Common Antigenic Sites Research Articles

Structural Biology Illuminates Molecular Determinants of Broad Ebolavirus Neutralization by Human Antibodies for Pan-Ebolavirus Therapeutic Development.

Monoclonal antibodies (mAbs) have proven effective for the treatment of ebolavirus infection in humans, with two mAb-based drugs Inmazeb™ and Ebanga™ receiving FDA approval in 2020. While these drugs represent a major advance in the field of filoviral therapeutics, they are composed of antibodies with single-species specificity for Zaire ebolavirus. The Ebolavirus genus includes five additional species, two of which, Bundibugyo ebolavirus and Sudan ebolavirus, have caused severe disease and significant outbreaks in the past. There are several recently identified broadly neutralizing ebolavirus antibodies, including some in the clinical development pipeline, that have demonstrated broad protection in preclinical studies. In this review, we describe how structural biology has illuminated the molecular basis of broad ebolavirus neutralization, including details of common antigenic sites of vulnerability on the glycoprotein surface. We begin with a discussion outlining the history of monoclonal antibody therapeutics for ebolaviruses, with an emphasis on how structural biology has contributed to these efforts. Next, we highlight key structural studies that have advanced our understanding of ebolavirus glycoprotein structures and mechanisms of antibody-mediated neutralization. Finally, we offer examples of how structural biology has contributed to advances in anti-viral medicines and discuss what opportunities the future holds, including rationally designed next-generation therapeutics with increased potency, breadth, and specificity against ebolaviruses.

Gene optimization for expression of Crimean-Congo haemorrhagic fever viral nucleoprotein

experiments showed that mAb 2A10G6 protected sucking mice from lethal dengue 1-4 viruses challenge in a dosedependent manner. Thus, we have established a novel flavivirus cross-reactive neutralizing mAb 2A10G6. Phagedisplayed random peptide library mapped the epitope of mAb 2A10G6 to a common antigenic site within the highly conserved N-terminal fusion loop peptide of flavivirus envelope protein. Functional assays confirmed that mAb 2A10G6 bind with the fusion peptide and blocks infection primarily at a step after viral attachment. Conclusion: Together, these experiments define the characteristics of a novel flavivirus cross-reactive neutralizing MAb 2A10G6 and make it a suitable candidate for humanization into a therapeutic antibody to treat severe flavivirus infections in human.

Cross-Protective Potential of a Novel Monoclonal Antibody Directed against Antigenic Site B of the Hemagglutinin of Influenza A Viruses

The hemagglutinin (HA) of influenza A viruses has been classified into sixteen distinct subtypes (H1–H16) to date. The HA subtypes of influenza A viruses are principally defined as serotypes determined by neutralization or hemagglutination inhibition tests using polyclonal antisera to the respective HA subtypes, which have little cross-reactivity to the other HA subtypes. Thus, it is generally believed that the neutralizing antibodies are not broadly cross-reactive among HA subtypes. In this study, we generated a novel monoclonal antibody (MAb) specific to HA, designated MAb S139/1, which showed heterosubtypic cross-reactive neutralization and hemagglutination inhibition of influenza A viruses. This MAb was found to have broad reactivity to many other viruses (H1, H2, H3, H5, H9, and H13 subtypes) in enzyme-linked immunosorbent assays. We further found that MAb S139/1 showed neutralization and hemagglutination-inhibition activities against particular strains of H1, H2, H3, and H13 subtypes of influenza A viruses. Mutant viruses that escaped neutralization by MAb S139/1 were selected from the A/Aichi/2/68 (H3N2), A/Adachi/2/57 (H2N2), and A/WSN/33 (H1N1) strains, and sequence analysis of the HA genes of these escape mutants revealed amino acid substitutions at positions 156, 158, and 193 (H3 numbering). A molecular modeling study showed that these amino acids were located on the globular head of the HA and formed a novel conformational epitope adjacent to the receptor-binding domain of HA. Furthermore, passive immunization of mice with MAb S139/1 provided heterosubtypic protection. These results demonstrate that MAb S139/1 binds to a common antigenic site shared among a variety of HA subtypes and neutralizes viral infectivity in vitro and in vivo by affecting viral attachment to cells. The present study supports the notion that cross-reactive antibodies play some roles in heterosubtypic immunity against influenza A virus infection, and underscores the potential therapeutic utility of cross-reactive antibodies against influenza.

Keratinization and ultrastructure of the epidermis of late embryonic stages in the alligator (Alligator mississippiensis).

Using specific anti-beta keratin and general anti-alpha keratin antibodies, keratins were located in the epidermis of the alligator during the final developmental stages by ultrastructural and immunocytochemical methods. The maturation of the bilayered periderm (= embryonic epidermis) coincides with the disappearance of cell organelles, including the 25-35-nm-thick coarse filaments, and the coalescing of alpha-keratin filaments into a compact mass. The plasmalemma of peridermal cells forms a 15-25-nm-thick electron-dense corneous envelope. These changes start at stage 25, about 3 weeks before hatching, and continue until hatching when the embryonic epidermis is shed. Immature beta-keratogenic cells beneath the embryonic epidermis accumulate immunolabelled beta-filaments which are packed into thin, electron-pale beta-keratogenic cells in the corneous layer. Together, electron-pale and electron-dense materials form a compact 3-4-nm filament pattern of beta-keratin. Melanosomes from epidermal melanocytes, incorporated into beta-cells, give rise to the banded skin pattern of hatchlings. Beta-keratin production is much reduced in the hinge regions, where many alpha-filaments remain packed together with lipid droplets or mucous granules into thinner, more electron-dense, alpha-cells. The keratinaceous material of the alpha-cells is mostly concentrated along the cell membrane, while the lipid/mucous material remains centrally located, as in sebokeratinocytes of the apteric areas of avian skin. Some lipid and mucus is also incorporated into typical beta-cells of the outer scale surface, so that lipids are part of the fully keratinized hard keratin layer of the alligator. Lipids within beta-cells of outer scale surfaces and alpha-cells of the hinge region are probably responsible for limiting water loss and ion movements across the skin. Neither typical mammalian keratohyalin granules nor lepidosaurian keratohyalin-like granules were detected anywhere in alligator epidermis. The combination of anti-beta and anti-alpha keratin antibodies revealed different distributions of beta- and alpha-keratins. In late embryonic stages (25-26 to hatching), beta-keratin occurs only in the upper suprabasal cells, in prekeratinized and keratinized layers, whereas alpha-keratin bundles (tonofilaments) remain only in the lowest layers. The cross-reactivity of the beta-antibody, produced against a chick scale keratin, further shows that avian and crocodilian hard (beta) keratins share common antigenic sites, reflecting a phylogenetic affinity between these taxa.

Generation of antibodies for soil aggregating basidiomycete detection as an early indicator of trends in soil quality

Polyclonal antisera were raised against cell walls of a soil aggregating basidiomycete isolated in eastern Montana (isolate BB1) which has been identified as a member of the russuloid clade using molecular genetic techniques. In cross-reactivity tests using quantitative enzyme-linked immunosorbent assay (ELISA), polyclonal antisera to BB1 cross-reacted significantly with fungal species representative of the russuloid clade and little or no reactivity was observed with fungal species of the polyporoid, euagaric, hymenochaetoid, bolete and gomphoid–phalloid clades of the Homobasidiomycetes. These results suggested that the cell walls of fungal species from the russuloid clade share common antigenic binding sites that are recognizable by the polyclonal antibodies and that these sites were not found or were present in small amounts in fungal species from the other clades. Experiments on the water stability of artificial aggregates amended with fungal species representative of these six homobasidiomycete clades indicated that many species of the russuloid clade were very efficient soil stabilizers. Fungal species from the other clades vary in their ability to aggregate soil particles but cross-react weakly with the antibodies. ELISA was used on water stable aggregates (WSA) from soil samples of three dryland locations under conventional tillage, no tillage and fallow management practices and on WSA from soil samples from grass barrier strips that were undisturbed for 30 years. Greater antigenic response was observed from WSA of undisturbed soils compared to cultivated soils and from WSA of soils under no till compared to till or fallow management practices. These results suggested that specific soil aggregating russuloids in WSA are sensitive to soil disturbance such as tillage. To our knowledge, this study is the first report of the detection and quantification of a taxonomic group of specific soil aggregating Homobasidiomycetes in dryland soils under diverse agricultural systems.

Deduced structural model for animal rhabdovirus glycoproteins.

The G protein sequences of fourteen animal rhabdoviruses, representing all four recognized genera (Vesiculovirus, Lyssavirus, Ephemerovirus and Novirhabdovirus) and the ungrouped sigma virus, were aligned using CLUSTAL W and adjusted to account for obvious sequence similarities not detected by the algorithm. Analysis of the alignment indicated remarkable preservation of G protein structural features including cysteine residues, antigenic sites and significant elements of secondary structure (alpha-helices, beta-strands and loops). Twelve highly conserved cysteine residues were assigned numbers (C(I) to C(XII)) according to their location in the alignment. Other cysteine residues were assigned numbers (C0 to C(XIIe)) according to their position relative to the conserved cysteines. The pattern of conservation of cysteine residues and the structural characteristics of identified discontinuous antigenic sites were used to deduce a model for G protein structure. Six absolutely conserved cysteines are predicted to associate in three disulphide bridges (C(I)-C(XII); C(VIII)-C(XI); C(IX)-C(X)) that form the core of the G protein structure and define the common discontinuous antigenic site. The associations of six other highly conserved cysteines (C(II)-C(IV); C(III)-C(V); C(VI)-C(VII)) are predicted by the absence of a specific pair in all viruses within a genus. Of the other cysteines, one pair occurs only in ephemeroviruses and novirhabdoviruses (C0-C(XIIa)); two pairs occur only in ephemeroviruses (C(Ib)-C(VIIIa); C(XIIb)-C(XIIe)); and two pairs occur only in lyssaviruses (C(Ia)-C(VIIIb); C(XIIc)-C(XIId)). The structures predicted by the model account for the preservation of conformational antigenic sites, accommodate genus-specific variations, and are generally consistent with previous observations of G protein structure.

Antibodies against lysophosphatidylcholine and oxidized LDL in patients with SLE.

Lysophosphatidylcholine (LPC) is present in oxidized low density lipoprotein (oxLDL), which is implicated in atherosclerosis. Antibodies to cardiolipin (aCL) and oxLDL (aoxLDL) have been shown to crossreact. LPC is formed by hydrolysis of phosphatidylcholine (PC) in LDL and cell membranes, induced by phospholipase A2 or by oxidation. We here demonstrate the presence of enhanced antibody levels to LPC in 184 patients with SLE as compared to 85 healthy, age-matched controls. The antibody reactivity to LPC was not specifically related to oxidation of the fatty acid moiety in LPC, since LPC containing only the saturated fatty acid palmitic acid showed equivalent antibody levels as LPC containing unsaturated fatty acids. aPC were significantly lower as compared to aLPC, indicating that hydrolysis of PC at the sn-2 position increases the antigenic potential of the molecule. Beta-glycoprotein 1 was a cofactor for aCL, but not for aoxLDL or aLPC, and the antigenicity of these compounds is therefore not directly related to beta2GP1. There was a close correlation between aoxLDL, aCL and aLPC and both LPC and oxLDL competitively inhibited aCL-binding to CL. LPC, oxLDL and CL thus display a common antigenic site, which could be formed by removal of a fatty acid at the sn-2 position, possibly due to the activity to phospholipase A2 and/or oxidation. This study indicates the potential role of LDL-oxidation and phospholipase A2 in SLE.

Identification of a common antigenic site in the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N protein genes from two PRRSV isolates Olot/91 (European) and Quebec 807/94 (North American) were cloned and expressed in Escherichia coli using the pET3x system. The antigenic structure of the PRRSV N protein was dissected using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E.coli. Three antigenic sites were found. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein or at least a large fragment comprising the first 78 residues, respectively. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50 to 66). This hydrophillic region is well conserved among different isolates of European and North American origin. However, since this epitope is not recognized by many pig sera, it is not adequate for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire N protein.

Epitope mapping of the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus.

Two major genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) have been described, which correspond to the European and North American isolates. PRRSV nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N genes from two PRRSV isolates, Olot/91 (European) and Québec 807/94 (North American), were cloned and expressed in: (i) baculovirus under the control of the polyhedrin promoter and (ii) Escherichia coli using the pET3x system. The N protein from both isolates was expressed much more efficiently in E. coli as a fusion protein than in baculovirus. The antigenicity of the protein was similar in both systems and it was recognized by a collection of 48 PRRSV-positive pig sera. The antigenic structure of the PRRSV N protein was investigated using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E. coli. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein, or at least a large fragment comprising the first 78 residues. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50-66). This region is well conserved among different isolates of European and North American origin and is the most hydrophilic region of the protein. However, this epitope, although recognized by the MAbs and many pig sera, is not useful for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire protein.

Detection by competitive enzyme-linked immunosorbent assay of a bovine serum albumin peptide (ABBOS) in infant formulas based on hydrolyzed cow's milk protein.

Our aim was to determine whether currently available products based on hydrolyzed milk protein and a small peptide (ABBOS) in bovine serum albumin (BSA) (positions 126-144) share common antigenic sites. The commercial products are primarily developed to reduce cow's milk protein-associated allergenicity, but whether they also could be used in intervention trials to elucidate the role of BSA in the etiology of IDDM is not known. A sensitive competitive enzyme-linked immunosorbent assay (ELISA) using a rabbit polyclonal antibody raised against the ABBOS peptide in BSA was developed. With this method, we determined cross-reactivity between the ABBOS peptide and commercially available products: four milk protein hydrolysates and eight infant formulas based on hydrolyzed milk protein. The products were further characterized by physicochemical techniques. Hydrolyzed milk protein products are found to inhibit competitively the binding of ABBOS peptide to antibody. The number of residual reactive sites varied considerably among products and was not strongly related to the degree of hydrolysis (DH) or the molecular mass distribution of the hydrolysates. The presence of possible immunoreactive peptides in infant formulas based on hydrolyzed cow's milk protein cannot be adequately predicted by the DH or molecular mass distribution of the hydrolysates. Its specific determination is needed to ensure infant formulas free of cross-reactive ABBOS antibody binding sites for use in ongoing and forthcoming intervention trials to elucidate the role of BSA as a possible environmental triggering agent of IDDM.

Epitope mapping of the Ro/SSA60KD autoantigen reveals disease-specific antibody-binding profiles.

Anti-Ro60KD autoantibodies are commonly found in sera from patients with primary Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). In order to identify the epitopes of this autoantigen, 22-mer, synthetic peptides overlapping by eight residues, and covering the entire sequence of the Ro60KD autoantigen were prepared. Three groups of sera were evaluated according to their autoantibody specificites. The first group consisted of monospecific anti Ro60KD sera from four patients with SLE and one with SS, the second one was composed of anti-Ro60KD+anti-La(SSB)-positive sera from four patients with SS and the third group included three normal sera and one anti Ro52KD serum. It was found that sera from SLE patients interact with a common antigenic site spanning the sequence TKYKQRNGWSHKDLLRSHLKP (169-190) of the Ro60KD protein. On the other hand, sera from SS patients recognise the ELYKEKALSVETEKLLKYLEAV (211-232) region of this autoantigen. Determination of the minimal required peptide length for optimal antibody recognition showed that the defined epitopes can be shortened to the NGWSHKDLLR (175-184) and KALSVETEKLLKYLEAV (216-232) sequences respectively. Inhibition experiments using the Ro60KD antigen and soluble peptides corresponding to the 175-184 and 216-232 segments further confirmed the specific antibody binding. These results, although only a small number of sera were used, indicate that the Ro60KD autoantigen, which is not characterized by disease specificity, contains two discrete epitopes specifically recognized from SLE and SS patient sera. Finally, the sequence similarity of the NGWSHKDLLR (175-184) epitope with some of the HLA haplotypes, associated with anti-Ro response, deserves to be noted.

Conformational analysis of the Campylobacter jejuni porin

The major outer membrane protein (MOMP) of Campylobacter jejuni was purified to homogeneity by selective solubilization and fast protein liquid chromatography. The amino acid composition of the MOMP indicates the presence of cysteine residues. The amino-terminal sequence, determined over 31 residues, shows no significant homology with any other porin from gram-negative bacteria except in a discrete region. Immunocross-reactivity between Escherichia coli OmpC and the MOMP was analyzed, and a common antigenic site between these two porins was identified with an anti-peptide antibody. From circular dichroism and immunological investigations, the existence of a stable folded monomer, containing a high level of beta-sheet secondary structure, is evident. Conformational analyses show the presence of a native trimeric state generated by association of the three folded monomers; the stability of this trimer is reduced compared with that of E. coli porins. This study clearly reveals that the C. jejuni MOMP is related to the family of trimeric bacterial porins.

TSH and TSH Receptor Antibody-Binding Sites in Fibroblasts of Pretibial Myxedema Are Related to the Extracellular Domain of Entire TSH Receptor

The role of TSH receptor antibodies in the pathogenesis of pretibial myxedema is still unclear. This study was designed to determine whether patients with pretibial myxedema had higher serum titers of TSH receptor antibodies, and whether there were TSH and TSH receptor antibody-binding sites on plasma membranes of fibroblasts derived from the skin of pretibial myxedema. If there were, were the binding sites similar to the TSH receptor? The TSH receptor antibodies were determined with radioreceptor assay in 20 normal subjects, 18 hyperthyroid Graves' disease patients without ophthalmopathy, 26 hyperthyroid Graves' disease patients with ophthalmopathy, and 11 patients with pretibial myxedema associated with Graves' ophthalmopathy. TSH and TSH receptor antibody-binding sites were studied on plasma membranes of fibroblasts cultured from the skin of pretibial myxedema with radioreceptor assay. RNA was also extracted from the fibroblasts of pretibial myxedema and reverse transcribed using random primers as the primers for cDNA synthesis. The resulting cDNAs were subjected to amplification by polymerase chain reaction with the use of a set of primers spanning the 5′ region (+256/+275 and 616/+635) and the 3′ region (+1819/+1838 and +2405/+2424) of the TSH receptor cDNA (+1 transcription start codon). They were further identified by Southern blot hybridization, with the probe spanning the 5′ region (+272/+612) and the 3′ region (+1908/+2268) of the TSH receptor cDNA (+1 transcription start codon), and sequencing. The results showed that patients with pretibial myxedema had higher titers of TSH receptor antibodies in the serum. TSH and TSH receptor antibody-binding sites were present on plasma membranes of fibroblasts derived from the skin of pretibial myxedema patients and related to the extracellular domain of the TSH receptor. These data suggest a common antigenic site in the skin and in the thyroid as a putative target for TSH receptor antibodies or lymphocytes of Graves' disease.

Immunity to Hantavirus challenge in Meriones unguiculatus induced by vaccinia-vectored viral proteins.

Vaccinia virus recombinants were constructed that incorporated genomic sequences coding for the nucleoprotein (N) and glycoproteins (G1 and G2) of the hantavirus R22 strain isolated from a rat in China, and designated as RNV and RMV9, respectively. The proteins expressed by RNV and RMV9 were identified by radioimmunoprecipitation and indirect immunofluorescence assay using a panel of monoclonal antibodies and polyclonal immune sera, and were found to be antigenically indistinguishable from authentic R22 viral proteins. Both RNV and RMV9 elicited an anti-R22 antibody response in Mongolian gerbils (Meriones unguiculatus) with titers ranging from 6,400 to 12,800 by enzyme-linked immunosorbent assay, but only RMV9 produced neutralizing antibodies to R22 virus (titer 1:200) and Hantaan (HTN) virus (titer 1:20). The ability of these recombinants to protect Mongolian gerbils against challenge with R22 and HTN viruses was examined. The RMV9 recombinant induced a complete protective immune response against challenge with 10(4) plaque-forming units (PFU) of both R22 and HTN viruses, while RNV induced partial protection against a challenge with the homologous R22 virus and the heterologous HTN virus at a dose of 10(3) PFU. Our data show that the common antigenic sites responsible for eliciting a protective response are located mainly on hantavirus glycoproteins, and that the nucleoprotein may also confer partial cross-protection that presumably involves cell-mediated as well as humoral mechanisms.

Asparagine-linked glycoprotein biosynthesis in rat epididymis. Presence of a mannosidase II-like enzyme

Previous studies from this laboratory using p-nitrophenyl alpha-D-mannoside (p-NPM) as substrate provided no evidence for the presence of mannosidase II in the rat epididymis [Skudlarek & Orgebin-Crist (1988) J. Reprod. Fertil. 84, 611-617]. However, rat epididymal epithelial cells cultured in the presence of swainsonine, an inhibitor of mannosidase II, produce abnormally processed N-linked glycoproteins containing hybrid-type oligosaccharides instead of complex-type [Tulsiani, Skudlarek & Orgebin-Crist (1990) Biol. Reprod. 43, 130-138], a result providing indirect evidence for the presence of mannosidase II-like enzyme in rat epididymis. In the studies described here, we present evidence for the occurrence of this processing enzyme in rat epididymal Golgi membranes. This enzyme is an integral Golgi membrane component. Like liver mannosidase II, the epididymal enzyme cleaves alpha 1,3- and alpha 1,6-linked mannosyl residues from GlcNAcMan5GlcNAc. However, unlike liver mannosidase II, the epididymal enzyme shows no activity towards the synthetic substrate, p-NPM. The epididymal mannosidase cross-reacts with liver anti-(mannosidase II) antibody, a result suggesting that the two enzymes share a common antigenic site(s). Immunoblotting studies following resolution of liver and epididymal Golgi membranes on SDS/PAGE show that, whereas the liver mannosidase II was resolved as a doublet of Mr 120,000 and 122,000, only the Mr 120,000 band was observed in the epididymal Golgi membranes. Immunoblotting of the Golgi-rich fractions, resolved under non-denaturing conditions, showed different patterns of charge and/or size isomers from the two tissues. These studies demonstrate tissue-specific differences in processing enzymes with similar function.

Characterization of multi-cellulases of Sporotrichum cellulophilum by using monoclonal antibodies

Cellulases of Sporotrichum cellulophilum, CII1, CII2, CII3, CII5, CIII1, CIII2, and CIV1 were purified to homogeneity by column chromatographies and preparative electrophoresis. The specific activity of CII3 was the highest (100 units/mg), while the activities of CII5, CIII1, and CII2 were low (12–17 units/mg). The molecular weight of CII2 was 19,000, while those of the others ranged from 46,000–63,000. All the cellulases were glycoproteins except for CII2. Three monoclonal antibodies (Mab CII11, Mab CII12, and MabCII13) were prepared against CII1, and Mab CIV11 against CIV1. From the immunological reactivities, it was found that CII1, CIII2, and CIV1 had a common antigenic site recognized by Mab CII11, Mab CII12, and Mab CII13, while CII12 and CVI1 had a different antigenic site that was recognized by Mab CIV11, MabCII11, MabCII12, MabCII13, and MabCIV11 did not react with CII2, CII3, CII5, and CIII1. The classification of these cellulases was discussed.

Identification and characterization of T helper cell epitopes of the major outer membrane protein of Chlamydia trachomatis.

Chlamydia trachomatis serovars A, B, and C are the causative agents of trachoma, the world's leading cause of preventable blindness. Immunoprophylaxis is a possible approach to control trachoma. The chlamydial major outer membrane protein (MOMP) is thought to play an important role in the development of protective immunity against chlamydial infection, and is therefore considered to be a promising candidate antigen in the development of a trachoma vaccine. Much effort has been focused on the molecular characterization of B cell sites of the MOMP that elicit neutralizing antibodies. Neutralizing sites have been identified as linear epitopes that reside within variable domains (VDs) of the protein whose primary sequences vary among different serovars. No information exists on MOMP T helper (Th) cell antigenic determinants, which are likely critical components for the development of a successful chlamydial vaccine. We used overlapping synthetic peptides (25 mers) representing the entire primary sequence of serovar A MOMP in T cell proliferation assays to identify T cell antigenic determinants of this molecule. Eight synthetic peptides (A-2, A-3, A-7, A-8, A-11, A-22, A-23, and A-24) stimulated proliferative responses of splenic T cells isolated from MOMP-immunized A/J mice. To ascertain if these peptides functioned as Th cell antigens, we determined their ability to prime A/J mice in vivo to produce an anamnestic IgG response specific to the MOMP. Mice primed with synthetic peptides A-8 (106-130) or A-23 (331-355) produced IgG antibodies reactive with the native MOMP and with the synthetic peptides corresponding to surface-accessible serovar-specific epitopes located in VD I and serogroup-specific epitopes located in VD IV of the protein. We synthesized the A-8 and A-23 peptides with the VD I sequence as colinear chimeric peptides. Immunization of mice with the T/B cell peptides produced high titered antibodies against the VD I sequence, and these antibodies reacted with the native MOMP and intact chlamydiae. The MOMP sequences containing these Th cell epitopes are conserved among the MOMP genes of different C. trachomatis serovars, indicating that they are common Th cell antigenic sites. Thus, the Th cell epitopes contained within these peptides, in combination with different trachoma serovar-specific B cell neutralizing determinants, may be useful in the development of a synthetic or recombinant trivalent trachoma vaccine.

Comparative study on cytochrome P‐450 of yeasts using specific antibodies to cytochromes P‐450alk and P‐45014DM

The occurrence of cytochrome P-450(14DM) (lanosterol 14 alpha-demethylase) and cytochrome P-450alk (long-chain alkane terminal hydroxylase) in various yeast strains was determined with immunological procedures. Cytochrome P-450(14DM), which is constitutive or housekeeping enzyme playing an essential role in ergosterol biogenesis, was found in all yeast strains so far tested. Cytochromes P-450(14DM) from different species of yeast were immunologically different, although they may have had a few common antigenic sites. In contrast, cytochrome P-450alk was detected only in the alkane-assimilating yeasts.

Biological function of bean pathogenesis-related (PR 3 and PR 4) proteins

Two of the four bean acidic PR proteins (PR 1, PR 2, PR 3 and PR 4), namely PR 3 and PR 4 are acidic chitinases. These two chitinases, localized in the intercellular spaces, share common antigenic sites, but possess no antigenic sites,similar to those of the vacuolar basic isoform (ethylene-induced bean chitinase). These two acidic enzymes account for about 10% of the total chitinase activity found in chemically stressed or virus-infected bean plants. In addition to the four previously characterized bean acidic PR proteins, two additional acidic PR proteins have been detected showing 1,3-β-glucanase activity. These two proteins cross-react with antisera raised against bean basic 1,3-β-glucanase and against a maize acidic glucanase.

'Salivary peptide P-C' of human pancreatic B-cells shares only partly immunoreactivity with salivary peptide P-C indicating a new B-cell protein which is different from insulin.

Salivary peptide P-C like immunoreactivity, originally isolated from human whole saliva has later been found in the human pancreatic B-cells. In the present work an indirect immunofluorescence technique using monoclonal antibodies against isolated salivary peptide P-C was applied to Bouin fixed pancreas and parotid glands to study the possible identity of the two substances. Positive P-C immunofluorescence was found in the serous cells of parotid glands but not in pancreatic B-cells, suggesting that pancreatic P-C substance is not salivary peptide P-C itself, but a substance sharing the common antigenic site with salivary peptide P-C. To examine this, an indirect immunofluorescence technique using polyclonal P-C antisera pre-absorbed with six kinds of synthetic fragments (1-22, 23-44, 23-29, 30-44, 30-38 and 38-44) of salivary peptide P-C was applied to the human pancreas. The result showed that pancreatic P-C substance was a substance which shares the common antigenic site with the 38-44 amino acid residue of salivary peptide P-C. Western blot analysis using extracts of human pancreata further showed that pancreatic P-C substance is not a precursor of insulin but a protein with molecular weight of 11,500 dalton, indicating the presence of a new protein in the insulin secretory granules of human pancreatic B-cells.