Analysis of the neutralizing antibody response to the measles virus using synthetic peptides of the haemagglutinin protein

Infection or immunization with measles virus induces a protective immune reaction including neutralizing antibodies against the haemagglutinin and fusion protein. The reactivity of the polyclonal IgG response of sera obtained from late convalescent donors was studied, using overlapping 15mer peptides covering the complete sequence of the measles virus haemagglutinin. Most sera reacted with a similar set of peptides generating a characteristic binding pattern. The reactive peptides correspond to a region mediating cell hemolysis (aa310-325), to regions which serve as targets to neutralizing antibodies and to a putative transmembrane region (aa35-58). The latter region contains also a human T-cell epitope providing evidence of a non-random association of T- and B-cell epitopes. We also immunized different strains of mice and rabbits with measles virus. In contrast to the human sera, animal sera with strong neutralizing activities did not react with any of the H-protein peptides. The mostly weak reactivities with the linear sequences contrast with the strong neutralizing activities of the human or animal antibodies, suggesting that these primarily recognize the fusion protein or conformational epitopes of the haemagglutinin protein.     

HIV-1 gp41 by a common immunological epitope induces increased levels of antibodies against human interferon-beta in HIV-1 positive individuals

Based on our finding that a similar epitope exists between human IFN-beta (aa128-134) and HIV-1 gp41 (aa586-595), we examined 20 sera from healthy and 20 from HIV-1 infected individuals for IFN-beta antibody levels by ELISA. The levels of anti-IFN-beta antibody in sera from HIV-infected individuals were increased by about 160% in comparison with HIV-negative. We affinity-purified anti-gp41 antibodies from sera of HIV-1-infected individuals using rsgp41-sepharose column. One of three antibodies could recognize human IFN-beta in comparison with antibodies from serum of a healthy individual. A mouse antiserum to human IFN-beta recognized rsgp41 (recombinant soluble gp41 Env amino acid 539-684), while the normal mouse serum (pre-immune serum) did not bind to rspg41. These results indicate that a common immunological epitope exists between human IFN-beta and HIV-1 gp41. The sequence-similarity suggests that this common immunological epitope may be located in the region aa128-134 of human IFN-beta and the immunosuppressive domain (aa583-599) of HIV-1 gp41. The increased levels of antibodies against interferon-beta in HIV-1 positive individuals may be explained by a common immunological epitope on human IFN-beta and HIV-1 gp41.     

Antigenic epitopes recognized by autoantibodies to calpastatin in patients with rheumatoid arthritis and their clinical significance

We have previously described that novel autoantibodies to calpastatin (endogenous inhibitor for calcium-dependent neutral protease, calpain) were detected in patients with rheumatoid arthritis (RA) and other disorders. Since calpain is thought to mediate inflammatory process and cartilage destruction, autoantibodies to its inhibitor protein, calpastatin, may be involved in the pathogenic mechanism of rheumatoid arthritis. In the present study, we analyzed antigenic epitopes reactive with autoantibodies to calpastatin and their clinical correlation. cDNA encoding the C-terminal 178 amino acids of human calpastatin (RA-6) was digested by restriction enzymes and ligated in to pEX expression vectors. These recombinant plasmids were tranfected into E. coli POP2136 and screened by colony blots using RA sera containing anticalpastatin antibodies and a mouse monoclonal antibody. RA patient sera recognized the C-terminus of domain IV (epitope C1 ; aa. 647-673) and C-terminus of domain III (epitope C2 ; aa. 496-571), whereas the mouse monoclonal antibody recognized an entirely different region containing the calpain-binding site (epitope B2 ; aa. 572-625). To evaluate epitope reactivity of patient autoantibodies, 15 RA sera containing anti-calpastatin were reacted with epitope fusion proteins. In immunoblotting, most RA sera recognized either C1 or C2 epitopes (67% and 40%, respectively), and only one patient recognized both epitopes. B2 epitope a more progressed and sever state of arthritis than those not reacting with C1. These results suggests that anti-calpastatin antibodies may play a role in the pathogenic mechanisms of RA and their epitope reactivity may be important for disease progression.     

Immunoreactivity and in-vitro effect on human sperm-egg binding of antibodies against peptides corresponding to bonnet monkey zona pellucida-3 glycoprotein

The following synthetic peptides were made as immunogens for development of a zona-based contraceptive vaccine: P1, KQPFWLLQGGASRAETSVQPVLVE [amino acids (aa) 23-45 with an additional K at the N-terminus]; P2, FSEEKLVFSLRLMEENC (aa 164-179 with an additional C at the C-terminus and T170 replaced by V); and P3, CSFSKSSNSWFPVEGPADICQCC (aa 300-322). The aa are numbered on the basis of bonnet monkey ZP3 precursor protein. Antibodies against an additional peptide P4, KGDCGTPSHSRRQPHVVSQWSRSA (aa 324-347), significantly inhibits human sperm-oocyte binding. In addition, antibodies against cocktail of peptide-diphtheria toxoid conjugates also significantly inhibit the binding of spermatozoa to zona pellucida in a hemizona assay. These results will further help in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to ZP3.     

Immunization with the "immunogenic peptide" of TSH receptor induces oligoclonal antibodies with various biological activities

Balb/c mice were immunized with a synthetic peptide (P354-14) corresponding to "the immunogenic peptide" of human thyrotropin receptor (hTSH-R). Through screening for binding to the peptide, we obtained several monoclonal antibodies with various biological activities: thyroid stimulation (SAb), inhibition of TSH stimulation (BAb) and no significant effect on cAMP production. One of the stimulatory clones was further studied. This clone enhanced cAMP production in Cos-7 cells transformed with the truncated TSH-R cDNA deleting the immunogenic peptide. These results indicated that the immunogenic peptide of the TSH-R induces oligoclonal anti-TSH-R antibodies, although the region is not essential for the functional epitope.     

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.     

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)     

Biological and immunochemical characterization of recombinant human thyrotrophin

Recombinant human thyroid-stimulating hormone (recTSH) has recently been engineered to detect metastatic lesions in patients operated on for thyroid cancer. In this report, we have compared the microheterogeneity, carbohydrate (CHO) content, mitogenic potency and immunoreactivity of the biotechnology product to those of human TSH of pituitary origin (pitTSH). Compositional analysis revealed that recombinant (rec) TSH produced in Chinese hamster ovary cells was overglycosylated compared with the native hormone (21 and 14%, respectively) with a higher amount of sialic acid and lack of N-acetylgalactosamine. Electrofocusing followed by immunoblotting resolved recTSH into six glycoforms with pIs ranging from 6.0 to 8.6, which were converted to a major species of pI 8.9 by sialidase treatment. pitTSH contained five main isoforms of pI 6.5-8.2 distinct from those of recTSH and partially resistant to sialidase. Binding activity of both human TSHs to porcine thyroid membrane receptors was found to be similar, but recTSH appeared to be 20% active compared to pitTSH in eliciting cAMP production and cell growth in rat FRTL-5 cells. Immunoreactivity of the recombinant hormone was investigated using polyclonal and monoclonal antibodies raised against the native hormone or synthetic peptide sequences of its subunits. While rec- and pitTSH were recognized to a similar extent by anti-protein antibodies, they exhibited a different binding pattern to antipeptide antibodies. Serial dilution of anti-alpha 1-25, anti-alpha 26-51, anti-beta 96-112 antisera bound recTSH to a greater extent than pitTSH, while anti-beta 31-51 and anti-beta 53-76 displayed similar recognition toward both preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine epitope specificity of antibodies to region II of the Plasmodium vivax circumsporozoite protein correlates with ability to bind recombinant protein and sporozoites

Recent work has suggested that important B- and T-cell epitopes on the circumsporozoite protein (CSP) of Plasmodium vivax lie external to the major repeat regions of the protein. We have studied two naturally exposed human populations (Caucasian and Papua New Guineans) and determined the antibody response to yeast-derived recombinant CSPs, overlapping synthetic peptides spanning amino acids 76 348 of the Belem P. vivax CSP and overlapping peptides representing the variant repeats of the VK247 strain of P. vivax. We have demonstrated that the P. vivax CSP-specific antibody response is directed towards areas within the repeat region as well as areas external to this; but the dominant epitopes recognized by the two populations studied, were distinct. One epitope, lying external to the repeats and recognized by both populations, partially overlaps an area of the protein referred to as region II-plus. Sera from malaria-exposed Papua New Guineans and Thais contained antibodies to this epitope (V22, single letter amino acid sequence TCGVGVRVRRRVNAANKKPE) which were capable of recognizing sporozoites, as determined by quantitative inhibition IFA. Seventeen percent of PNG sera had antibodies to this peptide compared with 33% who had antibodies to the central repeats of the protein. Immunization of mice with recombinant CSP did not induce antibodies to V22. However, immunization with overlapping peptide epitopes representing this region (V21 or V22) induced specific antibodies but only two sera recognized both V21 and V22 and, by inference, the overlapping peptide sequence (TCGVGVRVRR). Antibodies in these two sera could bind recombinant CSP in ELISA; however, in contrast, nine sera which recognized either V21 or V22 alone did not bind CSP. Only one of two sera containing antibodies recognizing CSP stained P. vivax sporozoites. This serum also recognized an epitope dependent upon two amino acids aminoterminal to V22. These data suggest that the fine specificity of antibodies is a critical determinant for binding to both recCSP and sporozoites.     

Accessibility of epitopes on UvrB protein in intermediates generated during incision of UV-irradiated DNA by the Escherichia coli Uvr(A)BC endonuclease

Structural intermediates generated during incision of damaged DNA by the Uvr(A)BC endonuclease were probed with monoclonal antibodies (mAbs) raised against the Escherichia coli UvrB protein. It was found that the epitope of B2C5 mAb, mapped at amino acids (aa) 171-278 of UvrB, is not accessible in any of the preformed Uvr intermediates. Preformed B2C5-UvrB immunocomplexes, however, inhibited formation of those intermediates. B2C5 mAb seems to interfere with the formation of the UvrA-UvrB complex due to overlapping of its epitope and the UvrA binding region of UvrB. Conversely, the epitope of B3C1 mAb (aa 1-7 and/or 62-170) was accessible in all Uvr intermediates. The epitope of B*2E3 mAb (aa 171-278) was not accessible in any of the nucleoprotein intermediates preceding UvrB-DNA preincision complex. However, B*2E3 was able to immunoprecipitate this complex and to inhibit overall incision. B2A1 mAb (aa 8-61) inhibited formation of those Uvr intermediates requiring ATP binding and/or hydrolysis by UvrB. B*2B9 mAb (aa 473-630) inhibited Uvr nucleoprotein complexes involving UvrB. B*2B9 seems to prevent the binding of the UvrA-UvrB complex to DNA. The epitope of the B*3E11 mAb (aa 379-472) was not accessible in Uvr complexes formed at damaged sites. These results are discussed in terms of structure-functional mapping of UvrB protein.     

Induction of native protein reactive antibodies by immunization with peptides containing linear B-cell epitopes defined by anti-porcine ZP3 beta monoclonal antibodies

To identify pertinent target epitopes for contraceptive vaccine development, rabbit polyclonal antibodies were raised against four peptides synthesized from the deduced amino acid (aa) sequence of porcine zona pellucida macromolecule ZP3 beta and coupled to diphtheria toxoid (DT). Synthetic peptides consisted of: P1, 23-37 aa; P2, 164-179 aa with an additional C-terminal cysteine; P3, 246-263 aa with an extra C-terminal cysteine; and P4, 310-321 aa residues corresponding to pZP3 beta precursor protein. Selected sequences were based upon B cell epitopes identified previously by monoclonal antibodies. Immune sera reacted with their respective peptides and DT in an ELISA, and also recognized porcine SIZP and pZP3 beta both in ELISA and Western blot and zona pellucida of porcine oocytes in an indirect immunofluorescence assay. None of the four anti-peptide sera recognized pZP3 alpha in Western blot, emphasizing the specificity of these antibodies to pZP3 beta. The anti-peptide sera, individually, failed to inhibit in vitro attachment of boar sperm to antibody treated zona encased porcine oocytes. However, combinations of immune sera against peptides such as P1 + P4, P2 + P4 and P1 + P2 + P4, did significantly inhibit porcine sperm-oocyte interaction. These results identify combinations of peptides that could potentially be used in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to pZP3 beta or its homologues in other species.     

Tobacco smoking, alcohol drinking and dietary factors as determinants of new primaries among male laryngeal cancer patients: a case-cohort study

The objective of the present study was to identify p24 antigenic domains recognized during natural human immunodeficiency virus type 1 (HIV-1) infection, the determination of the major epitopes of p24 having significant applications for both the improvement of diagnostic approaches and the development of vaccines. Reactivity of 20 HIV-1-infected patients and 8 HIV-1-negative patients was analyzed using an enzyme-linked immunosorbent assay (ELISA) developed with 45 overlapping synthetic pentadecapeptides, spanning amino acids 133 to 363 of HIV-1 p55gag precursor. Two peptides covering aa 178-192 and 288-302 of p55 were recognized by 40 and 45% of HIV-1 antibody-positive human samples, respectively. A peptide covering aa 272-322 of p55 was synthesized and recognized by most human sera in indirect ELISA. However, inhibition assays indicated that this sequence does not contain all of the immunodominant domains of p24 since it was not sufficient to block binding of human sera to whole p24. A three-dimensional model of p24 derived from the Mengovirus VP2 suggests that the two distant sequences recognized by human sera containing antibodies to HIV-1 could possibly be a part of a conformational epitope built up by two loops corresponding to aa 183-186 and 289-292.     

B-cell epitopes in HIV-1 Tat and Rev proteins colocalize with T-cell epitopes and with functional domains

We describe the characterization of the B-cell epitopes of HIV-1 regulatory proteins Tat and Rev. The prevalence of antibodies to these proteins among human immunodeficiency virus (HIV)-1-infected individuals was examined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The Tat and Rev antibody-positive sera were selected for epitope mapping performed with partially overlapping synthetic peptides bound to polyethylene pins. Eighteen and twelve percent of HIV-infected individuals had antibodies against Tat or Rev, respectively. In Tat, four epitopic regions were identified, situated within amino acids 6-10 (PRLEP), 21-37 (ACTNCYCKKCCFHCQVC), 39-58 (ITKALGISYGRKKRRQRRRA) and 74-82 (TSQSRGDPT). The most frequently recognized epitopic regions were located in the middle of the protein. In Rev, the two most frequently recognized epitopic regions were near the amino terminus of the protein within amino acids 12-20 (LIRTVRLIK) and 38-49 (RRNRRRRWRERQ). A third epitope was mapped around amino acids 55-62 (ISERILGT) and a fourth around amino acids 78-83 (LERLTU). To analyze the specificity of Tat and Rev epitopes, soluble synthetic peptides representing the identified epitopes were used in an ELISA assay, and the recognition of most epitopes was shown to be specific for HIV-1-infected individuals. In addition, many of the Tat and Rev epitopes were shown to overlap with regions having functional activity or with regions previously identified as T-cell epitopes.     

Interventional and intraoperative MRI: a general overview of the field

We have prepared a biotinylated thyrotropin receptor (TSHR-BIO), and characterized its activity in cells and when bound to solid phase (streptavidin agarose). TSHR-BIO consists of the N-terminal 725 amino acids of the human thyrotropin (TSH) receptor linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotinylation of the protein. TSHR-BIO was expressed using a vaccinia virus expression system. HeLa cells infected with recombinant virus produced large amounts of TSH receptor of approximately 120,000 molecules per cell. Vaccinia virus produced TSHR-BIO was fully functional interacting with TSH (Kd of 2.3+/-0.1 x 10(-10) M) and coupling to cyclic adenosine monophosphate (cAMP) second messenger system. The expressed protein was biotinylated with high efficiency; more than 90% of TSHR-BIO was bound to streptavidin. We have shown the application of streptavidin agarose immobilized TSHR-BIO for the detection of thyroid-binding inhibiting immunoglobulines in unfractionated sera. There was a good positive correlation between the results obtained in this assay and the commercially available TRAK assay performed with solubilized porcine TSH receptor (r = 0.71; p < 0.001, in 45 sera of patients with Graves' disease and 17 normal sera).     

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.     

Role of magnetic resonance in hemodialysis patients with amyloid shoulder arthropathy]

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.     

Mapping a conserved conformational epitope from the M protein of group A streptococci

The carboxyl terminus of the M protein of group A streptococci (GAS) is highly conserved and contains epitopes that have been shown to induce opsonic antibodies and protection against GAS infection. This region of the protein can also stimulate T cells, which can react in vitro with heart antigens. Since different segments of the carboxyl terminus may be involved in immunity to GAS and in the pathogenesis of autoimmune disease (rheumatic heart disease), it is important to precisely define critical epitopes. However, the M protein is known to be a coiled coil, and a critical immunodominant antibody-binding epitope within this region (peptide 145, a 20-mer with the sequence LRRDLDASREAKK-QVEKALE) is shown here to be conformational. Thus, small synthetic overlapping peptides of 8-12 amino acids in length that span peptide 145 (p145) were unable to capture antibodies present in p145-immune mouse sera or in endemic human sera, even though antibodies raised to these small peptides coupled to diphtheria toxoid could bind the smaller peptides and, in some cases, p145. A series of mutated peptides in which every residue of p145 was sequentially altered also failed to identify critical residues for antibody binding. We thus devised a strategy to produce chimeric peptides in which small peptides copying the M protein sequence were displayed within a larger 28-mer peptide derived from the sequence of the GCN4 leucine zipper DNA binding protein of yeast. A 12-amino-acid window of the p145 sequence was inserted into the GCN4 peptide in such a way as to preserve any potential helical structure. The window was moved along one residue at a time to give a series of peptides representing p145. Circular dichroism demonstrated that these larger chimeric peptides and p145, but not a shorter 12-mer peptide, displayed alpha-helical potential in 50% trifluoroethanol. Certain chimeric peptides efficiently captured antibodies specific for p145 and thus enabled us to map the minimal antibody-binding sequence. RRDLDASREAKK, referred to as J(1)2. The chimeric peptide containing this sequence, referred to as J2, was able to inhibit opsonization of GAS by human antisera containing anti-peptide 145 antibodies. The T-cell response from p145-immunized responder B10.BR mice to J2 and J(I)2 was much lower than the response to p145 and mapped to a different peptide.     

Protection against exotoxin A (ETA) and Pseudomonas aeruginosa infection in mice with ETA-specific antipeptide antibodies

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious and sometimes fatal infections in the compromised host, especially in patients with major trauma or thermal injuries. Exotoxin A (ETA) is the major and most lethal virulence factor produced by this ubiquitous microorganism. In a recent study (H. S. Elzaim, A. K. Chopra, J. W. Peterson, R. Goodheart, and J. P. Heggers, Infect. Immun. 66:2170-2179, 1998), we identified two major epitopes, one within the translocation domain (amino acid [aa] residues 289 to 333) of ETA and another within the enzymatic domain (aa 610 to 638), by using a panel of antipeptide antibodies. Synthetic peptides representing these two epitopes induced ETA-specific antibodies which were able to abrogate the cytotoxic activity of ETA, as measured by incorporation of [3H]leucine into 3T3 fibroblasts. In the present study, these antibodies were tested for the ability to provide protection against ETA and infection with a toxin-producing strain of P. aeruginosa in a mouse model. Antibodies to either of the synthetic peptides conferred protection against ETA. Also, when used for immunization, both peptides induced active immunity to ETA in mice. Antibodies to the peptide representing a region within the enzymatic domain of ETA, in combination with the antibiotic amikacin, enhanced the survival of mice infected with a toxin-producing strain of P. aeruginosa. Thus, antipeptide antibodies specific for ETA might be paired with antibiotic treatment for passive immunization of patients suffering from P. aeruginosa infection.     

Receptor binding sites and antigenic epitopes on the fiber knob of human adenovirus serotype 3

The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I beta-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells.     

Radiologic diagnosis of abdominal trauma

We have characterized 10 monoclonal antibodies (Mabs) to recombinant murine thyrotropin receptor extracellular domain (mTSHR-ecd). Affinity purified mTSHR-ecd (amino acids 22-415), expressed in a baculovirus-insect cell system, was refolded in vitro and used to hyperimmunize female Balb/c mice. Spleens were removed 10 days after a final boost of 25 microg mTSHR-ecd intraperitoneally and intravenously, and the cells were fused to SP-2 cells and cloned. Hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) with folded mTSHR-ecd antigen. Ten of 18 higher affinity hybridomas were selected at random and ascites fluids prepared. Nine of the monoclonals were of IgG 1 isotype, and one was IgM. Five Mabs (M3, M4, M5, M6, and M9) inhibited the binding of 125I-TSH to functional hTSHR expressed on Chinese hamster ovary (CHO) cells, and four (M1, M3, M5, and M9) blocked the TSH-stimulated generation of cyclic adenosine monophosphate (cAMP), using the same cells. The remaining Mabs appeared to be neutral in their interaction with native TSHR. The Mabs were also compared for their reactivity to mTSHR-ecd under folding (ELISA) and unfolding (reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) conditions. Most Mabs demonstrated reactivity to both conformational (folded) and linear (unfolded) forms of mTSHR-ecd, suggesting that they were generated primarily against linear epitopes although one Mab (M4) showed affinity for only folded antigen indicating a preference for a conformational epitope. Mapping the Mab epitopes using 26 overlapping peptides spanning the human (h)TSHR-ecd showed that 6 bound peptide 397-415, 1 bound peptide 352-371, and 1 peptide 22-41. These epitope mapped Mabs to the mTSHR-ecd, both receptor blocking and receptor neutral, will provide further insight into the structure-function of the TSHR ectodomain.