Functional activity of antibodies against the recombinant OpaJ protein from Neisseria meningitidis.

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.

Role of the polymorphic region 1 of the Bordetella pertussis protein pertactin in immunity.

In several countries pertussis is re-emerging, despite a high vaccination coverage. It is suggested that antigenic divergence between Bordetella pertussis vaccine strains and circulating strains, in particular with respect to pertactin, has contributed to pertussis re-emergence. Polymorphism in pertactin is essentially limited to region 1, which is composed of repeats and is located adjacent to an Arg-Gly-Asp motif implicated in adherence. Evidence is provided for the immunological relevance of polymorphism in region 1. Region 1 was found to contain a B-cell epitope recognized in both humans and mice. Furthermore, variation in region 1 affected antibody binding and, in a mouse respiratory infection model, the efficacy of a whole-cell vaccine. Moreover, passive and active immunization indicated that region 1 confers protective immunity. An mAb directed against a linear conserved epitope conferred cross-immunity against isolates with distinct pertactin variants. The results indicate an important role of region 1 of pertactin in immunity.

In vitro processing and presentation of a lipidated cytotoxic T-cell epitope derived from measles virus fusion protein.

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.

Relevance of the C-terminal Arg-Phe sequence in gamma(2)-melanocyte-stimulating hormone (gamma(2)-MSH) for inducing cardiovascular effects in conscious rats.

1. The cardiovascular effects by gamma(2)-melanocyte-stimulating hormone (gamma(2)-MSH) are probably not due to any of the well-known melanocortin subtype receptors. We hypothesize that the receptor for Phe-Met-Arg-Phe-amide (FMRFa) or Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide (neuropeptide FF; NPFFa), other Arg-Phe containing peptides, is the candidate receptor. Therefore, we studied various Arg-Phe containing peptides to compare their haemodynamic profile with that of gamma(2)-MSH(6 - 12), the most potent fragment of gamma(2)-MSH. 2. Mean arterial pressure (MAP) and heart rate (HR) changes were measured in conscious rats after intravenous administration of gamma(2)-MSH related peptides. 3. Phe-Arg-Trp-Asp-Arg-Phe-Gly (gamma(2)-MSH(6 - 12)), FMRFa, NPFFa, Met-enkephalin-Arg-Phe-amide (MERFa), Arg-Phe-amide (RFa), acetyl-Phe-norLeu-Arg-Phe-amide (acFnLRFa) and desamino-Tyr-Phe-norLeu-Arg-Phe-amide (daYFnLRFa) caused a dose-dependent increase in MAP and HR. gamma(2)-MSH(6 - 12) showed the most potent cardiovascular effects (ED(50)=12 nmol kg(-1) for delta MAP; 7 nmol kg(-1) for delta HR), as compared to the other Arg-Phe containing peptides (ED(50)=177 - 292 nmol kg(-1) for delta MAP; 130 - 260 nmol kg(-1) for delta HR). 4. Peptides, which lack the C-terminal Arg-Phe sequence (Lys-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Pro-Gly (gamma(2)-pro(11)-MSH), desamino-Tyr-Phe-norLeu-Arg-[L-1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid]-amide (daYFnLR[TIC]a) and Met-enkephalin (ME)), were devoid of cardiovascular actions. 5. The results indicate that the baroreceptor reflex-mediated reduction of tonic sympathetic activity due to pressor effects is inhibited by gamma(2)-MSH(6 - 12) and that its cardiovascular effects are dependent on the presence of a C-terminal Arg-Phe sequence. 6. It is suggested that the FMRFa/NPFFa receptor is the likely candidate receptor, involved in these cardiovascular effects.

HLA class I-restricted cytotoxic T-cell epitopes of the respiratory syncytial virus fusion protein.

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.

Intraocular T cells of patients with herpes simplex virus (HSV)-induced acute retinal necrosis recognize HSV tegument proteins VP11/12 and VP13/14.

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 HSV-1 VP11/12 and VP13/14 are major target antigens for T cells obtained from vitreous fluid samples of the HSV-induced ARN patients studied.

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.

Detection of T helper responses, but not of human papillomavirus-specific cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma.

Human papillomavirus type 16 (HPV16)-encoded E7 oncoprotein is constitutively expressed in cervical carcinoma cells and is required for cellular transformation to be maintained. The E7 protein, therefore, forms an attractive target for T-cell-mediated immune intervention to prevent or treat HPV16+ tumors. The authors performed a peptide-based phase I/II vaccination trial to induce anti-tumor immune responses in patients with recurrent or residual cervical carcinoma. Fifteen HLA-A*0201+ patients with HPV16+ cervical carcinoma received vaccinations with synthetic peptides representing 2 HPV16 E7-encoded, HLA-A*0201-restricted cytotoxic T lymphocyte epitopes and a pan-HLA-DR-binding T-helper epitope, PADRE, in adjuvant. No signs of toxicity were observed. Two patients had stable disease for more than 1 year after vaccination, 3 patients died of the disease during or shortly after the vaccination period, and 10 patients maintained progressive cervical carcinoma. Specific immune responses directed against the vaccine components were analyzed in peripheral blood samples. No cytotoxic T lymphocyte responses against the HPV16 E7 peptides were detectable. After vaccination, strong PADRE helper peptide-specific proliferation was detected in 4 of 12 patients. In conclusion, peptide vaccination with 2 HPV16 E7 cytotoxic T lymphocyte epitopes and a universal T helper epitope is well tolerated by patients with advanced cervical carcinoma. Despite a reduction of in vitro cytolytic or proliferative recall responses to some, but not all, conventional antigens in this patient group, peptide-specific proliferative responses were induced in 4 patients. Based on the current study, it is now feasible to perform peptide vaccination in earlier stages of HPV16-induced cervical disease.

Solid-phase synthesis and application of double-fluorescent-labeled lipopeptides, containing a CTL-epitope from the measles fusion protein.

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.

Immunogenicity of Streptococcus pneumoniae type 6B and 14 polysaccharide-tetanus toxoid conjugates and the effect of uncoupled polysaccharide on the antigen-specific immune response.

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.

Application of cystamine and N,N'-Bis(glycyl)cystamine as linkers in polysaccharide-protein 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%).

Identification and characterization of heparin binding regions of the Fim2 subunit of Bordetella pertussis.

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.

Thermodynamic analysis of the interaction between a bactericidal antibody and a PorA epitope of Neisseria meningitidis.

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.

Bactericidal antibody recognition of a PorA epitope of Neisseria meningitidis: crystal structure of a Fab fragment in complex with a fluorescein-conjugated peptide.

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.

Epitope specificity of murine and human bactericidal antibodies against PorA P1.7,16 induced with experimental meningococcal group B vaccines.

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.

Sequence variability of FrpB, a major iron-regulated outer-membrane protein in the pathogenic neisseriae.

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.

Distribution of Lys-gamma 2-melanocyte-stimulating hormone- (Lys-gamma 2-MSH)-like immunoreactivity in neuronal elements in the brain and peripheral tissues of the rat.

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.

T-cell responses to outer membrane proteins of Neisseria meningitidis: comparative study of the Opa, Opc, and PorA proteins.

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.

Polysaccharicb-conjugate vaccines.

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.

Cardiovascular effects of gamma-MSH/ACTH-like peptides: structure-activity relationship.

Intravenous administration of gamma2-melanocyte-stimulating hormone (gamma2-MSH) to conscious rats causes a dose-dependent increase in blood pressure and heart rate, while the structurally related peptide adrenocorticotropic hormone-(4-10) (ACTH-(4-10)) is 5-10 times less potent in this respect. This prompted us to investigate which amino acid sequence is determinant for the cardiovascular selectivity of peptides of the gamma-MSH family. Lys-gamma2-MSH, most likely the endogenously occurring gamma-MSH analog, was as potent as gamma2-MSH in inducing increases in blood pressure and heart rate. Removal of C-terminal amino acids resulted in gamma-MSH-fragments which were devoid of cardiovascular activities. Removal of amino acids from the N-terminal side of gamma2-MSH resulted in fragments which were less potent, but had an intrinsic activity not different from that of gamma-MSH. Surprisingly, gamma-MSH-(6-12) was more potent than gamma2-MSH. The shortest fragment which displayed pressor and tachycardiac responses was the MSH 'core', His-Phe-Arg-Trp (= gamma-MSH-(5-8)), which is identical to ACTH-(6-9). This was corroborated by testing fragments of ACTH-(4-10). We conclude that the message essential for cardiovascular effects resides in the gamma-MSH-(5-8)/ACTH-(6-9) sequence. Proper C-terminal elongation is required for full expression of cardiovascular activity of gamma2-MSH, as the sequence of Asp9-Arg10-Phe11 appears to play an important role in establishing intrinsic activity. The amino acids N-terminal to the MSH 'core' sequence appear to be essential for the potency of the peptides.