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Two different epitope mapping techniques were used to identify linear epitopes recognised by polyclonal IgG antibodies from rabbits immunised with bovine beta lactoglobulin (BLG), which is generally regarded as a major allergen in milk. The first, PEPSCAN, was used to investigate the binding of several rabbit polyclonal antisera to sequential overlapping peptides (12-mers) across the sequence of BLG. Each peptide was synthesized on a different polypropylene PIN, and a standard ELISA procedure was used to locate which of these peptides bound the antibodies under investigation. Comparisons of PEPSCANs for antisera from six different rabbits showed that each rabbit recognized a similar set of epitopes within BLG. PEPSCAN analysis also showed that polyclonal antibodies from the mouse recognize a set of epitopes similar to those recognized by the rabbit. The second epitope mapping technique is known as phage display and utilizes libraries of randomized short peptides fused to the coat proteins of filamentous phage as a source of epitopes for analysis. A gene VIII phage display library was used in this study with constrained nonapeptides, which were screened for epitopes recognized by affinity purified rabbit anti-BLG IgG. Immobilised rabbit anti-BLG IgG was screened in two separate experiments, each consisting of three rounds of panning. For each separate experiment, a sensitive phage ELISA was used to screen several hundred single phage clones for binding to anti-BLG IgG immobilised on microtiter plates. As a result, a number of positive phage were identified from the two separate screens of the library (19 different peptides were isolated, which resembled four different regions of BLG). The identified sequences were found to constitute a subset of the linear epitopes recognized by the PEPSCAN technique. The coordinates of the crystal structure of BLG were used to display mapped epitopes on its structure. This study has permitted detailed mapping of the major linear antigenic regions within BLG recognised by IgG antibodies from immunised rabbits and mice.

A converted form of the normal cellular prion protein (PrP) accumulates in the brains of sheep with scrapie. We describe an immunohistochemical method for identifying scrapie-associated PrP (PrPSc) in periodate-lysine-paraformaldehyde-fixed brain tissue, which provides adequate preservation of tissue morphology. After pretreatment of tissue sections with formic acid and hydrated autoclaving, we located PrPSc in the brains of 50 sheep with natural scrapie by use of antipeptide antisera raised against ovine PrP. No PrP was seen in 20 sheep without histopathologic signs of scrapie. PrP80 that did not stain for amyloid was present in the cytoplasm and at the cell membrane of both neurons and astrocytes. Large amounts of PrPSc were seen at the cell membrane of neurons in the medulla oblongata and pons, whereas PrPSc accumulated at the cell membrane of astrocytes of the glial limitans in all brain regions. PrPSc that stained for amyloid was located in the walls of blood vessels and perivascularly in the brains of 32 (64%) of 50 sheep, mainly in the thalamus and never in the pons or medulla oblongata. No apparent topographic relationship existed between PrPSc that stained for amyloid and PrPSc accumulation associated with neurons or astrocytes. In all scrapie-affected sheep, PrPSc was present in brain regions with vacuolation, but it could also be detected in regions with minimal or no vacuolation. We conclude that the immunohistochemical detection of PrP can be an important confirmative test in scrapie diagnosis.

The p190 protein (also called MSA1 or MSP1) of the asexual blood stage forms of Plasmodium falciparum, a human malaria vaccine candidate, shows polymorphism between different isolates. Mice were immunized with p190-3, a recombinant protein which contains mostly conserved sequences derived from the p190 protein of the K1 parasite isolate. Proliferative T-cell responses of lymph node cells from immunized mice were assessed by stimulation in vitro with p190-3 or preparations of parasitized red blood cells (PRBC) containing the native protein. The p190-3-specific T cells from C57BL/6 mice consistently responded to some P. falciparum isolates, representing either the K1 or MAD20 serotype of p190, but not to other P. falciparum strains or to rodent malaria parasite-infected red blood cells. p190-3-specific T-cell responses from other mouse strains (BALB/c, C3H/He) did not distinguish between P. falciparum isolates. The polymorphic epitopes which were preferentially recognized by T cells from C57BL/6 mice were identified.

In an effort to serologically differentiate syphilis from yaws, 69 monoclonal antibody species raised against Treponema pallidum subsp. pallidum were tested by immunoblotting for their reactivity with Treponema pallidum subsp. pertenue. All monoclonal antibodies reacted with antigens with the same molecular weight of both subspecies. Furthermore, no differences in reactivity between sera from yaws patients and from syphilis patients were found by Western blot (immunoblot) analysis of cell lysates of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue. We tried to exploit the only known molecular difference between the subspecies. The subunits of the 190-kilodalton multimeric proteins TpF1 and TyF1 of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue, respectively, have previously been shown to differ in one amino acid residue at position 40. In this study, no difference was found in immunoreactivity of TpF1 or TyF1 with either syphilis sera or yaws sera. Synthetic peptides based on the sequence of TpF1 and of TyF1 were used in an enzyme-linked immunosorbent assay with syphilis sera and yaws sera. Again, no difference in reactivity between the T. pallidum subsp. pallidum- and T. pallidum subsp. pertenue-derived peptides was observed.

A panel of murine monoclonal antibodies (MAbs) to the human immunodeficiency virus type 1 trans-activator tat protein were characterized. The anti-tat MAbs were mapped to the different domains of the tat protein by Western blot (immunoblot) and Pepscan analyses. One-half of the MAbs tested mapped to the amino-terminal proline-rich region, and one-third of the MAbs tested mapped to the lysine-arginine-rich region of tat. The individual MAbs were tested for inhibition of tat-mediated trans activation, using a cell-based in vitro assay system. MAbs which mapped to the amino-terminal region of the tat protein demonstrated the highest degree of inhibition, whereas MAbs reactive to other portions of the molecule exhibited a less pronounced effect on tat function.

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.

Rs-AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs-AFPs have been isolated (Rs-AFP1-4). The structure of Rs-AFP1 consists of three beta-strands and an alpha-helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure-function relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs-AFP2 is concentrated mainly in the beta2-beta3 loop. In this study, we synthesized linear 19-mer peptides, spanning the entire beta2-beta3 loop, that were found to be almost as potent as Rs-AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19-mer loop peptides, cysteines can be replaced by alpha-aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19-mer peptides, forced to adopt a hairpin structure by the introduction of one or two non-native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19-mer peptides, like Rs-AFP2 itself, cause increased Ca2+ influx in pregerminated fungal hyphae.

Three rabbit antibodies (R521, R505, R524) were produced, and raised to synthetic peptides corresponding to residues 94-105, 100-111, and 223-234, respectively, of the sheep prion protein (PrP). Epitope mapping analysis revealed the monospecific character of antisera R505 and R524. In addition to the amino acid sequence against which it was raised, R521 also recognized other small epitopes. ELISA and radio-immunoprecipitation were used to assess the relative immunoreactivities of the antisera to the normal sheep prion protein (PrP(c)). Highest reactivity was found for R521, followed by R505 and R524. According to Western blot analysis, all three sera specifically reacted with the prion proteins PrP(Sc) and PrP27-30, extracted from the brain stem of a scrapie-affected sheep. Yet, with R505 not all of the lower molecular weight deglycosylated forms could be detected. Contrary to the immunoreactivities found with the PrP(Sc) and PrP27-30 isoforms, only R521 recognised PrP(c) from a healthy sheep. The usefulness of all three anti-peptide sera in the immunohistochemical detection of PrP(Sc) in brain stem and tonsils of scrapie-affected sheep was demonstrated and compared with an established rabbit anti-PrP serum.

SUMMARY We have previously demonstrated that the Mycobacterium leprae 18-kD heat shock protein (HSP18) is represented among the antigenic targets of human T cell responses induced by M. leprae immunization and that the peptide 38–50 serves as an immunodominant epitope recognized by CD4+ T cell clones. By using peripheral blood mononuclear cells and T cell lines from the same donor group, we have in this study shown that the M. leprae HSP18 and peptide 38–50 were recognized by memory T cells 8 years after immunization with M. leprae. The finding that M. bovis BCG-induced T cell lines responded to M. leprae HSP18, but not to the peptide 38–50, suggested the existence of additional T cell epitopes of a cross-reactive nature. Consistent with this, testing of the T cell lines for proliferative responses to the complete HSP18 molecule, truncated HSP18 (amino acid (aa) residues 38–148) and overlapping synthetic peptides, made it possible to identify two cross-reactive epitope regions defined by aa residues 1–38 and 41–55. While peptide 38–50-reactive T cell clones showed limited cross-reactivity by responding to M. leprae, M. avium and M. scrofulaceum, the T cell lines specific to the epitopes 1–38 and 41–55 were broadly cross-reactive, as demonstrated by their response to M. leprae, M. tuberculosis complex, M. avium and other mycobacteria. MHC restriction analysis of the HSP18-responding T cell lines showed that the epitopes 1–38 and 38–50 were presented by one of the two HLA-DR molecules expressed from self HLA-DRB1 genes, whereas the epitope 41–55 was recognized in the presence of autologous as well as HLA-DR and HLA-DQ mismatched allogeneic antigen-presenting cells. The results obtained in this study made it possible to identify cross-reactive T cell epitopes of the M. leprae HSP18, and provide an explanation for T cell recognition of this antigen in individuals infected with species of the M. tuberculosis complex or environmental mycobacteria.

The murine natural resistance-associated macrophage protein, Nramp1, has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but precisely how this relates to macrophage activation and/or pathogen survival remains unclear. To gain insight into function, anti-Nramp1 monoclonal and polyclonal antibodies are used here to localise Nramp1 following activation and infection. Confocal microscope analysis in uninfected macrophages demonstrates that both the mutant (infection-susceptible) and wild-type (infection-resistant) forms of the protein localise to the membranes of intracellular vesicular compartments. Gold labelling and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electron-dense lysosomal compartments, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in both compartments, with macrosialin in late endosomes, and with BSA-5 nm gold in pre-loaded lysosomes. Nramp1 is upregulated with interferon-(gamma) and lipopolysaccaride treatment, coinciding with an increase in labelling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the periphery of the cytoplasm along the microtubular network. In both control and activated macrophages, expression of the protein is 3- to 4-fold higher in wild-type compared to mutant macrophages. In Leishmania major-infected macrophages, Nramp1 is observed in the membrane of the pathogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and activated macrophages, Nramp1-positive vesicles migrated to converge, but not always fuse, with pathogen-containing phagosomes. The Nramp1 protein is thus located where it can have a direct influence on phagosome fusion and the microenvironment of the pathogen, as well as in the more general regulation of endosomal/lysosomal function in macrophages.

The ultimate vaccine is an oral vaccine which given once protects against a multitude of diseases. Furthermore this ultimate vaccine needs to be very stable and inexpensive to produce. Probably this latter condition can be met only if the vaccines are produced in plants. Such vaccines are called 'edible vaccines'. Edible vaccines can be produced in plants in many ways. Using recombinant plantvirus, CPMV, it was shown that plants can produce massive amounts of chimaeric virus particles which protect after a single injection the target animal against disease. The final step, oral administration, is being addressed at present. Preliminary experiments by others suggest that this step may be solved sooner than expected.

There are few male contraceptive methods, and research is required to broaden the scope of available male antifertility methods. Two approaches toward hormonal contraception are currently being investigated. The first relies on elimination of testosterone while the second is based upon immunizations against FSH. However, most anti-whole FSH antisera cross-react with LH, thereby possibly inhibiting testosterone and leading to potential loss of libido. Therefore, a more effective alternative would be to define an FSH peptide that differs significantly from LH in order to prevent cross-reactivity between anti-FSH antisera and LH. Two peptides were selected from the beta subunit of FSH that were considered to be inducers of anti-FSH activity but not anti-LH activity. The first peptide (sequence beta33-53) is a linear antigenic site of human FSH found only in anti-FSH antisera that do not cross-react with LH. The second peptide (sequence beta81-95) is a part of FSH that confers receptor specificity. These peptides, in monomer and tandem form, were used to immunize rabbits. The antisera were tested for inhibition of FSH activity in a bioassay; they were also tested in a Leydig cell assay to detect anti-LH activity. It was found that antisera raised against the beta33-53 tandem could inhibit the FSH bioactivity but not that of LH. Antisera against the beta33-53 monomer or the beta81-95 monomer or tandem did not inhibit FSH. Thus, the tandem peptide beta33-53 is an attractive candidate for use as antigen in a male contraceptive vaccine. The better results obtained with tandem vaccinations might be related to the ability of the tandem peptide to direct the antibody response toward the N-terminal end of the peptide and to raise antisera with the ability to react with shorter chains of amino acids.

The mycobacterial 60,000 MW heat shock protein (hsp 60) is a major antigen recognized by mycobacteria-reactive human CD4+ T cells with lymphokine profiles and effector functions consistent with protective immunity. In addition, the presence of a large number of T-cell epitopes presented by several HLA class II molecules makes this antigen relevant to subunit vaccine design. However, the results from animal models as well as human studies suggest that the mycobacterial hsp 60 may induce T-cell-mediated autoimmune conditions. In humans, the expression of HLA-DR4 represents a risk factor for some autoimmune diseases. These observations suggest that the epitopes from the mycobacterial hsp 60 presented to T cells in the context of HLA-DR4 could be relevant to autoimmunity. This is the first report on identification of HLA-DR4-restricted T-cell epitopes from the mycobacterial antigen hsp 60. In total, five epitopes recognized in the context of HLA-DR4 by the M. leprae hsp 60-reactive CD4+ T-cell clones from a subject immunized with M. leprae were defined by synthetic peptides. Two of the epitopes were M. leprae-specific (aa 343-355, aa 522-534), whereas three epitopes were common to M. leprae and M. tuberculosis (aa 331-345, aa 441-455, aa 501-515). However, all of these epitopes belong to the regions that are highly divergent between the mycobacterial hsp 60 and the homologous human hsp 60 sequence, suggesting that the T cells recognizing the mycobacterial hsp 60 in the context of HLA-DR4 may not necessarily induce autoreactivity.

Recently, we developed a panel of monoclonal antibodies (MoAbs) to rat IL-1 beta and found that MoAbs binding to the aminoacid sequences 66-85 and 123-143 of mature rIL-1 beta inhibited the binding of rIL-1 beta to murine EL4 cells. Here we study whether MoAbs to these and other domains of IL-1 interfere with the biological effects of rIL-1 beta in adult male rats in vivo. Administration of rIL-1 beta (1 or 5 micrograms/kg i.v.) enhanced the plasma concentrations of ACTH, corticosterone (CORT) and of IL-6 in a time- (0.5-4 h) and dose-dependent manner. Because 2 h after 5 micrograms/kg i.v., all three parameters were consistently elevated, this dose and time interval was used for further studies. Prior to injection, rIL-1 beta was incubated alone or in the presence of a MoAb (10 mg/kg) for 30 min at 37 degrees C or at 4 degrees C. Plasma ACTH, CORT and IL-6 responses to these mixtures are compared to those obtained after preincubation of rIL-1 beta with a non-IL-1 binding MoAb (PEN7). SILK 3, a MoAb that binds to the 66-85 domain of rIL-1 beta, reduced the ACTH and IL-6 responses by 48 and 45% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

By combining a DNA subclone and synthetic-peptide approach, we mapped epitopes of the immunogenic mycobacterial 70-kDa heat shock protein (HSP70) recognized by human CD4+ T-cell clones and lines. In addition, we identified the respective HLA-DR molecules used in antigen presentation. The donor groups used were healthy persons immunized with killed Mycobacterium leprae and tuberculoid leprosy patients. The results show that the N-terminal part of the HSP70 molecule contains three different T-cell epitopes, of which two were presented by DR7 (amino acids [aa] 66 to 82 and 210 to 226) and one was presented by DR3 (aa 262 to 274). The C-terminal part contains one epitope (aa 413 to 424) presented by HLA-DR2. The C-terminal epitope shows extensive homology to the corresponding region of the human HSP70 sequence. All of the T-cell epitopes identified were presented by only one particular HLA-DR molecule. We also found that HLA-DR5 and DRw53 can present HSP70 to T cells, demonstrating the presence of additional epitopes not yet defined at the peptide level. On the basis of the donors used in this study, recognition of HSP70 at the epitope level seems to be ruled by the restriction elements expressed by the donor rather than by any difference in reactivity between healthy individuals and patients. In conclusion, mycobacterial HSP70 is relevant to subunit vaccine design since it contains a variety of T-cell epitopes presented in the context of multiple HLA-DR molecules.

Three monoclonal antibodies, LF-hCG-6, -26 and -28, raised against holo-human chorionic gonadotropin and directed against its beta-subunit, have been used to design synthetic peptides with improved affinity. By PEPSCAN analyses with pinbound, overlapping nonapeptides, it was shown that the peptide consisting of amino acids (125-133) of the beta-subunit (i.e., PPSLPSPSR) reacted with monoclonal antibody (LF-hCG-6; the sequence (135-143) (i.e., PGPSDTPIL), with LF-hCG-28; and the C-terminal nonapeptide (137-145) (i.e., PSDTPILPQ), with LF-hCG-26. To determine amino acids essential for binding and those comprising the necessary amino acid core, and to establish the optimal length for binding reactivity, sets of replacement nonapeptides and overlapping octa- to dodecapeptides derived from the beta-subunit sequences (122-134) (i.e., KAPPPSLPSPSRL) and (132-145) (i.e., SRLPGPSDTPILPQ) were synthesized. Amino acid cores were as follows: beta-hCG (125-131) (i.e., PPSLPSP) for monoclonal antibody LF-hCG-6, beta-hCG (135-142) for LF-hCG-28 and beta-hCG (139-145) for LF-hCG-26. Based on the optimal length, parent peptide sequences [beta-hCG (125-133), (137-145) and (135-145)] were synthesized by conventional solid-phase procedures. In addition, based on the results with the replacement peptides, peptide derivatives were produced in which specific single improvements had been combined. The affinities of the monoclonal antibodies for the peptide derivatives, compared to the parent peptide sequences, were at least 700 times better for LF-hCG-6 reactive peptides (6.9 x 10(6) M-1 and10(4) M-1, respectively) and 130 times better for LF-hCG-26 reactive peptides (1.3 x 10(6) M-1 and10(4) M-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

CD4 cross-linking by antibodies or its natural ligands triggers a tyrosine kinase activity that is one of the necessary steps in the mechanism of human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and full Th-cell activation. In this study we mapped a part of the dimerization site of human CD4 to amino acids 87-98 using a bivalent CD4 immunoadhesin and a series of overlapping 12-mer peptides of the D1 domain. The dimerization site we found is part of the complementary determining region (CDR) 3-like region of CD4. Using the three-dimensional structure of other immunoglobulin dimers as a basis, a molecular modeling study was performed to dimerize the D1 domains of CD4. Both the peptide binding studies and molecular modeling studies independently led to the conclusion that the CDR3-like region is part of the CD4 dimerization site. The suggested dimerization of CD4 through its CDR3-like region explains the important role that has been ascribed to this region in Th-cell activation and HIV-1-mediated fusion. Based on this model of the CD4 dimer and published results of different mutational analysis studies, a model was proposed for the complex of the CD4 dimer with two MHC-II molecules. The CD4 dimer allows tight binding to a large surface of MHC-II and the complex of CD4 and MHC-II reconciles mutational analysis studies that were previously incompatible. Moreover, the complex suggests how CD4 can dimerize through ligand binding.

Phospholipase A2 (PLA) is a biochemically fully defined glycoprotein, representing the main allergen of bee venom. We have established CD4+ T cell clones specific to PLA from subjects allergic and nonallergic to bee sting. By screening the epitope specificity of these clones with 62 synthetic overlapping dodecapeptides representing the PLA molecule, two immunogenic epitopes, PLA81-92 and PLA113-124, were identified. Additional screening, using longer peptides of up to 18 residues, revealed a third epitope at position PLA 45-62. These epitopes are recognized by specific T cell clones in association with HLA-DP and -DQ molecules, although HLA-DR-associated responses to PLA exist. Primary in vitro proliferation of unfractionated PBMC from bee sting allergic, hyposensitized, or hyperimmune subjects to PLA-derived peptides revealed the same immunogenic sites as found in the T cell clones. Among the different groups of individuals, proliferative responses to the PLA molecule and fragments thereof were generally higher in allergic patients than in nonallergic subjects. Thus, at least three linear epitopes are involved in T cell recognition of PLA, independently whether or not subjects are allergic.

An immunodominant region on baculovirus-produced parvovirus B19 VP2 capsids was localized between amino acids 259 and 426 by mapping the binding sites of a panel of monoclonal antibodies which recognize determinants on the particles. The binding sites of three monoclonal antibodies were fine-mapped within this antigenic domain. Six VP2-specific monoclonal antibodies recognized determinants common to both the empty capsids and native parvovirus. The defined antigenic region is most probably exposed on the native B19 virion and corresponds to part of the threefold spike on the surface of canine parvovirus particles.

We have previously shown that p3-13 (KTIAY-DEEARR) of the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis and Mycobacterium leprae is selected as an important T cell epitope in HLA-DR17+ individuals, by selectively binding to (a pocket in) DR17 molecules, the major subset of the DR3 specificity. We have now further studied the interaction between p3-13, HLA-DR17 and four different TCR (V beta 5.1, V beta 1, and V beta 4) by using T cell stimulation assays, direct peptide-DR binding assays, and a large panel (n = 240) of single amino acid substitution analogs of p3-13. We find that residues 5(I) and 8(D) of p3-13 are important DR17 binding residues, whereas the residues that interact with the TCR vary slightly for each DR17-restricted clone. By using N- and C-terminal truncated derivatives of p2-20 we defined the minimal peptide length for both HLA-DR17 binding and T cell activation: the minimal peptide that bound to DR17 was seven amino acids long whereas the minimal peptide that activated T cell proliferation was eight amino acids in length. Furthermore, two new DR17-restricted epitopes were identified on hsp70 and hsp18 of M. leprae. Alignment of the critical DR17-binding residues 5(I) and 8(D) of p3-13 with these two novel epitopes and two other DR17-binding peptides revealed the presence of highly conserved amino acids at positions n and n + 3 with I, L, and V at position n and D and E at position n + 3. D and E are particularly likely to interact with the DR17-specific, positively charged pocket that we have defined earlier. Based on these results, a set of single amino acid substituted analogs that failed to activate these T cell clones but still bound specifically to DR17 was defined and tested for their ability to inhibit T cell activation by p3-13 or other DR17-restricted epitopes. Those peptides were able to inhibit the response to p3-13 as well as other DR17-restricted mycobacterial epitopes in an allele-specific manner, and are anticipated to be of potential use for immunotherapeutic and vaccine design strategies.

Based on PEPSCAN analyses, a peptide derived from the 25-kDa surface protein of P. falciparum sexual stages (LDTSNPVKT, amino acids 122-130) was recently shown to react with transmission-blocking monoclonal antibodies. Subsequently, a pinbound construct (FDDTDPIKK; six amino acids substituted) was designed that reacted 1000 times better with the transmission-blocking monoclonal antibody 32F81 than with the parent LDTSNPVKT peptide. While this construct was obtained through single amino acid replacement studies, it now is shown that the combined contribution of the various substitutions is necessary to give the total effect. Free peptides comprising LDTSNPVKT or FDDTDPIKK were shown to inhibit the interaction of the transmission-blocking monoclonal antibody with plate-bound peptides, when prepared by conventional synthesis procedures. Compared with peptides that contained LDTSNPVKT, similar levels of inhibition could be achieved with an average 500-fold lower molar amount of peptides containing the FDDTDPIKK sequence. Affinity constants of the peptides for the transmission-blocking monoclonal antibody ranged from 1.3 x 10(5), for peptides that contained LDTSNPVKT, to 2.6 x 10(8), for peptides that contained FDDTDPIKK. The structural relationship between the epitope of the 25-kDa surface protein and the peptides was demonstrated by competition experiments with the transmission-blocking monoclonal antibody 32F81. Again, peptides comprising the newly designed sequence FDDTDPIKK competed about 200 times better than peptides with the parent LDTSNPVKT sequence.

Antibody-binding sites were mapped on all overlapping nonapeptides of the major core protein p24 of human immunodeficiency virus type 1 (HIV-1) using murine monoclonal antibodies (MAbs) and sheep and rabbit polyclonal antibodies raised against HIV-1/H9 (strain IIIB) viral lysate and antibodies obtained from humans infected with HIV-1. The binding sites were mapped to various distinct regions of this protein. After superimposition of the antibody-binding sites on a proposed model of p24 of HIV-1, these sites appeared to be located on the surface of the protein on loops, turns and coils of p24 but, unexpectedly, not on the major part of the predicted 'puff'. Little reaction was found with the inaccessible anti-parallel beta-barrel. These results are the first experimental evidence for the validity of the structure proposed for p24 of HIV-1.

We analyzed the antigenic fine-structure of an immunodominant region in the peplomer protein of infectious bronchitis virus. This region near the N-terminus of the S2 subunit is recognized by polyclonal antisera and by the majority of mAb that cross-react with denatured protein. Despite their involvement in neutralization, epitopes in this region were conserved in different serotypes. Epitopes of four mAb and two chicken antisera were localized by using prokaryotic expression of cDNA fragments, and overlapping peptides with lengths increasing from 3 to 12 residues (PEPSCAN). We found overlapping epitopes with lengths of 6, 9, 11, and more than 17 residues. The results indicate that the expression products are antigenically equivalent to denatured protein fragments. This suggests a general strategy for the localization of sequential epitopes in large proteins. We propose that the immunodominance of the N-terminal region of S2 is explained by features of the protein structure. Presumably, this region is a protruding protein segment of about 20 residues with a high local mobility, as indicated by the antigenicity of the peptides. The conservation of the sequence points to an involvement in a molecular recognition process during infection.

Antisera were raised against the chemically synthesized peptide corresponding to each epitope of three foot-and-mouth disease virus strains. Peptide synthesis was further used to determine which amino acid residues in each epitope are important for the specificity of antisera raised against the whole virus. The specificity of the antibody paratope for its epitope was shown to depend on structure as well as sequence. Anti-virus sera demonstrated a greater specificity for the homologous peptide than did the anti-peptide sera. Two of the three peptides were able to induce neutralizing antibodies against the homologous virus. The specificities of the antibodies present in the anti-peptide sera were also inferred from the reactions of each with related sets of peptides. The cross-reactions observed for the anti-peptide sera were readily explained in terms of the antibody specificities determined to be present. The findings also suggest that the diversity of antibodies raised against small peptides is limited and is determined by the immune system. A similar limited response to the native protein was observed, which may account for the high frequency with which anti-peptide sera react with the native homologous protein.

A procedure is described for rapid concurrent synthesis on solid supports of hundreds of peptides, of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner an immunogenic epitope of the immunologically important coat protein of foot-and-mouth disease virus (type O1) is located with a resolution of seven amino acids, corresponding to amino acids 146-152 of that protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope was synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. It was found that the leucine residues at positions 148 and 151 were essential for reaction with antisera raised against intact virus. A lesser contribution was derived from the glutamine and alanine residues at positions 149 and 152, respectively. Aside from the practical significance for locating and examining epitopes at high resolution, these findings may lead to better understanding of the basis of antigen-antibody interaction and antibody specificity.

Sera from three chimpanzees infected with a primary lymphadenopathy-associated virus (LAV-1) or human T-lymphotropic virus type III (HTLV-IIIB) passage, from two chimpanzees infected with blood from the primary infected chimpanzees, and from one chimpanzee infected with blood from a secondary passage animal all bound the peptides 3B and 3B/RF, sharing the sequence IQRGPGR, with equally high titers. Pepscan analysis confirmed the amino acids Q, R, G, P, and G as irreplaceable in order to retain antigenicity.

PEPSCAN analysis, performed using 536 overlapping nonapeptides derived from the HTLV-III B nucleotide sequence of the region encoding the external envelope protein of 120 kDa (gp120), identified in the V3 region of gp120 a major binding site for antibodies of HIV-1-infected humans. The minimal amino acid sequence of this antibody binding site was demonstrated by multiple length scanning to be five to eight amino acids in length: (G)PGRAF(VT), i.e. amino acids 312-319. A peptide (Neu 21) containing this binding site for human antibodies (KSIRIQRGPGRAFVTIG) was synthesized and shown to induce HTLV-III B cell fusion-inhibiting antibodies in rabbits and mice. Antibodies binding to this HTLV-III B/LAV-1-specific peptide were shown to be primarily of the IgG 1 subclass, appeared within 6 months after HIV-1 antibody seroconversion in six out of 14 men studied, and persisted throughout the follow-up period of 10-24 months. The other eight seroconverting men did not develop antibodies to Neu 21 during the observation period. The appearance of antibodies to Neu 21 paralleled the capacity of the serum to inhibit HTLV-III B in cell fusion. HIV-1-infected men with Kaposi's sarcoma exhibited a similar frequency of antibodies to the synthetic peptide Neu 21 (14 out of 39, 36%) as asymptomatic HIV-1-infected men (112 out of 319, 35%). Adults with Pneumocystis carinii pneumonia had a significantly lower frequency (11 out of 78, 14%) of antibodies to Neu 21. Similarly, a low prevalence of antibodies to Neu 21 (8 out of 43, 19%) was observed among symptomatic HIV-1-infected children.

Plant defensins are a class of cysteine-rich peptides of which several members have been shown to be potent inhibitors of fungal growth. A series of overlapping 15-mer peptides based on the amino acid sequence of the radish antifungal protein Rs-AFP2 have been synthesized. Peptides 6, 7, 8 and 9, comprising the region from cysteine 27 to cysteine 47 of Rs-AFP2 showed substantial antifungal activity against several fungal species (minimal inhibitory concentrations of 30-60 micrograms/mL), but no activity towards bacteria (except peptide 6 at 100 micrograms/mL). The active peptides were shown to be sensitive to the presence of cations in the medium and to the composition and pH of the medium. When present at a subinhibitory concentration (20 micrograms/mL), peptides 1, 7, 8 and 10 potentiated the activity of Rs-AFP2 from 2.3-fold to 2.8-fold. By mapping the characteristics of the active peptide on the structure of Rs-AFP2 as determined by nuclear magnetic resonance, the active region of the antifungal protein appears to involve beta-strands 2 and 3 in combination with the loop connecting those strands. A cyclized synthetic mimic of the loop, cysteine 36 to cysteine 45, was shown to have antifungal activity. Substitution of tyrosine 38 by alanine in the cyclic peptide substantially reduced the antifungal activity, indicating the importance of this residue for the activity of Rs-AFP2 as demonstrated carrier by mutational analysis.