A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.

We studied the natural MHC class I display of measles virus (MV) epitopes. Peptide ligands associated with HLA-A*0201 were purified from a B lymphoblastoid cell line prior to and after infection with MV. Infection-induced peptides were revealed using microcapillary reversed phase high performance liquid chromatography electrospray ionization/mass spectrometry (microLC-ESI/MS) by subtraction of the "infected" and "uninfected" ion traces. Three naturally processed viral epitopes derived from different MV proteins were identified through tandem MS sequencing. These peptides were expressed at widely divergent levels of HLA-peptide complexes, but had similar binding capacities to HLA-A*0201. The most abundant viral peptide species, identified as residues 84-92 (KLWESPQEI) of the MV nonstructural C protein, was expressed at an unprecedented high density (> 10(5) copies per cell) and was immunogenic in HLA-A2/Kb-transgenic mice. Furthermore, natural mutants of this epitope, occurring in persistent lethal MV strains, were shown to have lost their HLA-A*0201 binding capacity. Thus, here we report for the first time the direct discovery through microLC-ESI/MS of a uniquely dominant viral HLA class I ligand, KLWESPQEI, with features eligible for immune selection pressure.

Modified methods for the enumeration of spores of mesophilic Clostridium species in dried foods.

Clostridia constitute markers of limited though definite importance for the microbiological integrity of particular foods processed for safety, provided their application and the results obtained are meticulously considered and guided by proper ecological awareness. Their selective diagnostic enumeration in food specimens relies on their ability to reduce sulphite in agar media, visualised by the presence of ferrous cations leading to the production of black colonies. The composition of the medium used substantially affects the productivity of the procedure. We established that (1) the sulphite activity and the ferrous ion should be rigorously standardised; (2) tryptose is one of the appropriate nitrogen sources for a limited number of clostridia; (3) the basal medium should be free of added acetate and lactate. Black colonies obtained in the newly elaborated medium, termed Differential clostridial agar (DCA) should be further examined for morphology and metronidazole sensitivity, since some bacilli might mimic clostridia under the conditions of the procedure. An elegant variant of the technique relies on using a bottom-layer of mannitol/egg yolk/polymyxin/bromocresol purple agar, inoculated with macerates of food in buffered cysteine hydrochloride peptone saline, immediately liberally overlayered with freshly prepared DCA. Plates are incubated and read in tightly closed bags of plastic with a low oxygen permeability coefficient, which eliminates the need for using anaerobic jars. Colony identification is relying on assessment of sulphite reduction, egg yolk dissimilation, the mode of attack on mannitol and when required to be supported by classical other physiological traits. The mandatory precautions to be observed in this procedure call for extreme caution when introducing reference ranges ("standards") for clostridial spores in foods, particularly in the international commerce.

Conjugates of synthetic cyclic peptides elicit bactericidal antibodies against a conformational epitope on a class 1 outer membrane protein of Neisseria meningitidis.

Bactericidal antibodies directed against surface loops of class 1 outer membrane proteins play a crucial role in protection against meningitis and sepsis caused by Neisseria meningitidis. So far, all efforts to obtain protective antibodies against these apparently conformational epitopes by using linear peptide analogs have been in vain. In this study, conjugates of head-to-tail cyclic peptides encompassing the predicted top of a protective surface loop were used for immunization. A series of 18 cyclic peptides with a ring size ranging from 7 to 17 residues, conjugated to tetanus toxoid, was investigated. Antipeptide and anti-whole-cell immunoglobulin G (IgG) titers elicited by the conjugates were determined. Conjugates of three peptides, containing 14, 15, and 17 amino acid residues (peptides 7, 12, and 13, respectively), induced an anti-whole-cell titer when Quillaja saponin A was used as the adjuvant. When alum was used as the adjuvant, the conjugate of peptide 12 did not elicit an anti-whole-cell response. From the Quillaja saponin A group, some of the sera obtained with conjugates of peptides 7 and 12 and all sera obtained with the peptide 13 conjugate were bactericidal in vitro. None of the sera evoked with alum as the adjuvant showed bactericidal activity. Nonbactericidal sera contained IgG1 primarily, whereas bactericidal sera showed significant titers of IgG2a and IgG2b. Class 1 protein-derived synthetic cyclic peptides which are capable of eliciting bactericidal antibodies, such as peptide 13 derived from meningococcal strain H44/76, represent potential candidates for a (semi)synthetic vaccine against meningococcal disease.

Identification of T cell epitopes occurring in a meningococcal class 1 outer membrane protein using overlapping peptides assembled with simultaneous multiple peptide synthesis.

The meningococcal class 1 outer membrane protein (OMP) plays an important role in the development of protective immunity against meningococcal infection, and is therefore considered to be a promising candidate antigen (Ag) for a meningococcal vaccine. The induction of an effective antibody response entirely depends upon T helper cells. To identify T cell epitopes of the OMP, we prepared 45 overlapping synthetic peptides representing the entire sequence of the class 1 protein of reference strain H44/76. Fully automated simultaneous multiple peptide synthesis (SMPS) was used to assemble the 45 twenty mer which overlapped by 12 amino acid residues on a 12 mumol scale. The peptides were tested for recognition by peripheral blood mononuclear cells (PBMC) obtained from 34 volunteers. Surprisingly, all synthetic peptides induced proliferative responses of PBMC isolated from one or more human histocompatibility leukocyte antigen (HLA)-typed immune adults. With PBMC from seven nonimmune donors, no proliferative response was observed. Immunodominant regions were found, recognized by PBMC from many volunteers, irrespective of their HLA type. Most of the immunodominant T cell epitopes are located outside the variable regions and, thus, will be conserved among different meningococcal (and gonococcal) strains. Furthermore, the overlapping peptides could be used to identify the epitopes recognized by OMP-specific T cell clones with known HLA restriction. It is interesting that the epitopes defined with the clones occur in highly conserved areas, shared by all neisserial porin proteins. In summary, this analysis of the T cell response to the meningococcal class 1 OMP constitutes a complete study of reactivity to a foreign protein, and illustrates some important features of Ag recognition by T cells. Our data demonstrate unexpected diversity in the T cell recognition of the OMP, and imply that the T cell repertoire against foreign Ag may be greater than previously assumed. This observation is supported by recent data on the interaction of peptide and major histocompatibility complex (MHC) class II, the latter being much less selective than MHC class I. Finally, a comparative analysis pointed out the limitations of algorithms predicting T cell determinants, and the importance of the empirical methodology provided by SMPS.

T cell recognition of Neisseria meningitidis class 1 outer membrane proteins. Identification of T cell epitopes with selected synthetic peptides and determination of HLA restriction elements.

No vaccine is yet available against serogroup B meningococci, which are a common cause of bacterial meningitis. Some outer membrane proteins (OMP), LPS, and capsular polysaccharides have been identified as protective Ag. The amino acid sequence of the protective B cell epitopes present within the class 1 OMP has been described recently. Synthetic peptides containing OMP B cell epitopes as well as capsular polysaccharides or LPS protective B cell epitopes have to be presented to the immune system in association with T cell epitopes to achieve an optimal Ir. The use of homologous, i.e., meningococcal, T cell epitopes has many advantages. We therefore investigated recognition sites for human T cells within the meningococcal class 1 OMP. We have synthesized 16 class 1 OMP-derived peptides encompassing predicted T cell epitopes. Peptides corresponding to both surface loops and trans-membrane regions (some of which occur as amphipathic beta-sheets) of the class 1 OMP were found to be recognized by T cells. In addition, 10 of 11 peptides containing predicted amphipathic alpha-helices and four of five peptides containing T cell epitope motifs according to Rothbard and Taylor (Rothbard, J. B., and W. R. Taylor. 1988. EMBO J 7:93) were recognized by lymphocytes from one or more volunteers. Some of the T and B cell epitopes were shown to map to identical regions of the protein. At least six of the peptides that were found to contain T cell epitopes show homology to constant regions of the meningococcal class 3 OMP and the gonococcal porins PIA and PIB. Peptide-specific T cell lines and T cell clones were established to investigate peptide recognition in more detail. The use of a panel of HLA-typed APC revealed clear HLA-DR restriction patterns. It seems possible now to develop a (semi-) synthetic meningococcal vaccine with a limited number of constant T cell epitopes that cover all HLA-DR locus products.

Thyrotropin dependent and independent thyroid cell lines selected from FRTL-5 derived tumors grown in nude mice.

FRTL-5 cells were used to set up a thyroid tumor model system in C3H nu/nu mice. FRTL-5 tumors could be grown in nude mice provided serum TSH levels were elevated. Persistent TSH elevation was obtained by administration of Na131I, rendering the mice hypothyroid. After 4 weeks FRTL-5 cells were injected sc resulting in tumor growth within 2 weeks in eight out of eight mice. Although the tumors showed an apparently undifferentiated histology, lacking normal follicular structures, they were functional since the tumors were capable of concentrating [131]iodine, as demonstrated by nuclear imaging. From one of the tumors a new cell line was isolated (FRTL-5/T) that, like the parental FRTL-5 cell line, was TSH dependent for growth. In a control group of six euthyroid nude mice FRTL-5 tumor growth could not be obtained with one exception. After 3 months one animal developed a small tumor that grew rapidly thereafter. This tumor was easily transplantable in other euthyroid nude mice, showed an undifferentiated histology, and was nonfunctional, as it could not concentrate [131]iodine. From this tumor two cell lines were derived: one cultured in the presence of TSH (FRTL-5/TP) and one in the absence of TSH (FRTL-5/TA). Both cell lines were found to be TSH independent for growth. The cell lines were analyzed for TSH responsive functions and TSH receptor expression. Responsiveness to TSH in FRTL-5/T and the parental FRTL-5 cell line were similar for most thyroid specific functions tested. However, FRTL-5/T was less sensitive than FRTL-5 for TSH induced [3H]thymidine incorporation. Both cell lines had two classes of TSH binding sites with high and low affinity respectively, as determined by Scatchard analysis. FRTL-5/TP and FRTL-5/TA were both able to grow in TSH free medium and were nonresponsive to TSH in vitro, as tested for [3H]thymidine and [3H]uridine incorporation, iodine uptake, thyroglobulin iodination, and thyroglobulin secretion. This correlated with an approximately 100-fold decreased number of TSH binding sites compared to FRTL-5. The latter was caused by a complete absence of low affinity binding sites, whereas high affinity receptors were still detectable. The FRTL-5/TA cell line was the least differentiated one as thyroglobulin mRNA was detectable in only minute amounts and thyroid peroxidase expression could not be measured. These in vivo selected FRTL-5 cell lines offer a suitable model to investigate several aspects of TSH responsiveness, including signal transduction and postreceptor events, thyroid differentiation, and thyroid tumorigenesis.

Iodination of newly synthesized thyroglobulin by FRTL-5 cells is selective and thyrotropin dependent.

This study shows that the Fisher rat thyroidal cell line (FRTL-5) can iodinate newly synthesized thyroglobulin. Iodinated thyroglobulin was found intra- and extracellularly. Both the synthesis of thyroglobulin and its subsequent iodination were found to be thyrotropin (TSH) dependent, with optimal activity at 10-100 microU TSH/ml. Thyroglobulin was the only protein in the culture medium, that was iodinated with high specificity and in a TSH-dependent fashion. Albumin, which was abundantly present in the culture medium, was only weakly iodinated. Various proteins, including thyroglobulin, were found to be iodinated intracellularly. Of these iodoproteins only thyroglobulin appeared in the medium suggesting selective secretion of iodinated thyroglobulin. It was shown that the other intracellular iodoproteins were no thyroglobulin breakdown products. Their function is as yet unknown.

Efficient selection of high-affinity B cell hybridomas using antigen-coated magnetic beads.

A method for the selection of antigen-specific B cell hybridomas using antigen-coated magnetic beads is described. Stable B cell hybridoma cell lines directed against human thyroglobulin were incubated with thyroglobulin-coated beads. 2 h of incubation at 4 degrees C using bead-to-cell ratios of at least 3:1 were found to be the optimal conditions for rosette formation. Rosettes were efficiently isolated with a strong magnet. Rosette formation was antigen-specific since irrelevant hybridoma cell lines could not form rosettes, nor could BSA-coated or uncoated beads form rosettes. Free antibodies produced by the hybridoma cells were able to block rosette formation. Blocking of rosette formation permitted the identification of different and overlapping epitopes recognized by four different hybridomas. Using six stable hybridoma cell lines with different affinities for thyroglobulin, rosette formation appeared to be dependent on the affinity of the immunoglobulin membrane receptor for antigen. A correlation was observed between the affinity of the secreted antibodies and the capacity of the hybridomas to form rosettes, suggesting that this method is suitable for the selection of hybridomas producing antibodies with a high affinity for the antigen. Antigen-coated magnetic beads were found to be suitable for the efficient selection of thyroglobulin-specific hybridoma cells from bulk cultures shortly after fusion. A 300-fold enrichment of thyroglobulin-specific cells was obtained using this method.

Growth of Candida famata and Trichosporon cutaneum on uric acid as the sole source of carbon and energy, a hitherto unknown property of yeasts.

Yeast strains capable of utilizing uric acid as the sole source of carbon and energy were isolated from soil by the enrichment culture method. The strains were identified as Candida famata (Harrison) Meyer et Yarrow and Trichosporon cutaneum (De Beurm., Gougerot et Vaucher) Ota. On the subcellular level growth of yeasts on uric acid was accompanied with the development of a number of large microbodies in the cells.