Precise location of sequential dengue virus subcomplex and complex B cell epitopes on the nonstructural-1 glycoprotein

The reactions of a panel of 34 mouse monoclonal antibodies (MAbs) specific for the dengue-2 virus nonstructural-1 glycoprotein (NS1), were analysed using 174 overlapping synthetic nonameric peptides covering the entire sequence. Using this methodology, four epitopes were identified. One pair of MAbs, which defined a dengue-2/4 virus subcomplex epitope (24C: amino acids 299-309) using native NS1 proteins, showed the same reaction pattern with synthetic peptides containing the corresponding NS1 sequences of each virus serotype. One amino acid substitution, present in the sequences from the dengue-1/3 virus subcomplex abrogated almost all reaction by these MAbs. A dengue complex epitope (LX1: amino acids 111-121) was also located and peptides containing the sequences of each serotype were shown to contain only antigenically silent amino-acid substitutions. In contrast, MAbs which defined a dengue type-specific epitope (LD2: amino acids 25-33) and another dengue subcomplex epitope (24A: amino acids 61-69) failed to show the same reaction profiles using peptides of each serotype, suggesting that these determinants were partially dependent upon conformation. The LX1 epitope is a good candidate for further trials aimed at generating cross-protective immune responses to these viruses without the risk of antibody-dependent enhancement.     

Cross-priming of CTL responses in vivo does not require antigenic peptides in the endoplasmic reticulum of immunizing cells

It has been proposed that the cross-priming of CTL responses in vivo involves the transfer to host APCs of heat shock protein glycoprotein 96-chaperoned antigenic peptides released from the endoplasmic reticulum (ER) of dying or infected cells. We have tested this possibility directly using TAP-deficient cell lines lacking antigenic ER peptides derived from two model Ags, the human adenovirus type 5 early regions E1A and E1B. Although both proteins were well expressed, the cells were not recognized by E1A- or E1B-specific CTLs unless the relevant epitope was either provided exogenously as a synthetic peptide or targeted to the ER in a TAP-independent fashion. Despite the absence of these ER peptides, the TAP1-/- cells were able to efficiently cross-prime E1A- and E1B-specific CTLs following immunization of syngeneic mice. These results indicate that, although purified peptide/glycoprotein 96 complexes are potent immunogens, the mechanism of CTL cross-priming in vivo does not depend upon antigenic peptides in the ER of immunizing cells.     

Cell binding sequences in mouse laminin alpha1 chain

Laminin-1, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha1, beta1, and gamma1 chains. Previously, we used synthetic peptides to screen for biologically active sequences in the laminin alpha1 chain C-terminal globular domain (G domain) and identified several cell binding sequences (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Song, S. Y., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). Here, we identify new cell binding sequences on the remainder of the laminin alpha1 chain by systematic peptide screening, using 208 overlapping synthetic peptides encompassing the central and N-terminal portions of the alpha1 chain. HT-1080 cell attachment activity to the peptides was evaluated using peptide-coated plastic substrates and peptide-conjugated Sepharose beads. Twenty five peptides showed cell attachment activities on either the peptide-coated plastic substrates and/or the peptide-conjugated Sepharose beads. A-13 (RQVFQVAYIIIKA) showed strongest cell attachment activity in both the assays. Cell attachment to 14 of the peptides was inhibited by heparin. EDTA and integrin antibodies inhibited cell adhesion to two of the peptides, A-13 and A-25, suggesting that these sites likely bind to integrins. These peptides inhibited cell attachment to laminin-1 but not to collagen I, suggesting these active sites are available on the intact molecule. Most of active sequences were localized on globular domains suggesting that these structures play a critical role in binding to cell-surface receptors.     

Olive pollen allergy: searching for immunodominant T-cell epitopes on the Ole e 1 molecule

BACKGROUND: The amino-acid and nucleotide sequence of Ole e 1 (the major antigen of olive pollen) has been described and the IgE antibody response to this major allergen was associated with DR7/DQ2 antigens. With this previous data we try to define the T-cell epitopes implicated in Ole e 1 reactivity. OBJECTIVES: To study the recognition of T cells (derived from allergic and non-allergic Ole e 1 patients) to Ole e 1 synthetic peptides in order to define immunodominant T-cell epitopes. METHODS: We have compared the proliferative response of the peripheral blood mononuclear cells from Ole e 1 sensitized patients vs. non-sensitized controls, induced by 14 Ole e 1 synthetic peptides. Thirty subjects were classified in two groups: group 1 (non-responders against Ole e 1, n=16) and group 2 (Ole e 1 responders, n=14), according to their clinical parameters and the presence or not in their sera of the significant Ole e 1 IgE antibody levels. RESULTS: Our results shown that it is possible to find T cells reactive to Ole e 1 peptides in patients with and without significant levels of Ole e 1 IgE antibodies. However, the percentage of response was higher in patients with IgE antibodies 71.4% vs 25%), and the recognition profile was different: the control group showed a broad reactivity pattern, in contrast, the response by the 'Ole e 1 responders' group was mainly directed against three peptides of the carboxi-terminal region, peptides 10 (91-102), 12 (109-120) and 13 (119-130), with a response frequency of 35.7, 28.5 and 28.5%, respectively. By direct and inhibition test no antibody response was found against the synthetic peptides. CONCLUSIONS: Our data suggest that the regions between 91 and 102 and 109-130 aminoacids on the Ole e 1 molecule are immunodominant T-cell epitopes. These epitopes are not recognized by IgE antibodies.     

Accelerated (malignant) hypertension: a study of 121 cases between 1974 and 1996

DNA-based immunization is one of the most promising strategies to induce protective immunity against a variety of pathogens, presenting clear advantages as compared to the use of recombinant antigens. One of these advantages might be the ability to induce antibodies directed primarily against conformational determinants, as compared to immunization with recombinant proteins. To test this possibility, we have analyzed the antibody responses induced in mice by immunization with either recombinant soluble CD4 (rCD4) or by immunization with plasmid DNA-encoding CD4 (CD4-DNA). Mice immunized with CD4-DNA had lower titers of antibodies able to recognize rCD4 than mice immunized with rCD4. However, immunization with CD4-DNA induced antibodies reactive with the native cell surface CD4 molecule in all mice, whereas only two out of five mice immunized with rCD4 produced antibodies reactive with cell surface CD4, thus demonstrating that the genetic immunization approach may lead to an antibody response more consistent and superior at a qualitative level as compared to immunization with the corresponding recombinant protein. In addition, differences in the kinetics of appearance of antibodies directed against the native CD4 molecule were observed between mice immunized with CD4-DNA or rCD4. In the first case, antibodies reacting with cell surface CD4 were present 28 days after the first immunization, whereas mice immunized with rCD4 produced antibodies directed against the native molecule only following a booster injection. Finally, the two groups of mice produced antibodies with a different isotype distribution. No clear predominance of a specific IgG subclass was detected in the antibody population produced in response to DNA immunization. Conversely, mice immunized with rCD4 produced predominantly antibodies of the IgG1 isotype, indicating generation of a TH2 response. Together, results from this study indicate that the CD4 molecule endogenously produced following DNA immunization is expressed, at least partially, in a native conformation. This feature confers a major advantage to the DNA immunization approach as compared to immunization with the corresponding recombinant protein, which seems to elicit antibodies predominantly directed to epitopes uniquely expressed on the recombinant molecule.     

Biochemical and immunocytochemical characterization of antipeptide antibodies to a cloned GluR1 glutamate receptor subunit: cellular and subcellular distribution in the rat forebrain

Antibodies were made to synthetic peptides corresponding to residues 253-367, 757-771 and 877-889 of the published amino acid sequence of the rat brain glutamate receptor GluR1 subunit [Hollmann et al. (1989) Nature 342, 643-648]. The peptides were synthesized both as multiple copies on a branching lysyl matrix (multiple antigenic peptides) and conventional linear peptides using solid-phase synthesis. Rabbits were immunized with these peptides either without conjugation (multiple antigenic peptides) or following coupling to ovalbumin with glutaraldehyde (monomeric peptides). The antibodies from immune sera were then purified by affinity chromatography using reactigel coupled monomeric peptides. All the rabbits produced good antipeptide responses, and were characterized by immunoprecipitation of solubilized alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and kainate binding activity and by their staining patterns on immunoblots. Antibody to peptide 253-267 specifically immunoprecipitated 12 +/- 3, 50 +/- 3 and 44 +/- 4% of solubilized alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding activity from cortex, hippocampus and cerebellum, respectively. Under identical conditions, antibody against the 877-889 peptide removed 23 +/- 4, 9 +/- 4 and 15 +/- 9% of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding sites from these areas. On immunoblots of rat brain membrane samples separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, antibodies labelled a 105,000 mol. wt immunoreactive band. GluR1 was immunoaffinity-purified using subunit-specific antibodies against both N-terminal (253-267) and C-terminal (877-889) residues, covalently attached to protein A-agarose. Analysis of the purified product from each column showed a major immunoreactive band, recognized by both sera at 105,000 mol. wt and silver staining identified the same major protein. After exhaustive immunoprecipitation of solubilized membrane samples with antibody against the C-terminal of the subunit, a subpopulation of GluR1 was labelled with antibodies specific for the N-terminal part of the receptor. These observations suggest that the GluR1 subunit consists of at least two isoforms possessing a common N-terminal region but a distinct C-terminus. Immunocytochemistry, using immunoperoxidase staining, was performed for the GluR1 subunit in rat forebrain with antisera raised against the N-terminal (253-267) and the C-terminal parts (877-889) of the molecule. Both antisera gave a similar distribution of immunoreactivity at the light-microscopic level. Immunoreactivity for the GluR1 subunit was selectively distributed throughout the rat forebrain. The hippocampus, septum, amygdala and olfactory bulb exhibited the strongest immunoreactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of GM-CSF antagonist peptides

Granulocyte/macrophage colony stimulating factor (GM-CSF) is both a hematopoietic growth factor and a cytokine implicated in inflammatory disease. The development of GM-CSF antagonist peptides corresponding to the GM-CSF native sequence should allow their modification into higher affinity analogs, but this is hampered by the low affinity of linear peptides. To adequately evaluate such low affinity peptides, the use of several independent assays should allow specific versus nonspecific inhibitors to be distinguished. In this study, inhibition of GM-CSF-dependent cell growth, inhibition of GM-CSF binding and immunologic cross-reactivity between GM-CSF-derived peptides and native protein by neutralizing antibodies have been used to evaluate peptide analogs with potential bioactivity. The GM-CSF sequence was divided into 6 peptides ranging in size from 15-24 amino acids. Antisera were raised to these peptides in mice and assayed for immunologic cross-reactivity. 4/6 anti-peptide antisera bound GM-CSF on ELISA and 3/6 on immunoprecipitation. Antisera to two of the peptides (corresponding to residues 17-31 and 96-112) inhibited GM-CSF-dependent cellular proliferation in two cell lines, with one peptide derived from residues 17-31 demonstrating inhibition of GM-CSF binding and direct biological inhibitory activity. A peptide that did not elicit native GM-CSF reactive antibodies, corresponding to residues 54-78, was recognized by two neutralizing monoclonal antibodies. It exhibited inhibition of GM-CSF binding and direct biological antagonist activity. These studies implicate two sites in mediating GM-CSF biological activity, and indicate that biological antagonists can be developed based on these sites.     

Localization of a domain in the FimH adhesin of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection

The FimH subunit of type 1-fimbriated Escherichia coli has been implicated as an important determinant of bacterial adherence and colonization of the urinary tract. Here, we sought to localize the functionally important domain(s) within the FimH molecule and to determine if antibodies against this domain would block adherence of type 1-fimbriated E. coli to the bladder mucosa in situ and in vivo in an established mouse model of cystitis. We generated translational fusion proteins of disparate regions of the FimH molecule with an affinity tag MalE, and tested each of the fusion products in vitro for functional activity. The minimum region responsible for binding mouse bladder epithelial cells and a soluble mannoprotein, horseradish peroxidase, was contained within residues 1-100 of the FimH molecule. We validated and extended these findings by demonstrating that antibodies directed at the putative binding region of FimH or at synthetic peptides corresponding to epitopes within the binding domain could specifically block type 1 fimbriae-mediated bacterial adherence to bladder epithelial cells in situ and yeast cells in vitro. Next, we compared the ability of mice passively immunized intraperitoneally with antisera raised against residues 1-25 and 253-264 of FimH or 1-13 of FimA to resist bladder colonization in vivo after intravesicular challenge with type 1-fimbriated E. coli. Only the antibody directed at the putative binding region of FimH (anti- s-FimH1-25) significantly reduced E. coli bladder infections in the experimental mouse model of urinary tract infections. Similar results were obtained when the mice were actively immunized with synthetic peptides corresponding to residues 1-25 and 253-264 of FimH or 1-13 of FimA. The mechanism of protection was attributed, at least in part, to inhibition of bacterial adherence to the bladder surface by s-FimH1-25-specific antibody molecules that had filtered through the kidneys into the urine. The level of FimH antibodies entering the bladder from the circulatory system of the immunized mice was found to be markedly enhanced upon bacterial challenge. The potential broad spectrum activity of the protective FimH antibody was indicated from its serologic cross-reactivity with various urinary tract bacterial isolates bearing type 1 fimbriae. These findings could be relevant in the design of an efficacious and broadly reactive FimH vaccine against urinary tract infections.     

Primary diffuse large B-cell lymphoma of the mediastinum: outcome following high-dose chemotherapy and autologous hematopoietic cell transplantation

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.     

Determination of antibody affinity using threshold values of serum activity in the passive hemagglutination test

Agglutination of erythrocytes of the antigenic diagnostic agents with the IgG antibodies is attributed to the fact that the reactive centres of the same molecule of the antibody attached to the antigenic determinants located on different erythrocytes. A possibility of agglutination of erythrocytes under conditions of the maximal dilution of the immune serum depended on the number of molecules located on a single erythrocyte. When the erythrocytes are sensitized with the optimal dose of the antigen those serum antibodies are involved in the passive hemagglutination test (PHAT) which are characterized by the mean integrated association constant. When the antibodies participate in the PHAT with several batches of the erythrocytic diagnostic agent characterized by an average number of the antigen molecules on a single erythrocyte there appears a possibility of determining the extent of heterogeneity of the antibodies. Not only the amount of antibodies, but also their affinity can be ascertained by the PHAT, and also the heterogeneity of antibodies can be evaluated.     

The NH2-terminal region of the sickle hemoglobin beta chain. II. Characterization of monospecific antibodies

We have previously shown that antibodies specific for hemoglobin S could be fractionated by absorption of an antiserum to hemoglobin S to Sepharose containing a synthetic oligopeptide. betaS (1-13), corresponding to the first 13 amino acid residues of the beta chain of hemoglobin S. We report here that this antibody population, anti-betaS (1-13), shows considerable restriction of heterogeneity in isoelectric focusing studies and monospecificity on velocity ultracentrifugation in the presence of hemoglobin S. The binding of various hemoglobin species to anti-betaS (1-13) was studied using a double antibody radioimmunoassay with [14C]carbamoylated hemoglobin S. Carbonmonoxy-, oxy-, met-, and cyanmethemoglobin S reacted equally with the antibody, but deoxyhemoglobin (with or without organic phosphates) reacted differently. Hemoglobin A and several hemoglobin mutants with alterations in the NH2-terminal region of the beta chain did not displace labeled hemoglobin S from anti-betaS (1-13). BETAS chains reacted with the antibody, but less well than hemoglobin S, while betaA and alpha chains, and globins did not react with the antibody. The synthetic peptide, betaS (1-13), used for fractionation, reacted with the antibody about 300-fold less efficiently than hemoglobin S. BetaS (3-13) was even less reactive, while smaller peptides which included the valine residue at position 6 displaced little of the tracer [14C]carbamoylated hemoglobin S at concentrations as high as 10(-2) M. We interpret these results to indicate that this method of immunoabsorption has produced a monospecific subfraction of antibodies which is specific for the NH2-terminal region of the beta chain of hemoglobin S in its native conformation.     

Residues near the amino and carboxyl termini of staphylococcal enterotoxin E independently mediate TCR V beta-specific interactions

Previous studies identified three COOH-terminal residues in staphylococcal enterotoxin E (SEE; Asp200, Pro206, and Asp207) that in part mediate TCR V beta recognition. We have identified an additional three residues near the NH2-terminus of SEE (Arg20, Asn21, and Ser24 that are needed in conjunction with these COOH-terminal residues to fully restore native levels of V beta-specific T cell proliferation. A staphylococcal enterotoxin A SEA-SEE hybrid molecule containing the NH2-terminal V beta determinants of SEE to activate alone exhibited V beta specificities of both SEA and SEE, indicating that these residues of SEE independently contribute to V beta recognition and do not obscure the native V beta determinants of SEA. These findings suggest that the ability of SEE to activate certain V beta-specific T cell subsets may result from multiple interactions with a single TCR beta-chain or perhaps by cross-linking two TCR. High affinity binding to HLA-DR1, a property of native SEA, was not altered in the SEA-SEE hybrid enterotoxins containing amino acid substitutions in regions 20 to 24 and 200 to 207, indicating that residues comprising the V beta determinants of SEE are separate from residues that contribute to HLA-DR1 binding affinity. Computer models of the predicted structure of SEE revealed that the V beta determinants of SEE are located on two adjacent solvent-exposed loops. Thus, the residues of SEE that mediate V beta recognition may coalesce to form a TCR binding site with specificities for multiple TCR beta-chains.     

Dissecting the structure of a partially folded protein. Circular dichroism and nuclear magnetic resonance studies of peptides from ubiquitin

The nature and interaction of structural elements in a partially ordered form of ubiquitin, the A-state, which is populated at low pH in 40 to 60% aqueous methanol, have been investigated. Two synthetic peptides have been studied under the same conditions: U(1-21), corresponding to the N-terminal beta-hairpin in the native (N) and A-states of ubiquitin and U(1-35), which includes this hairpin plus an alpha-helix. Circular dichroism studies indicate that, although these peptides are largely unfolded in water, their structural content in 30 and 60% methanol is comparable with the corresponding native secondary structure. Sequence-specific assignments of the 1H n.m.r. spectra of U(1-35) in aqueous methanol and subsequent secondary structure determination confirm the conservation in detail of native-like secondary structure. Corresponding resonances in spectra of U(1-35), U(1-21) and the A-state itself were found to have closely similar chemical shifts, suggesting that the beta-hairpin exists independently in the partially folded protein, with little or no influence from the rest of the molecule. This is confirmed by the virtual absence in nuclear Overhauser enhancement spectroscopy and rotating frame nuclear Overhauser enhancement spectroscopy spectra of nuclear Overhauser enhancement effects between structural elements. c.d. and n.m.r. evidence suggests that structure in the C-terminal half of the molecule in the A-state is largely non-native. Thus, although methanol is necessary to assure its stability in the absence of wider native interactions, the structure of the beta-hairpin, including the register of its hydrogen bonding, appears to be determined entirely by its own sequence. This intrinsic structural preference in the first part of the ubiquitin sequence is much stronger than in the C-terminal half, a conclusion reflected in the results from a variety of secondary structure prediction algorithms.     

Generation of neutralizing antipeptide antibodies to the enzymatic domain of Pseudomonas aeruginosa exotoxin A

Burn patients suffer a break in the physical barrier (skin), which, when combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly with Pseudomonas aeruginosa. Infection of the burn wound has always been a major factor in retardation of wound healing, and sepsis remains the leading cause of death in burn patients. Because studies have shown that topical treatment with antiexotoxin A (ETA) antibodies significantly increases survival in rats infected with toxin-producing strains of P. aeruginosa, we examined 11 synthetic peptides encompassing 12 to 45 amino acid (aa) residues, representing what were predicted by computer analysis to be the most hydrophilic and antigenic regions of ETA. These synthetic peptides were injected into rabbits for antibody production. Different groups of rabbits were immunized with a combination of peptides, with each combination representing one of the three distinct domains of ETA. Animals immunized with various peptide combinations produced peptide-specific antibodies that exhibited cross-reactivity to ETA. Two major epitopes were identified on the ETA molecule by experiments with peptide-specific antibodies in enzyme-linked immunosorbent assay and immunoprecipitation. One of these epitopes was located in the translocation domain (II) (aa 297 to 310), while the other was mapped to the last 13 aa residues at the carboxy-terminal end of the enzymatic domain (III) (aa 626 to 638). Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that abrogated the cytotoxic activity of ETA in vitro. Antibodies to this epitope blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enzymatic active site of the ETA molecule. We conclude that polyclonal, as well as monoclonal, antibodies to short peptides, representing small regions of ETA, may have therapeutic potential in passive immunization or topical treatment of burn patients infected with toxin-producing strains of P. aeruginosa.     

Outcome in general medical patients presenting with common symptoms: a prospective study with a 2-week and a 3-month follow-up

Human T-cell-mediated autoimmune diseases are often genetically linked to particular alleles of HLA class II genes. Vogt-Koyanagi-Harada's (VKH) disease, which is regarded as an autoimmune disorder in multiple organs containing melanocytes, has been found to be associated with HLA-DR4 (DRB1(*)0405) and HLA-DR53 (DRB4(*)0101). Tyrosinase is a melanoma antigen (Ag) expressed by normal melanocytes as well as melanoma cells against which responses by autologous T cells have been detected. We established a T-cell line from the peripheral blood of a patient with VKH disease which responded to synthetic peptides corresponding to tyrosinase. The T-cell line was generated which recognized the tyrosinase p188 - 208 peptide when presented by the HLA-DR4 (DRB1(*)0405) molecule on the surface of HLA class II-expressing L-cell transfectants. The minimal antigenic peptide which induced T-cell responses was an 11-amino-acid sequence and located at tyrosinase p193 - 203 (E-I-W-R-D-I-D-F-A-H-E). This peptide contained the DRB1(*)0405-binding peptide motif (hydrophobic residues (Y, F, W) at position 1 as an anchor residue, and negatively charged residues (D, E) at position 9), which corresponded to the W at p195 and the D at p203. These observations demonstrate that tyrosinase peptides are immunogenic, and may be a candidate for an autoantigen in VKH disease, suggesting that probing the T-cell responses against synthetic peptides is a productive approach for identifying the autoantigenic peptides associated with autoimmune diseases including VKH disease.     

Dependence of the murine antibody response to an anti-CDR2 VH peptide on immunogen formulation

A peptide corresponding to the second complementarity determining region of the heavy chain (CDR2 VH) from a murine anti-CD4 monoclonal antibody, designated L202, was synthesized by solid phase methodology in a number of different antigenic forms, for the purpose of comparing the effectiveness of different adjuvant-carrier systems in the induction of a murine antibody response against the immunizing peptide and parent antibody molecule. Two of the synthetic constructs contained the palmitoyl and N-palmitoyl-cysteinyl-S-(2,3-palmitoyloxy)-propanediol (PAM3Cys) moieties, respectively, attached to the peptide amino terminus with the immunogen comprising liposomal formulations of each. A third immunogen consisted of the CDR2 VH peptide admixed with the PAM3Cys non-covalently and incorporated into liposomes (PAM3Cys + CDR2 VH). A fourth composition comprised the CDR2 VH peptide conjugated to KLH via the sulfhydryl of an added N terminal cysteine (KLH-CDR2 VH) and injected with Complete Freund's adjuvant (CFA). A fifth immunogen consisted of the CDR2 VH peptide synthesized on an octameric, branched polylysine core as a multiple antigenic peptide (MAP-CDR2 VH) injected in the presence of Freund's adjuvant. Groups of five mice were injected intramuscularly with each of these immunogens and bled at two week intervals. The highest anti-peptide gamma-immunoglobulin (IgG) responses (against uncoupled peptide by ELISA) after 56 days were obtained with mice receiving the PAM3Cys-CDR2 VH peptide. However, when screened against the CDR2 V(H) peptide present as the MAP derivative by ELISA, IgG raised against the cognate MAP-CDR2 peptide was much more reactive than IgG raised against the liposomal PAM3Cys-CDR2 VH immunogen. In either case, IgG raised against the KLH-CDR2VH conjugate was poorly reactive. These differences in reactivity to the two forms of the CDR2 VH peptide by ELISA did not correspond to major differences in reactivities to the intact L202 Ab by ELISA. Although the IgG against the MAP immunogen was slightly more reactive than the other antisera against the l202 Ab, all titers were less than 1:100. These data illustrate some limitations of using anti-peptide responses as indicators of potential reactivity against the native protein, but suggest that alternate formulations including lipoidal peptides are more effective than corresponding KLH-peptide conjugates in eliciting Ab responses against poorly immunogenic epitopes.     

Characterization of monoclonal and polyclonal antibodies to human choline acetyltransferase and epitope analysis

Choline acetyltransferase (ChAT) was partially purified from human placenta and brain. In order to raise monoclonal antibodies, Balb/c mice were immunized with a preparation from placenta or with a mixture of eight synthetic peptides that were deduced from the primary structures of porcine and human ChAT. Polyclonal antibodies were raised in rabbits against five synthetic peptides deduced from the amino acid sequence of human ChAT. The monoclonal and polyclonal antibodies were characterized by their ability to recognize ChAT in various immunoassays: immunoblot, enzyme-linked immunosorbent assay (ELISA), two-side ELISA and immunohistochemistry. With one exception all monoclonal antibodies recognized ChAT on immunoblots, some were particularly sensitive; one bound active ChAT in ELISA when used as capture reagent; most antibodies recognized immobilized ChAT in ELISA. Two monoclonal antibodies out of nine gave particularly excellent results in staining cholinergic neurons and fibers on sections from rat and primate brain. With the help of nine synthetic peptides it was possible to evaluate two major binding sites for the monoclonal antibodies on the ChAT molecule, comprising amino acids 167-189 and 57-76, respectively.     

Detailed characterization of the binding site of the lipoprotein lipase-specific monoclonal antibody 5D2

Monoclonal antibody (MAb) 5D2 recognizes lipoprotein lipases (LPL) from different species but not related lipases. This MAb is a unique reagent, used world-wide, because it differentiates between monomeric inactive and dimeric active LPL, inhibits human LPL enzyme activity, and binds to C-terminal LPL sequences involved in interactions with lipoproteins, lipoprotein receptors, and heparin. In this study we have analyzed the fine specificity of the MAb epitope recognition in order to better understand its functional properties and species-specific LPL immune reactivity. In peptide scan assays, MAb 5D2 reacted with all, except two, 13 amino acid-long peptides located between positions 380 and 410. Peptides from the amino terminal end of this region reacted more strongly than those from the carboxyl terminal end. Furthermore, only a peptide from the amino terminal end competed effectively with the binding of MAb 5D2 to native LPL bound to microtiter plates or nitrocellulose. A systematic peptide mutagenesis study indicated that 8 amino acids of the reactive region, mainly located in the amino terminal end, are critical for binding and probably directly interact with MAb 5D2. The experimentally determined antigenicities of species-specific LPL peptides and of the corresponding denatured full-length LPL proteins on immunoblots were consistent with these findings. According to a proposed 3D-model for LPL, only the amino terminal end of the antigenic region is easily surface-accessible. These data combined with 3D-modelling of monoclonal antibody (MAb)-lipoprotein lipase (LPL) protein interaction provide new insight into the known biological effects of MAb 5D2 on LPL and the antigenic determinants that are recognized.     

Identification of immunoreactive epitopes in proteins coded by gag, env, and pol genes of the type I human T-lymphotropic virus (HTLV-I) using synthetic peptides

Reactivity of 26 synthetic peptides that comprise 12 to 26 amino acid residues corresponding to segments of the gag p19, env gp46, and pol proteins of human T-lymphotropic virus type I toward 31 positive sera was studied using enzyme-linked immunosorbent assay. Specific reactivity with high titers of antibodies (presented in reciprocal dilution values) was detected for the synthetic peptides corresponding to fragments 110-130 and 100-130 (titers up to 4050) of p19, 174-197 (up to 800), 186-201 (up to 4050), 191-215 (up to 1350), 242-257 (up to 800), and 272-292 (up to 450) of gp46. Immunoreactivity of seven peptides, fragments of pol-proteins, was weak. New linear epitopes in the regions 145-158, 272-277, and 292-300 of gp46 were detected. In addition, location of the known linear epitopes in p19 and gp46 was refined on the basis of comparative study of overlapping peptides from these proteins.     

Mapping of the linear antigenic determinants from the Leishmania infantum histone H2A recognized by sera from dogs with leishmaniasis

Antibodies reacting against the H2A histone protein were frequently observed in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum. Using synthetic peptides covering the complete sequence of the protein we have identified the amino terminal region, comprising from amino acids 1 to 20, and the carboxyl terminal region, comprising from amino acids 106 to 132, as conforming the antigenic determinants of the protein. Those regions, exposed in the nucleosome surface, are highly divergent in sequence relative to the mammalian H2A histones. The anti-H2A histone antibodies present in the sera of these dogs specifically recognize the L. infantum H2A histone and they do not react with mammalian histones. The present data indicate that, in spite of the evolutionary conservation of the H2A histone protein among eukaryotic organisms, the humoral response against this protein during natural infection is specifically triggered by the parasite protein antigenic determinants.