosylated using MCF7 extracts as the ppGalNAc-T source was able to induce immunoglobulin G (IgG) antibodies that recognized a human tumor cell line. In conclusion, the large-scaled production of MUC6 with tumor-relevant glycoforms holds considerable promise for developing effective anticancer vaccines, and further studies of their immunological properties are warranted.     

The use of immunoenzyme analysis and immunoblotting for the serological characterization of mycobacterial antigens]

The enzyme immunoassay and immunoblotting were used for the study of the serological activity of different mycobacterial antigens and the spectrum of antibodies to them in patients with different forms of tuberculosis and healthy persons. Antibodies in patients' sera were shown to bind antigens with different molecular weight. The level and spectrum of antibodies to purified protein fraction I made it possible to differentiate between patients with various forms of tuberculosis and healthy persons.     

Preliminary study of thyroid and colon cancers-associated antigens and their cognate autoantibodies as potential cancer biomarkers

Background: Autoantibodies, which are produced against tumor-associated antigens, are potential tumor markers and attract a growing interest for cancer detection, differential diagnostics and prognosis.

Objective: To evaluate the diagnostic significance of 40 antigens identified by immunoscreening of cDNA libraries from thyroid and colon cancers by allogenic screening with different tumor types patients’ sera.

Method: Plaque-spot serological assay.

Results: Increased frequency of antibody response in sera of cancer patients compared with that of healthy donors was shown toward 14 antigens, 8 of which (CG016, BTN3A3, FKBP4, XRCC4, TSGA2, ACTR1A, FXYD3 and CTSH) have revealed exclusively cancer-related serological profile.

Conclusion: Allogenic screening of 40 SEREX-antigens with sera from cancer patients and healthy donors allowed us to reveal 14 antigens with potential diagnostic significance. These antigens and their cognate autoantibodies could be considered as valuable targets for further analysis as potential cancer biomarkers.     

A case of Graves' disease with anti-triiodothyronine antibodies

A case of Graves' disease with high serum thyroxine (T4) and low triiodothyronine (T3) levels which was therefore initially diagnosed as a T4-thyrotoxicosis is reported. Examination of the serum from the patient showed the presence of unusual protein which bound T3. It was later confirmed as IgG class anti-T3 antibodies. In addition to treatment with methylmercaptoimidazole (MMI), the patient was treated with prednisolone for 30 days (total amount 500 mg). Titers of anti-T3 antibodies in the sera were unchanged before and after prednisolone treatment. Our present case indicates that it is clinically important to bear the presence of autoantibodies in mind to account for a possible error in measuring T3 and T4 by radioimmunoassay (RIA). In the case that RIA determination gives an unexpectedly high or low T3 and/or T4 value, the presence of autoantibodies to them should be considered and a test for them is recommended.     

T cell and antibody reactivity with the Borrelia burgdorferi 60-kDa heat shock protein in Lyme arthritis.

The reactivity of cloned T cells and serum antibodies, obtained from patients with chronic Lyme arthritis, with expressed recombinant B. burgdorferi 60-kDa heat shock protein homologue (HSP60) was analyzed. The expressed recombinant Borrelia burgdorferi HSP60 was bound by antibodies in the sera of patients with Lyme arthritis, but not by control sera. A T cell clone (CR253), isolated from one of four patients examined, exhibited an HLA-DR2 restricted proliferative response to the expressed recombinant B. burgdorferi HSP60. This T cell clone specifically recognized the HSP60 of B. burgdorferi and did not proliferate in response to the human, mycobacterial, or Escherichia coli HSP60 homologues. The epitope recognized by this cloned T cell, located between amino acids 260 and 274, is in a region of the spirochetal HSP60 that is not conserved between bacteria and eukaryotes.     

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。     

Human monoclonal antibodies derived from pleural effusion lymphocytes of a patient with breast carcinoma react with human breast cancer-associated antigens and mouse mammary tumor virus polypeptides

Four human hybridoma cell lines (PEB1-4) were established from a fusion of pleural effusion lymphocytes isolated from a breast cancer patient with metastatic disease, 6 years postmastectomy. The hybridomas secreted IgG-k (3 micrograms/ml/10(6) cells). These monoclonal antibodies (PEB1-4) reacted to different degrees with mouse mammary tumor virus (MMTV) and T47D particles (HuMTV). Immunological cross-reaction was also detected with antigens isolated from body fluids of breast cancer patients (BF-Ag). The binding capacity of the monoclonal antibodies (MAbs) PEB1-4 to the above-mentioned antigens was measured by RIA. The specificity of these antibodies was further demonstrated by radioimmunoprecipitation of MMTV, T47D (HuMTV) and BF-Ag. The binding of PEB1-4 to surface antigens of intact cells grown in culture was measured by RIA. Some of the MAbs were shown to bind more avidly to breast cancer cells than to nonbreast cancer cells or nonmalignant cells. The PEB1-4 human monoclonal antibodies may be found useful in analyzing the virus-breast cancer relationship.     

Comparison of serological specificity of anti-endotoxin sera directed against whole bacterial cells and core oligosaccharide of Escherichia coli J5-tetanus toxoid conjugate

The rough mutants of Gram-negative bacteria are widely used to induce protective antisera but the nature of the target epitope for such antibodies is not precisely defined. Endotoxin is one of several antigens present on the surface of bacterial cells, which are able to elicit specific antibodies. We studied the specificity of antibodies produced against a conjugate of E. coli J5 endotoxin core oligosaccharide with tetanus toxoid. The use of chemically defined antigen for immunisation excludes the possibility of production of antibodies against other cell surface antigens. A comparison of this monospecific anti-endotoxin serum with antiserum against E. coli J5 whole cells was performed in order to distinguish the role that endotoxin core oligosaccharide plays in the interaction with humoral host defences from that of other potentially important Gram-negative bacterial surface antigens. The reactivity of both sera with smooth and rough lipopolysaccharides was determined in ELISA, immunoblotting and by flow cytometry. Both antisera reacted with similar specificity with most lipopolysaccharides of identical or related core type. Less distinct reactions with endotoxins of the antibacterial serum in comparison with the anti-conjugate serum were found in all serological tests. LPS of E. coli O100 that showed the strongest reactions with both sera was used to stimulate IL-6, TNFalpha and nitric oxide production by the J-774A.1 cell line. Both sera were used to inhibit that stimulation and no inhibitory effects of the examined sera in comparison with non-immune serum were observed.     

Expression and purification of recombinant 38-kDa and Mtb81 antigens of Mycobacterium tuberculosis for application in serodiagnosis

Availability of genome sequence of Mycobacterium tuberculosis has accelerated identification of antigens for serodiagnosis of tuberculosis and a number of new antigens are being tested in various combinations to produce cocktails with high sensitivity and specificity. For producing a highly specific diagnostic test, it is important that the recombinant antigens be highly pure, free of host protein, and correctly folded so that they bind only to specific antibodies. Also, for commercial viability they need to be produced in high yields. We have cloned, expressed, and purified a number of mycobacterial antigens in Escherichia coli. This paper describes, expression and purification of two important mycobacterial proteins with serodiagnostic potential, namely, 38-kDa and Mtb81 antigens, in monomeric form. The protocol involves using a T7 promoter based expression vector under conditions of regulated and slow expression followed by three-step column chromatography procedure to obtain highly purified proteins. The yields of the two proteins were several folds higher than previously reported. The purified proteins were useful in detecting antibodies in sera of tuberculosis patients (smear positive, smear negative, and extra-pulmonary categories).     

Substrate specificity for the detection of autoantibodies to anterior pituitary cells in human sera

Using the indirect immunofluorescence test for the detection of pituitary autoantibodies in human serum, the results obtained with human fetal and non-human pituitary antigens were compared. Of the sera that were positive on human fetal substrate, 4% were recovered with adult baboon, 0% with fetal cymologous, 20% with porcine, 11% with bovine, 11% with ovine, and 7% with rat tissue. The rate of heterophilic antibodies to the above animal substrates was 5% to 14%. In contrast to human adult pituitaries, normal human sera did not bind to Fc receptors on ACTH-cells of human fetal pituitaries. This allowed the demonstration of ACTH-cell antibodies. The specificity of the reaction was proven by absorption studies with purified Fc fragments. These results suggest that human fetal tissue is the best source of antigen for the detection of pituitary autoantibodies. The use of animal tissue including non-human primate pituitary yields results that bear no clinical significance.     

Trichinella spiralis shares epitopes with human autoantigens

Like other helminths, Trichinella spiralis has evolved strategies to allow it to survive in the host organism, including the expression of epitopes similar to those present in either expressed or hidden host antigens. To identify T. spiralis-derived antigens that are evolutionarily conserved in the parasite and its host and that could be responsible for its evasion of the host immune response, we examined the reactivity of six different types of autoantibodies to T. spiralis larvae from muscle. T. spiralis antigens that share epitopes with human autoantigens were identified by assessing the cross-reactivity of autoantibody-containing serum samples with T. spiralis antigens in the absence of specific anti-parasite antibodies. Of the 55 autoantibody-containing human serum samples that we analysed by immunohistological screening, 24 (43.6%) recognised T. spiralis muscle larvae structures such as the subcuticular region, the genital primordium or the midgut. Using Western blots, we demonstrated that the same sera reacted with 24 protein components of T. spiralis muscle larvae excretory-secretory L1 antigens. We found that the human autoantibodies predominantly bound antigens belonging to the TSL1 group; more specifically, the autoantibody-containing sera reacted most frequently with the 53-kDa component. Thus, this protein is a good candidate for further studies of the mechanisms of T. spiralis-mediated immunomodulation.     

Identification of in vivo induced protein antigens of Salmonella enterica serovar Typhi during human infection

During infectious disease episodes, pathogens express distinct subsets of virulence factors which allow them to adapt to different environments. Hence, genes that are expressed or upregulated in vivo are implicated in pathogenesis. We used in vivo induced antigen technology (IVIAT) to identify antigens which are expressed during infection with Salmonella enterica serovar Typhi. We identified 7 in vivo induced (IVI) antigens, which included BcfD (a fimbrial structural subunit), GrxC (a glutaredoxin 3), SapB (an ABC-type transport system), T3663 (an ABC-type uncharacterized transport system), T3816 (a putative rhodanese-related sulfurtransferase), T1497 (a probable TonB-dependent receptor) and T3689 (unknown function). Of the 7 identified antigens, 5 antigens had no cross-immunoreactivity in adsorbed control sera from healthy subjects. These 5 included BcfD, GrxC, SapB, T3663 and T3689. Antigens identified in this study are potential targets for drug and vaccine development and may be utilized as diagnostic agents.     

Serodiagnosis of infection with verocytotoxin-producing Escherichia coli

Human sera (167) were screened for antibodies to lipopolysaccharide (LPS) prepared from strains of Verocytotoxin-producing Escherichia coli (VTEC) belonging to a range of serogroups, secreted proteins expressed by attaching and effacing VTEC, enterohaemolysin and H = 7 flagellar proteins. Twelve sera (about 7%) contained antibodies to the LPS of E. coli 05 (one), 026 (two), 0115 (two), 0145 (one), 0163 (one) and 0165 (five). Sera containing antibodies to the LPS of E. coli O26 and O145 also contained antibodies to secreted proteins of 100 and 40 kDa. An additional 34 sera, known to contain antibodies to the lipopolysaccharide of E. coli O157, were examined for antibodies to enterohaemolysin, H = 7 flagellar antigens and bacterial cell surface-associated proteins of 5, 6 and 22 kDa. Three sera contained antibodies to enterohaemolysin and one serum contained antibodies to flagellar proteins. Antibodies to membrane-associated proteins were not detected. It was concluded that enterohaemolysin, H = 7 flagellar proteins and the cell surface-associated proteins were unsuitable for use in immunoassays for providing evidence of infection with VTEC.     

Immunochemistry of Yersinia enterocolitica O3 grown at different temperatures.

Comparative agglutinations of homogeneous stable suspensions prepared with Yersinia enterocolitica growth at 37 degrees C and at 25 degrees C were performed with anti-sera prepared in rabbits with the bacteria grown at both these temperatures. Sera prepared with live Y. enterocolitica grown at 37 degrees C agglutinated both suspensions at a much lower titre than the sera prepared with formaldehyde-treated bacteria is grown at 25 degrees C. All the sera in which strongly precipitating antibodies were induced reacted, in agar-gel, against native and heated proteins. The small amounts of antipolysaccharides induced in all the sera reacted only in the ring test against the bacterial polysaccharides. The absorption of the sera prepared with live Y. enterocolitica grown at 37 degrees C, with antigens synthesized at 25 degrees C did not remove all the homologous antibodies; apparently, some determinants are specific for the bacteria grown at 37 degrees C. Morphological changes of the small rods to elongated bacilli and filamentous forms were observed in most cultures of the Y. enterocolitica grown at 37 degrees C; these changes coincided with a low yield of proteins and point to an inhibitory effect of the 37 degrees C temperature.     

Trypanosoma lewisi: effects of immunosuppression on the serological reactivities of exoantigens and cellular antigens of bloodstream parasites.

Groups of rats were immunosuppressed with antithymocyte serum (ATS) and infected with Trypanosoma lewisi. Immunodiffusion studies were performed which demonstrated that trypanosome exoantigens, present in the plasma of these animals, were precipitated by antibodies in the sera of rats undergoing a typical primary T. lewisi infection; extracts of trypanosomes which had been collected from ATS-treated rats contained antigens which also were precipitated by antibodies in these sera. These precipitating antibodies could not be detected using either the plasma of untreated infected rats or extracts of trypanosomes which had been collected from untreated rats. With the exoantigens, precipitating antibodies were detected in serum samples collected from rats 14 to 250 days after infection. With the extract, precipitating antibodies were found as early as 5 days after infection and could be detected as late as 90 days after infection. Antigens of trypanosome extracts partially blocked the precipitin reactions between antisera and exoantigens, suggesting the presence of common antigens in the two preparations. Intact trypanosomes were serologically more reactive when collected from immunosuppressed rats. Trypanosomes collected from ATS-treated rats were agglutinated by antisera at titers fourfold higher than trypanosomes collected from untreated hosts. Absorption with exoantigens from immunosuppressed infected rats blocked trypanosome agglutination, indicating that these antigens are of cell surface origin. The experiments suggest that a likely result of immunosuppressing the host is a trypanosome antigen preparation that is a more reactive serodiagnostic reagent.