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Background: Soluble oligomeric amyloid-β (Aβ), rather than Aβ plaques, seems to be the culprit in Alzheimer's disease (AD). Accordingly, a new concept vaccine of small cyclic peptide conjugates, selectively targeting oligomeric Aβ, has been developed.Objective: Study the therapeutic potential of this new vaccine in a mouse model for AD.Methods: J20 mice, overexpressing human amyloid precursor protein, were validated for an AD-like phenotype. Then, J20 mice were vaccinated at 2, 3, and 4 months of age and AD phenotype was evaluated at 6, 9, and 12 months of age; or at 9, 10, and 11 months with evaluation at 12 months. Effects on Aβ pathology were studied by plaque load (immunohistochemistry; 6E10) and antibody titers against Aβ (ELISA). AD behavioral phenotype was evaluated by performance in a battery of cognitive tests.Results: J20 mice displayed age-related Aβ plaque development and an AD-like behavioral phenotype. A consistent antibody response to the cyclic peptides was, however, not extended to Aβ, leaving plaque load unaffected. Nevertheless, immunization at young ages prevented working- and short-term spatial memory loss, but deteriorated long-term spatial learning and memory, at 12 months of age. Immunization at later ages did not affect any measured parameter.Conclusion: J20 mice provide a relevant model for AD to study potential anti-Aβ treatment. Early vaccination prevented short-term memory loss at later ages, but deteriorated long-term spatial memory, however without affecting Aβ pathology. Later vaccination had no effects, but optimal timing may require further investigation.

Background: Soluble oligomeric (misfolded) species of amyloid-beta (A beta) are the main mediators of toxicity in Alzheimer's disease (AD). These oligomers subsequently form aggregates of insoluble fibrils that precipitate as extracellular and perivascular plaques in the brain. Active immunization against A beta is a promising disease modifying strategy. However, eliciting an immune response against A beta in general may interfere with its biological function and was shown to cause unwanted side-effects. Therefore, we have developed a novel experimental vaccine based on conformational neo-epitopes that are exposed in the misfolded oligomeric A beta, inducing a specific antibody response.Objective: Here we investigate the protective effects of the experimental vaccine against oligomeric A beta(1-42)-induced neuronal fiber loss in vivo.Methods: C57BL/6 mice were immunized or mock-immunized. Antibody responses were measured by enzyme-linked immunosorbent assay. Next, mice received a stereotactic injection of oligomeric A beta(1-42) into the nucleus basalis of Meynert (NBM) on one side of the brain (lesion side), and scrambled A beta(1-42) peptide in the contralateral NBM (control side). The densities of choline acetyltransferase-stained cholinergic fibers origination from the NBM were measured in the parietal neocortex postmortem. The percentage of fiber loss in the lesion side was determined relative to the control side of the brain.Results: Immunized responders (79%) showed 23% less cholinergic fiber loss (p = 0.01) relative to mock-immunized mice. Moreover, fiber loss in immunized responders correlated negatively with the measured antibody responses (R-2 = 0.29, p = 0.02).Conclusion: These results may provide a lead towards a (prophylactic) vaccine to prevent or at least attenuate (early onset) AD symptoms.

International audience; Conjugation chemistry is among the most important parameters governing the efficacy of glycoconjugate vaccines. High robustness is required to ensure high yields and batch to batch reproducibility. Herein, we have established a robust bioconjugation protocol based on the thiol-maleimide addition. Major variables were determined and acceptable margins were investigated for a synthetic pentadecasaccharide-tetanus toxoid conjugate, which is a promising vaccine candidate against Shigella flexneri serotype 2a infection. The optimized process is applicable to any thiol-equipped hapten and provides an efficient control of the hapten:carrier ratio. Moreover, comparison of four S. flexneri 2a glycoconjugates only differing by their pentadecasaccharide:tetanus toxoid ratio confirmed preliminary findings indicating that hapten loading is critical for immunogenicity with an optimal ratio here in the range of 17 ± 5. In addition, the powerful influence of alum on the immunogenicity of a Shigella synthetic carbohydrate-based conjugate vaccine candidate is demonstrated for the first time, with a strong anti-S. flexneri 2a antibody response sustained for more than one year.

β-Propiolactone is often applied for inactivation of viruses and preparation of viral vaccines. However, the exact nature of the reactions of β-propiolactone with viral components is largely unknown. The purpose of the current study was to elucidate the chemical modifications occurring on nucleotides and amino acid residues caused by β-propiolactone. Therefore, a set of nucleobase analogues was treated with β-propiolactone, and reaction products were identified and quantified. NMR revealed at least one modification in either deoxyguanosine, deoxyadenosine, or cytidine after treatment with β-propiolactone. However, no reaction products were found from thymidine and uracil. The most reactive sides of the nucleobase analogues and nucleosides were identified by NMR. Furthermore, a series of synthetic peptides was used to determine the conversion of reactive amino acid residues by liquid chromatography-mass spectrometry. β-Propiolactone was shown to react with nine different amino acid residues. The most reactive residues are cysteine, methionine, and histidine and, to a lesser degree, aspartic acid, glutamic acid, tyrosine, lysine, serine, and threonine. Remarkably, cystine residues (disulfide groups) do not react with β-propiolactone. In addition, no reaction was observed for β-propiolactone with asparagine, glutamine, and tryptophan residues. β-Propiolactone modifies proteins to a larger extent than expected from current literature. In conclusion, the study determined the reactivity of β-propiolactone with nucleobase analogues, nucleosides, and amino acid residues and elucidated the chemical structures of the reaction products. The study provides detailed knowledge on the chemistry of β-propiolactone inactivation of viruses.

The synthesis of analogue 14f (Scheme 2) of a fragment of the S. pneumoniae type 1 C-substance antigen (Fig. 1, Ia) is presented. The protected disaccharide 5 and trisaccharide 7 (Scheme 1) were prepared by silver trifluoromethanesulfonate-catalyzed α-glycosylation of 2c with 4b (54%) and 2d with 6b (59%), respectively. A silver-silicate-catalyzed β-glycosylation of ribitol derivative 9 with trisaccharide bromide 8b gave 10a (55%). The selectively deprotected trisaccharideribitol 10b was coupled with spacer 11 (Scheme 2) using phosphorylating reagent 12 to give the fully protected phosphotriester derivative 14a (98%). The stepwise deprotection of 14a, including conversion of the azide functions into acetamido groups, turned out to be troublesome and afforded 14f in low yield.

The syntheses of two (spacer-containing and choline-phosphate-deficient) analogues (12e and 12h, Scheme 2) of a fragment of the C substance from S. pneumoniae type 1 are presented. Starting from 1,6:2,3-dianhydro-s-D-talopyranose (la), the “building block” 1-O-(3,6-di-O-acetyl)-2-azido-4-O-benzyl-2-deoxy-s-D-galactopyranosyl)-2,3,4-tri-O-benzyl-D-ribitol (6b) was obtained in six steps (Scheme 1). A second “building block” (9e), containing a protected propanolamine spacer, was prepared in five steps, starting from 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (7). The building blocks 6b and 9e were coupled (Scheme 2) using bis(1-benzotriazolyl) 2-chlorophenyl phosphate (10) to give the fully protected phosphotriester derivative 12a. Complete deprotection of compound 12a via two different routes afforded β-D-GalNH2p-(1 ŕ l)-ribitol-5-phosphate-6-β-D–Glcp-[1 ŕ O-(CH2)3-NH2] (12h, an analogue of structure Ia) and its β-D-GalNAcp derivative 12e (corresponding to Ib).

P.69 pertactin (P.69 Prn), an adhesion molecule from the causative agent of pertussis,Bordetella pertussis, is present in cellular and most acellular vaccines that are currently used worldwide. Although both humoral immunity and cellular immunity directed against P.69 Prn have been implicated in protective immune mechanisms, the identities of CD4+T-cell epitopes on the P.69 Prn protein remain unknown. Here, a single I-Ad-restrictedB. pertussisconserved CD4+T-cell epitope at the N terminus of P.69 Prn was identified by using a BALB/c T-cell hybridoma. The epitope appeared immunodominant among four other minor strain-conserved P.69 Prn epitopes recognized after vaccination andB. pertussisinfection, and it was capable of evoking a Th1/Th17-type cytokine response.B. pertussisP.69 Prn immune splenocytes did not cross-react with natural variants of the epitope as present inBordetella parapertussisandBordetella bronchiseptica. Finally, it was found that the immunodominant P.69 Prn epitope is broadly recognized in the human population by CD4+T cells in an HLA-DQ-restricted manner. DuringB. pertussisinfection, the epitope was associated with a Th1-type CD4+T-cell response. Hence, this novel P.69 Prn epitope is involved in CD4+T-cell immunity afterB. pertussisvaccination and infection in mice and, more importantly, in humans. Thus, it may provide a useful tool for the evaluation of the type, magnitude, and maintenance ofB. pertussis-specific CD4+T-cell mechanisms in preclinical and clinical vaccine studies.

Starting from the alpha-(2,4-dimethoxybenzyl) ester of N-(9-fluorenylmethoxycarbonyl)aspartic acid [Fmoc-Asp-ODmb], side-chain-protected resin-bound Fmoc-peptides containing an N epsilon-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl lysyl [Lys(Dde)] residue were prepared. The C-terminal dimethoxybenzyl esters of aspartic acid were removed with 1% trifluoroacetic acid and 10% anisole in dichloromethane, followed by Fmoc-cleavage in the usual manner. The resin-bound peptides were then cyclized using 1-benzotriazolyloxy-tris-[N-pyrrolidino]phosphonium hexafluorophosphate (PyBOP) in the presence of N-methylmorpholine. The (dimethyldioxocyclohexylidene)ethyl groups of lysine were removed with 1% hydrazine hydrate in N,N-dimethylacetamide, and the liberated side-chain amino functions were modified by reaction with pentafluorophenyl S-acetylmercaptoacetate (SAMA-OPfp). Finally, the peptides were side-chain deprotected, with exception of the Lys(SAMA) residue, and cleaved from the solid support with trifluoroacetic acid/anisole/water, 95/2.5/2.5. Cyclic peptides comprising 7-14 amino acid residues were obtained employing this procedure. As a model conjugation, cyclo[Thr-Asn-Asn-Asn-Leu-Lys(SAMA)-Thr-Lys-Asp] was coupled with bromoacetamide. The same peptide was also coupled with a bromoacetylpeptide to give a well defined peptide/peptide conjugate. All peptides were conjugated to bromoacetylated tetanus toxoid for immunization purposes.

The bacterial cell-wall-associated teichoic acids contain predominantly D-ribitol residues interconnected by phosphodiester linkages. Because of their location, these antigens may be vaccine candidates as part of conjugate vaccines. Here, we describe the synthesis of extended oligomers of D-ribitol-1-phosphate linked to a spacer having an amino group at its terminus. The synthesis utilized a fully protected D-ribitol-phosphoramidite that was oligomerized in a stepwise fashion followed by deprotection. The free oligomers were connected to bovine serum albumin using oxime chemistry. Thus, the ribitol phosphate oligomers were converted into keto derivatives, and the albumin counterpart was decorated with aminooxy groups. Reaction of the functionalized saccharide and protein moieties afforded conjugates having up to 20 ribitol phosphate chains.

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4+ and CD8+ T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/ 6 background, we characterized the specificity, level and functional capabilities of CD8+ T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes were recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8+ T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice. © 2006 Elsevier Inc. All rights reserved. Keywords: Respiratory syncytial virus; C57BL/6; CD8+ T cells; Inactivation; Tetramer

In various western countries, subtype P1.4 of Neisseria meningitidis serogroup B causes the greatest incidence of meningococcal disease. To investigate the molecular recognition of this subtype, we crystallised a peptide (P1HVVVNNKVATH(P11)), corresponding to the subtype P1.4 epitope sequence of outer membrane protein PorA, in complex with a Fab fragment of the bactericidal antibody MN20B9.34 directed against this epitope. Structure determination at 1.95 A resolution revealed a unique complex of one P1.4 antigen peptide bound to two identical Fab fragments. One Fab recognises the putative epitope residues in a 2:2 type I beta-turn at residues P5NNKV(P8), whereas the other Fab binds the C-terminal residues of the peptide that we consider a crystallisation artefact. Interestingly, recognition of the P1.4 epitope peptide is mediated almost exclusively through the complementarity-determining regions of the heavy chain. We exploited the observed turn conformation for designing conformationally restricted cyclic peptides for use as a peptide vaccine. The conformational stability of the two peptide designs was assessed by molecular dynamics simulations. Unlike the linear peptide, both cyclic peptides, conjugated to tetanus toxoid as a carrier protein, elicited antibody responses in mice that recognised meningococci of subtype P1.7-2,4. Serum bactericidal assays showed that some, but not all, of the sera induced with the cyclic peptide conjugates could activate the complement system with titres that were very high compared to the titres induced by complete PorA protein in its native conformation administered in outer membrane vesicles.

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.

The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.

Lipopeptidic formulations have been described as efficient activators of cytotoxic T lymphocytes (CTL). To better understand the pathway via which lipopeptides reach the MHC class I molecules we studied the intracellular processing and presentation of a measles virus-derived CTL epitope, to which a palmitoyl moiety was added synthetically. The palmitoyl group was conjugated to the N-terminus either directly or via a spacer sequence. The use of single or double fluorescent-labeled lipopeptides allowed the visualization of intracellular processing of these antigens using confocal microscopy. Our data indicate that the spacer composition influences internalization of the conjugate into the cell, proteasomal degradation, translocation into the ER by the transporter associated with antigen processing (TAP), and the intracellular trafficking of lipopeptides.

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.

The mechanism which enables lipopeptides to induce cytotoxicity is not known. By preparing fluorescent-labeled lipopeptides one might unravel the mechanism of their entry into the cell and their intracellular pathway. A method of preparing double-fluorescent-labeled peptides by solid-phase chemistry is described. As model peptides we have chosen analogs of the sequence RRYPDAVYL, which occurs in the measles fusion protein (F438-446) and is an epitope for cytotoxic T lymphocytes. The peptides Pal-K(TMR)KKKRRYPDAVK(FL)L (7) and Pal-K(FL)KKKRRYPDAVK(TMR)L (8), in which Pal is palmitoyl and K(TMR) and K(FL) are Nepsilon-carboxytetramethylrhodamine- and Nepsilon-carboxyfluorescein-labeled lysyl residues, respectively, were prepared and obtained in approximately 30% yield after purification by high-performance liquid chromatography. The fluorescence of fluorescein and tetramethylrhodamine in lipopeptide Pal-K(TMR)KKKRRYPDAVK(FL)L (7) was quenched to 98-99% due to intramolecular interaction of the labels. On incubation with trypsin (i.e. cleavage at the KKKRR-site) the fluorescence of both labels was restored. The intracellular routing of lipopeptide Pal-K(TMR)KKKRRYPDAVK(FL)L was studied with human melanoma cell line, Mel/J, which was transfected with human leukocyte antigen B*2705. It appeared that the double-fluorescent-labeled lipopeptide was able to induce antigen-specific cytotoxicity. Furthermore, preliminary confocal microscopical studies indicated that this lipopeptide is observed intracellularly.

The immunogenicity of two types of Streptococcus pneumoniae capsular polysaccharide-tetanus toxoid conjugates (PS6BTT and PS14TT) was evaluated in mice. Both conjugates induced high titres of high avidity type-specific anti-PS IgG, which include all IgG isotypes except IgG2a. Repeated immunization resulted in booster responses in both cases. The antibodies induced exhibited opsonic activity, as measured in an in vitro opsonophagocytosis assay, using the mouse macrophage cell line RAW-264. Furthermore, the influence of spiking PS6BTT with free PS6B of either 1000 kDa (native) or 37 kDa was investigated. The results indicate that not only the amount but also the molecular weight of the free PS6B present in the conjugate vaccine affect the anti-PS6B immune response. Large amounts of free PS6B of both molecular weights decrease each anti-PS6B IgG isotype response. However, unlike admixture of the low molecular weight PS6B, addition of the high molecular weight PS6B leads to a rather persistent state of unresponsiveness.

An antibody-peptide model system was used to study the binding characteristics between a bactericidal antibody (MN12H2) and the P1. 16 epitope of class 1 outer membrane protein PorA of Neisseria meningitidis by means of a thermodynamic approach. A series of four linear peptides and three "head-to-tail" cyclic peptides (with ring sizes of 9, 15 and 17 amino acids) were synthesized and evaluated as ligands. The peptides contain a fluorescein label and the core determinant amino acid sequence TKDTNNN (residues 180-186) of the PorA P1.16 epitope of meningococcal strain H44/76. Thermodynamic data of the binding of the peptide homologs of the epitope by MN12H2 were assessed by measuring affinity constants (Ka) over a temperature range of 4-55 degrees C, using fluorescence spectroscopy. Curvilinear plots of ln Ka versus T (K) revealed strong temperature dependencies of enthalpy (DeltaH) and entropy (DeltaS). The Gibbs free energy change (DeltaG) was only weakly temperature dependent. The large negative enthalpy value indicated the importance of polar interactions in the binding of both linear and cyclic peptides by MN12H2. Sturtevant's analysis of the thermodynamic parameters showed large unfavorable vibrational contributions to the binding for all linear peptides [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci.U.S.A. 74, 2236-2240]. The large hydrophobic contribution compensating these vibrational modes was partially attributed to aspecific interaction of the fluorescein label with the antibody. Binding of MN12H2 to conformationally restricted epitope sequences was characterized by a dramatic reduction in the size of unfavorable vibrational components of the thermodynamic parameters. Substitution of individual charged amino acids of the P1.16 epitope sequence revealed that aspartate-182 was essential for the binding. The pH profile observed for the MN12H2-peptide complexes with a midpoint pH of approximately 8.5 suggests a positively charged histidine from the antibody binding site to be involved in a charge interaction with Asp-182. These findings are consistent with the results from the crystal structure of the Fab fragment of MN12H2 in complex with a linear fluorescein-conjugated peptide homolog of the P1.16 epitope [van den Elsen et al. (1997) Proteins (in press)], thereby identifying the basis of an increased incidence of endemic disease in England and Wales since 1981 caused by a mutant meningococcal strain.

Class 1 outer membrane protein PorA of Neisseria meningitidis is a vaccine candidate against bacterial meningitis. Antibodies against PorA are able to induce complement-mediated bacterial killing and thereby play an important role in protection against meningococcal disease. Bactericidal antibodies are all directed against variable regions VR1 and VR2 of the PorA sequence, corresponding to loops 1 and 4 of a two-dimensional topology model of the porin with eight extracellular loops. We have determined the crystal structure to 2.6 A resolution of the Fab fragment of bactericidal antibody MN12H2 against meningococcal PorA in complex with a linear fluorescein-conjugated peptide TKDTNNNL derived from the VR2 sequence of sero-subtype P1.7,16 (residues 180-187) from meningococcal strain H44/76. The peptide folds deeply into the binding cavity of the Fab molecule in a type I beta-turn, with the minimal P1.16 epitope DTNNN virtually completely buried. The structure reveals H-bonds and van der Waals interactions with all minimal epitope residues and one essential salt bridge between Asp-182 of the peptide and His-31 of the MN12H2 light chain. The key components of the recognition of PorA epitope P1.16 by bactericidal antibody MN12H2 correspond well with available thermodynamic data from binding studies. Furthermore, they indicate the structural basis of an increased endemic incidence of infection by group B meningococci in England and Wales since 1981 associated with the occurrence of an Neisseria meningitidis escape mutant (strain-MC58). The observed three-dimensional conformation of the peptide provides a rationale for the development of a synthetic peptide vaccine against meningococcal disease.

Using an antiserum raised against Lys- gamma 2-melanocyte-stimulating hormone (Lys- gamma 2-MSH), with a high specificity for this peptide and its des-Lys derivative, gamma 2-MSH, we found Lys- gamma 2-MSH-like immunoreactivity to have a widespread distribution in the rat brain. In colchicine-treated rats, groups of immunopositive cell bodies were found in the intermediate and anterior lobes of the pituitary gland, in the hypothalamic arcuate nucleus and in the commissural part of the nucleus of the solitary tract (NTS). Immunopositive fibers were found to originate from the latter two cell body regions. The distribution of these fibers was similar to that of the pro-opiomelanocortin containing cell bodies and projections as it has been described previously. Immunopositive terminals were found in brain region containing neurons which have been shown to express mRNA for melanocortin receptors, though the distribution of Lys-gamma 2-MSH-like immunoreactivity is considerably more widespread than that of mRNA for the 'gamma-MSH receptor' (the melanocortin MC3 receptor), which has been reported to be mainly expressed in the hypothalamus. In the periphery Lys-gamma 2-MSH immunoreactivity was localized in the adrenal medulla and in neuronal fibers and varicosities in the heart. The vascular system, the bronchi and kidney were immunonegative. The occurrence of Lys-gamma 2-MSH immunoreactivity in many of the brain regions which are involved in cardiovascular regulation offers leads for further studies on the putative role of gamma-MSHs in cardiovascular control. The occurrence in the rat heart of Lys-gamma 2-MSH-containing fibers suggests a role of the gamma-MSHs in cardiac function.

Former studies have shown that the class 5 outer membranes proteins (Opa and Opc proteins) of Neisseria meningitidis are at least as immunogenic as meningococcal porin proteins. High antibody titers to class 5 proteins have been observed in sera obtained during convalescence after meningococcal infection. A strong increase in anti-class 5 antibodies has also been observed in vaccinees who received a meningococcal outer membrane vesicle preparation. The enhanced B-cell response to class 5 proteins may be due to the presence of immunodominant helper T-cell epitopes in these proteins. In order to investigate this hypothesis, we tested purified Opa, Opc, and class 1 proteins for recognition by human T cells. a hierarchy of T-cell immunogenicity was observed among the outer membrane proteins, the Opa protein being more immunogenic than the other proteins. In most cases, the proliferative responses elicited by Opc were higher than the responses observed for the class 1 protein. The epitopes recognized by the immune T cells were identified by using overlapping synthetic peptides spanning the protein sequences of OpaB, Opa5d, and Opc.

β-Propiolactone is often applied for inactivation of viruses and preparation of viral vaccines. However, the exact nature of the reactions of β-propiolactone with viral components is largely unknown. The purpose of the current study was to elucidate the chemical modifications occurring on nucleotides and amino acid residues caused by β-propiolactone. Therefore, a set of nucleobase analogues was treated with β-propiolactone, and reaction products were identified and quantified. NMR revealed at least one modification in either deoxyguanosine, deoxyadenosine, or cytidine after treatment with β-propiolactone. However, no reaction products were found from thymidine and uracil. The most reactive sides of the nucleobase analogues and nucleosides were identified by NMR. Furthermore, a series of synthetic peptides was used to determine the conversion of reactive amino acid residues by liquid chromatography-mass spectrometry. β-Propiolactone was shown to react with nine different amino acid residues. The most reactive residues are cysteine, methionine, and histidine and, to a lesser degree, aspartic acid, glutamic acid, tyrosine, lysine, serine, and threonine. Remarkably, cystine residues (disulfide groups) do not react with β-propiolactone. In addition, no reaction was observed for β-propiolactone with asparagine, glutamine, and tryptophan residues. β-Propiolactone modifies proteins to a larger extent than expected from current literature. In conclusion, the study determined the reactivity of β-propiolactone with nucleobase analogues, nucleosides, and amino acid residues and elucidated the chemical structures of the reaction products. The study provides detailed knowledge on the chemistry of β-propiolactone inactivation of viruses.

The trimolecular interaction of T cell receptor (TcR), antigen and major histocompatibility complex (MHC) class II was analyzed using a panel of HLA-DR2-restricted T cell clones recognizing the 49-61 region of a meningococcal class I outer membrane protein (OMP). The clones, all CD3+CD4+CD8-TcR alpha/beta+, were selected by restimulation with the synthetic peptide OMP(49-61), which contains an immunodominant T helper determinant. Using a series of peptides that were sequentially truncated from the N or C terminus, four different epitope fine-specificity patterns were identified. Furthermore, each clone was found to exhibit a distinct recognition pattern for a panel of 20 single-residue substitution analogues of the minimal epitope OMP(50-58). Most substitutions that were not tolerated in the nonamer were allowed when the analogues were prepared departing from the native peptide OMP(49-61). Obviously, the residues outside the minimal epitope contribute to stabilization of the trimolecular complex. These findings suggest that defining the minimal size of T cell determinants may be of limited value. By performing proliferation competition assays putative MHC and TcR contact residues were identified in the peptide. Most likely, Ile 51 and Phe 54 act as MHC-anchoring residues, whereas Asp 53 represents a critical TcR contact residue for all of the clones. MHC anchoring may be provided by other residues as well, since Ile 51 and Phe 54 can be substituted by conservative residues [as OMP(50-58) and OMP(49-61) analogues] and with Ala [as OMP(49-61) analogues only]. Some evidence was found for interaction of particular side chains at other positions with TcR molecules, but this contribution was not equally important for all clones. Apparently, the clonotypic TcR can see a single epitope in different ways in the context of the same MHC restriction element. Since most clones use different V alpha and V beta genes (which encompass the putative MHC-binding regions first and second complementarity-determining regions, CDR1 and CDR2) different modes of interaction with the HLA-DR2 molecule indeed are likely to occur.

A simple and rapid method for determining the affinity constant of a monoclonal antibody-peptide complex under equilibrium conditions is presented. A peptide corresponding to sequence 178-185 of meningococcal strain MC50 class 1 outer membrane protein, which is recognized by monoclonal antibody MN12 (mouse IgG2a), was synthesized. After fluorescein was coupled to the peptide, the peptide-fluorescein conjugate was used for binding studies with MN12, employing fluorescence polarization of the fluorescein label to probe the bound fraction of the peptide. Scatchard analysis showed that the affinity constant was pH dependent. Storage of MN12 under alkaline conditions resulted in a loss of antigen-binding sites, but did not alter the affinity constant. Sips plots showed a homogeneity index of unity.

CD8+ T lymphocytes play a major role in the clearance of respiratory syncytial virus (RSV) infections. To be able to study the primary CTL response in RSV-infected children, epitopes presented by a set of commonly used HLA alleles (HLA-A1, -A3, -B44 and -B51) were searched for. Five epitopes were characterized derived from the matrix (M), non-structural (NS2) and second matrix (M2) proteins of RSV. All epitopes were shown to be processed and presented by RSV-infected antigen-presenting cells. HLA-A1 tetramers for one of these epitopes derived from the M protein were constructed and used to quantify and phenotype the memory CD8+ T cell pool in a panel of healthy adult donors. In about 60 % of the donors, CD8+ T cells specific for the M protein could be identified. These cells belonged to the memory T cell subset characterized by expression of CD27 and CD28, and down-regulation of CCR7 and CD45RA. The frequency of tetramer-positive cells varied between 0·4 and 3 per 104 CD8+ T cells in PBMC of healthy asymptomatic adult donors.

Bordetella pertussis is reemerging in several countries with a traditionally high vaccine uptake. An analysis of clinical isolates revealed antigenic divergence between vaccine strains and circulating strains with respect to P.69 pertactin. Polymorphisms in P.69 pertactin are mainly limited to regions comprised of amino acid repeats, designated region 1 and region 2. Region 1 flanks the RGD motif, which is involved in adherence. Although antibodies against P.69 pertactin are implicated in protective immunity, little is known about the structure and location of its epitopes. Here we describe the identification by pepscan analysis of the locations of mainly linear epitopes recognized by human sera and mouse monoclonal antibodies (MAbs). A total of 24 epitopes were identified, and of these only 2 were recognized by both MAbs and human antibodies in serum. A number of immunodominant epitopes were identified which were recognized by 78 to 93% of the human sera tested. Blocking experiments indicated the presence of high-avidity human antibodies against conformational epitopes. Human antibodies against linear epitopes had much lower avidities, as they were unable to block MAbs. Pepscan analyses revealed several MAbs which bound to both region 1 and region 2. The two regions are separated by 289 amino acids in the primary structure, and we discuss the possibility that they form a single conformational epitope. Thus, both repeat regions may serve to deflect the immune response targeted to the functional domain of P.69 pertactin. This may explain why the variation in P.69 pertactin is so effective, despite the fact that it is limited to only two small segments of the molecule.

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.

It was recognized early this century that small molecules, called haptens, can be made immunogenic after conjugation to carrier proteins (1), This principle was thereafter applied successfully to improve the rmmunogenicity of (poly)saccharides (2, 3). We now know that the carrier proteins ensure the involvement of T-helper lymphocytes in the activation of the haptenor polysaccharide-specific antibody producing B lymphocytes (Fig. 1). In contrast to small molecules or haptens, polysaccharides (or other macromolecules with a repeating structure) are able to induce an immune response, most likely by directly activating B lymphocytes. Antigens that are able to induce an immune response without the involvement of T-helper lymphocytes are named TI (thymus independent) antigens (4) (Table 1). TI-2 antigens, such as plain polysaccharides, are not able to activate relatively immature B-cells. This is in contrast to TI-1 antigens, which can activate immature B-cells because of their mitogenic activity. Lipopolysaccharides are examples of TI-1 antigens. T-cells with specificity for saccharide structures that are recognized in association with the major histocompatibility complex (MMC) structures have never been found nor described; binding to MHC and stimulation of T-cells appears to be limited to peptides. The findings of T-cell regulation of the immune response against polysaccharides (5-7) without biochemical demonstration of the specificity of the molecular interactions can best be explained by assuming a role for antiidiotypic antibodies and T-cells.

The absolute and relative abundance of major histocompatibility complex class I-presented viral epitopes is important in the induction and maintenance of antiviral cytotoxic-T-lymphocyte (CTL) responses. We demonstrate that the supra-abundant HLA-A*0201-restricted peptide KLWESPQEI of the measles virus nonstructural C protein induces strong gamma interferon CD8+-T-cell responses in children with acute measles. However, longitudinal analysis indicates that these responses are only short-lived. Thus, some viral epitopes that can be immunodominant during primary infection may fail to establish memory CTL responses.

Memory CD4 T-cell responses against respiratory syncytial virus (RSV) were evaluated in peripheral blood mononuclear cells of healthy blood donors with gamma interferon enzyme-linked immunospot (Elispot) assays. RSV-specific responses were detected in every donor at levels varying between 0.05 and 0.3% of CD4 T cells. For all donors tested, a considerable component of the CD4 T-cell response was directed against the fusion (F) protein of RSV. We characterized a set of 31 immunodominant antigenic peptides targeted by CD4 T cells in the context of the most prevalent HLA class II molecules within the Caucasian population. Most antigenic peptides were HLA-DR restricted, whereas two dominant DQ peptides were also identified. The antigenic peptides identified were located across the entire sequence of the F protein. Several peptides were presented by more than one major histocompatibility complex class II molecule. Furthermore, most donors recognized several F peptides. Detailed knowledge about immunodominant antigenic peptides will facilitate the ability to monitor CD4 T-cell responses in patients and the measurement of correlates of protection in vaccinated subjects.

Virus-specific cytotoxic T lymphocytes (CTL) play a major role in the clearance of respiratory syncytial virus (RSV) infection. We have generated cytotoxic T-cell clones (TCC) from two infants who had just recovered from severe RSV infection. These TCC were functionally characterized and used to identify HLA class I (B57 and C12)-restricted CTL epitopes of RSV.

It has previously been shown that T cells specific for the triggering virus infiltrate the eye of patients with herpes simplex virus type 1 (HSV-1)‐induced acute retinal necrosis (ARN). The T cells were mainly directed against 0.67‐0.73 HSV-1 map region encoded antigens. The fine specificities of genetically different T cell clones (TCC), obtained from affected eyes of 3 patients with HSV-induced ARN and reactive toward this genomic region of HSV-1, were analyzed with recombinant HSV viruses and synthetic peptides. For 1 patient, the HSV-1 UL46 gene encoded tegument protein VP11/12 was identified as the target antigen. Two separate CD4 + T cell epitopes were defined in VP11/12. TCC from the other 2 patients recognized the HSV-1 UL47 gene encoded tegument protein VP13/14. Two separate CD4 + VP13/ 14 T cell epitopes were identified in these patients. Analysis of the data indicates that HSV1 VP11/12 and VP13/14 are major target antigens for T cells obtained from vitreous fluid samples of the HSV-induced ARN patients studied. Acute retinal necrosis (ARN) is a rare, rapidly progressing, and potentially blinding inflammatory eye disease initiated by an ocular infection with herpesviruses like herpes simplex virus (HSV) or varicella zoster virus [1]. Both the direct cytopathic effect of the virus and, in particular, the intraocular T cell response against the triggering virus have been suggested to play a role in the local inflammatory response of ARN [2‐4]. The percentage of activated T cells in ocular fluid samples of patients with ARN is higher than that in peripheral blood, suggesting that these T cells are actively involved in the local inflammatory response [5]. Studies in the experimental mouse model of ARN support the immunopathogenetic role of ocular infiltrating T cells [2, 3]. However, the antigen specificity of these T cells has not been defined. We have recently described

For many applications, peptides should be conjugated to carriers. An important example is the conjugation of peptides to proteins, carbohydrates, or lipids for immunological studies. Further examples are the preparation of peptide affinity media and the conjugation of peptides to suitable coating compounds on surfaces for enzyme-linked immunosorbent assay and plasma resonance. The most important consideration in designing conjugates is that the peptide part of the conjugate must retain its biological activity. This means that the site of activity of the peptide must not be involved in the conjugation reaction. However, the active part of the peptide is often not precisely known, which complicates the design of a proper conjugation strategy. In addition, if the peptide is a fragment of a larger biologically active protein, the small peptide will frequently not adopt the conformation (for instance, a loop structure) of the corresponding sequence in the native protein. This might also abolish desired biological properties, such as the possibility to induce functional antibodies recognizing the native protein. In that case, appropriate artificial conformational restrictions should be introduced into the peptide - if possible. From the numerous conjugation methods available, only a few are described in this chapter. One example concerns the application of the homobifunctional cross-linker glutaraldehyde. Heterobifunctional cross-linking is illustrated by coupling of thiol-containing peptides or carriers to sulphydryl-reactive carriers or peptides, respectively. A general and easy method of conjugating peptides to proteins is to make use of homobifunctional cross-linkers. An example of a homobifunctional cross-linker is glutaraldehyde. This bis-aldehyde reacts with amine groups at neutral or basic pH to yield enamines, which can be reduced optionally to amines with sodium cyanoborohydride. The peptide and the protein react selectively via their N-terminus and/or lysine side chains to give not only a very complex mixture of products—peptide-peptide, peptide-protein, and protein-protein conjugates—but also large constructs containing peptide and protein. Typically, several peptide molecules per protein molecule are coupled. In view of the complex reaction pathway, the batch-to-batch reproducibility of the conjugate is difficult to control. If the peptide contains a lysine in the active part, it is recommended to extend the peptide N- or C-terminally with some additional lysines during synthesis in order to provide more amine groups for conjugation.

Pneumococcal polysaccharide type 6B, 14, or 23F (35-70 kDa) was activated with cyanogen bromide and modified with cystamine. After reduction of the spacer, the thiol-containing (i.e. cysteamine-modified) polysaccharide obtained was added in a 5-10-fold molar excess to bromoacetylated tetanus toxoid to give thioether-linked polysaccharide-protein conjugates in a yield of 10-20%. This approach failed for preparing a type 19F polysaccharide-protein conjugate, possibly due to intramolecular elimination of cysteamine from the reduced 19F polysaccharide. When N,N'-bis(glycyl)cystamine was introduced as a spacer molecule, the elimination of the reduced spacer was suppressed, thus allowing preparation of a 19F polysaccharide-tetanus toxoid conjugate (15%).

Bordetella pertussis fimbriae bind to sulfated sugars such as heparin through the major subunit Fim2. The Fim2 subunit contains two regions, designated H1 and H2, which show sequence similarity with heparin binding regions of fibronectin, and the role of these regions in heparin binding was investigated with maltose binding protein (MBP)-Fim2 fusion proteins. Deletion derivatives of MBP-Fim2 showed that both regions are important for binding to heparin. The role of H2 in heparin binding was confirmed by site-directed mutagenesis in which basic amino acids were replaced by alanine. These studies revealed that Lys-186 and Lys-187 are important for heparin binding of MBP-Fim2, whereas Arg-179 is not required. Peptides derived from H1 and H2 (pepH1 and pepH2) also showed heparin binding activity. Using a series of peptides, in each of which a different basic amino acid was substituted for alanine, we demonstrated that the structural requirements for heparin binding differ significantly among pepH1 and pepH2 peptides. A Pepscan analysis of Fim2 revealed regions outside H1 and H2 which bind heparin and showed that not only basic amino acids but also tyrosines may be important for binding to sulfated sugars. A comparison of the heparin binding regions of Fim2 with homologous regions of Fim3 and FimX, two closely related but antigenically distinct fimbrial subunits, showed that basic amino acids and tyrosines are generally conserved. The major heparin binding regions identified in Fim2 are part of epitopes recognized by human antibodies, suggesting that the heparin binding regions are exposed at the fimbrial surface and are immunodominant. Since B. pertussis fimbriae show weak serological cross-reactivity, the differences in primary structure in the heparin binding regions of Fim2, Fim3, and FimX may affect antibody binding but not heparin binding, allowing the bacteria to evade antibody-mediated immunity by switching the fimbrial gene expressed.

Synthetic peptides derived from the predicted loops 1 and 4 of meningococcal PorA, sero-subtype P1.7,16, were used to study the epitope specificity of murine and human PorA P1.7,16 bactericidal antibodies. The predicted loops 1 and 4 are surface exposed and carry in their apices the sero-subtype epitopes P1.7 (loop 1) or P1.16 (loop 4), respectively. Peptides were synthesized as mono- and multimeric peptides. Murine monoclonal and polyclonal antibodies were induced with meningococcal whole cell preparations. Polyclonal antibodies were evoked in volunteers after one immunization with 50 micrograms or 100 micrograms protein of a hexavalent meningococcal PorA vesicle vaccine. The induction of PorA antibodies was determined in ELISA using purified PorA P1.7,16. The epitope specificity of anti-PorA antibodies for both murine and human antibodies could be demonstrated by direct peptide ELISA using overlapping multimeric peptides almost spanning the entire loops 1 or 4 of the protein. The capacity of peptides to inhibit the bactericidal activity of murine and human antibodies was investigated using meningococcal strain H44/76 (B:15:P1.7,16) as a target strain. Bactericidal activities could be inhibited with both monomeric and multimeric peptides derived from epitopes P1.7 and P1.16.

The FrpB protein from pathogenic neisseriae is a 77 kDa iron-regulated outer-membrane protein that belongs to the family of TonB-dependent receptors and may have potential as a vaccine component. Comparison between the frpB gene from three different meningococcal strains and a published gonococcal one revealed that the region from residues 350 to 390 displays pronounced sequence variability. In a model for the topology of FrpB in the outer membrane, this region corresponds to loop 7, the longest of the predicted 13 surface-exposed loops. Binding of four out of a total of eight bactericidal monoclonal antibodies to synthetic peptides corresponding to loop 7 showed that their epitopes are located here. The frpB genes from five additional meningococcal strains were cloned and sequenced in this region. Pairwise comparisons showed different degrees of similarity.

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.

The transmembrane fusion (F) glycoprotein of measles virus is an important target antigen of human HLA class I- and class II-restricted cytotoxic T lymphocytes (CTL). Genetically engineered F proteins and nested sets of synthetic peptides spanning the F protein were used to determine sequences of F recognized by a number of F-specific CTL clones. Combined N- and C-terminal deletions of the respective peptides revealed that human HLA class I and HLA class II-restricted CTL efficiently recognize nonapeptides or decapeptides representing epitopes of F. Three distinct sequences recognized by three different HLA class II (DQw1, DR2, and DR4/w53)-restricted CTL clones appear to cluster between amino acids 379 and 466 of F, thus defining an important T-cell epitope area of F. Within this same region, a nonamer peptide of F was found to be recognized by an HLA-B27-restricted CTL clone, as expected on the basis of the structural homology between this peptide and other known HLA-B27 binding peptides.

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.

In Escherichia coli, membrane-spanning amphipathic beta-sheet structures are characteristic of many outer membrane proteins. By applying the principles that have been recognized for them to the four classes of neisserial porins, we have constructed a model for the topology of the porins within the outer membrane. This model predicts eight surface-exposed loops, both in the meningococcal class 1 and 2 proteins and in the gonococcal PIA and PIB proteins. The transmembrane sequences are highly conserved among these porins and are able to form an amphipathic beta-sheet structure. The surface-exposed hydrophilic loops show extensive variation in both length and sequence. Experimental evidence in support of this model has been obtained by using antisera against synthetic peptides which correspond to surface-exposed loops in class 1 and 2 proteins. Thus, binding to the cell surface was observed with antibodies against loops 1, 4, and 5 of class 1 and loops 1 and 5 of class 2. In class 1, these loops are the longest ones and show the highest sequence diversity among strains of different subtypes. Mapping of epitopes recognized by monoclonal antibodies with bactericidal activity has also provided strong support for the model. The epitopes are located in loops 1 and 4 of class 1 protein, loop 5 of PIB, and loop 6 of PIA. A nonbactericidal antibody that binds only weakly to whole cells was shown to recognize loop 3 of PIB. These results suggest that the longest loops are immunodominant, provide the binding sites for bactericidal antibodies, and display the greatest variation among different strains.

The reagent pentafluorophenyl S-acetylmercaptoacetate was used to modify the N-terminus of resin-bound side-chain-protected peptides. The modification was carried out in an automated cycle in the final stage of fluorenylmethoxycarbonyl (Fmoc)/polyamide-mediated solid-phase synthesis. Side-chain deprotection and cleavage from the resin with aqueous trifluoroacetic acid gave the N-(S-acetylmercaptoacetyl) peptides. The S-acetylmercaptoacetyl peptides were transformed into reactive thiol-containing peptides by incubation with hydroxylamine at neutral pH. The S-deacetylation was performed in the presence of a sulfhydryl-reactive compound (or intramolecular group) to enable immediate capture of the sensitive thiol. Three applications were investigated. An S-acetylmercaptoacetyl peptide, containing a sequence of a meningococcal membrane protein, was incubated with hydroxylamine in the presence of 5-(iodoacetamido)fluorescein to give the corresponding fluorescein-labeled peptide in 62% yield. The same peptide was also S-deacetylated in the presence of bromoacetylated poly- l -lysine to afford a peptide/polylysine conjugate. Finally, a peptide corresponding to a sequence of herpes simplex virus glycoprotein D was prepared. This peptide, containing an N-terminal S-acetylmercaptoacetyl group and an additional C-terminal S-(3-nitro-2-pyridinesulfenyl)cysteine residue, was converted into a cyclic disulfide peptide (20%).

The cesium salts of N-[(9-fluorenylmethoxy)carbonyl]-L-serine (Fmoc-Ser, 1a)*** and -L-threonine (Fmoc-Thr, 1b) were treated with 2-(bromomethyl)-9,10-anthraquinone (Maq-Br), yielding Fmoc-Ser-OMaq (2a, 79%) and Fmoc-Thr-OMaq (2b, 74%). HgBr2-catalyzed glycosylation with 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide gave Fmoc-Ser(Ac4βGal)-OMaq (3a, 89%) and Fmoc-Thr(Ac4βGal)-OMaq 13b, 53%). Fmoc-Ser(Ac4βGal)-OH (4), obtained by reaction of 1a with Na2S2O4 (88%), was condensed with H-Ala-OMaq using the DCC/HOBt method (83%). The Fmoc-Ser(Ac4βGal)-Ala-OMaq(5), thus obtained, was Nα-deprotected with morpholine and coupled with Fmoc-Ala using water soluble carbodiimide in the presence of HOBt to yield Fmoc-Ala-Ser(Ac4βGal)-Ala-OMaq (6, 59%). The fully deprotected glycotripeptide (8) was obtained from 6 by successive treatment with Na2S2O4, morpholine and hydrazine hydrate (81%). The compounds 3a, 6 and 8 were characterized using correlated 300 MHz 1H NMR spectroscopy (2D-COSY).

1,2-O-Carbonyl-4,6-O-ethylidene- (4), 4,6-O-benzylidene-1,2-O-carbonyl- (6) and 1,2-O-carbonyl-4,6-O-(p-methoxybenzylidene)-α-D-galactopyranose (7) were prepared from the corresponding 4,6-O-alkylidene-D-galactopyranoses (1, 2 and 3) by reaction with 1,1′-carbonyldiimidazole and the isomerization of 4, 6 and 7 into the 3,4-acetals 8, 10 and 11 (endo/exo mixtures) by various (Lewis) acids studied.