Identification of a surface-exposed immunodominant epitope on outer membrane protein P1 of Haemophilus influenzae type b.

Eight murine monoclonal antibodies (MAbs) directed against outer membrane protein P1 of Haemophilus influenzae type b were generated and characterized. Seven of the eight MAbs reacted with recombinant P1 and purified P1 protein from H. influenzae type b strains MinnA and 1613; MAb P1.8 was specific for the latter strain. A panel of 32 nontypeable and 140 encapsulated Haemophilus strains recovered worldwide representing the major clonal families of serotypes a, b, and d was used to evaluate the distribution among Haemophilus strains of the epitopes identified by the P1-specific MAbs. The epitope reactive with the seven MAbs which recognized P1 from strains MinnA and 1613 was shared by 92% of the encapsulated Haemophilus isolates tested. The epitope is present in the H. influenzae type b strains from clonal families commonly recovered from cases of invasive disease in North America and Europe. A series of nested 5' and 3' deletions of the P1 gene were constructed and analyzed to localize the determinants on P1 recognized by the MAbs. MAbs P1.2, P1.4, P1.5, P1.6, and P1.7 recognized an epitope localized to the carboxy-terminal portion of P1. Murine MAbs P1.1 and P1.3 and two human MAbs, HiH-7 and HiH-10, recognized a complex epitope which was partially localized to the carboxy-terminal portion of the P1 protein. These data indicate that an immunodominant surface-exposed epitope is present on the carboxy-terminal portion of the P1 protein of type b Haemophilus isolates responsible for the majority of invasive disease in North America.     

Characterization of monoclonal antibodies against the Escherichia coli hemolysin.

Twelve monoclonal antibodies (MAbs) produced against the Escherichia coli hemolysin (HlyA) encoded by the hemolysin recombinant plasmid pWAM04 were studied. HlyA derivatives from recombinant strains with different plasmids encoding HlyA amino-terminal and carboxy-terminal truncates, HlyA in-frame deletions, and HlyA frameshift mutations were used in immunoblots to localize the antigenic determinants for the anti-HlyA MAbs. The mapping of the MAb epitopes was also facilitated by immunoblotting analysis of HlyA polypeptide fragments derived by cyanogen bromide cleavage. The HlyA epitopes for 11 of the MAbs were mapped to relatively small linear regions of the cytolysin ranging from 28 to 160 amino acids. Five of the MAbs (C10, G8, E2, B7, and D12) neutralized HlyA hemolytic activity to varying degrees. The epitopes for these neutralizing MAbs were found to reside within the following HlyA regions: C10 and G8, amino acids 2 to 160; E2, amino acids 161 to 194; B7, amino acids 518 to 598; and D12, amino acids 626 to 726. Hemolytically active HlyA was dependent on the action of the hlyC gene product. The D12 MAb recognized only HlyA produced by strains with an intact hlyC function. MAb A10 recognized an epitope within the HlyA region from amino acids 728 to 829 where a glycine-rich repeat domain exists; however, this MAb did not neutralize HlyA hemolytic activity. A HlyA domain map showing the anti-HlyA epitope location was constructed.     

Mapping of bactericidal epitopes on the P2 porin protein of nontypeable Haemophilus influenzae.

The P2 porin protein is the major outer membrane protein of nontypeable Haemophilus influenzae and is a potential target of a protective immune response. Nine monoclonal antibodies (MAbs) to P2 were developed by immunizing mice with nontypeable H. influenzae whole organisms. Each MAb reacted exclusively with the homologous strain in a whole-cell immunodot assay demonstrating exquisite strain specificity. All nine MAbs recognized abundantly expressed surface-exposed epitopes on the intact bacterium by immunofluorescence and immunoelectron microscopy. Each MAb was bactericidal to the homologous strain in an in vitro complement-mediated killing assay. Immunoblot assay of cyanogen bromide cleavage products of purified P2 indicated that MAb 5F2 recognized the 10-kDa fragment, and the other eight MAbs recognized the 32-kDa fragment. Competitive ELISAs confirmed that 5F2 recognized an epitope that is different from the other eight MAbs. To further localize epitopes, MAbs 5F2 and 6G3 were studied in protein footprinting by using reversed-phase high-performance liquid chromatography. Three potential epitope-containing peptides which were reactive in an enzyme-linked immunosorbent assay with both 5F2 and 6G3 were isolated. These peptides were identified by N-terminal amino acid sequence and localized to loops 5 and 8 of the proposed model for P2. Fusion proteins consisting of glutathione S-transferase fused with variable-length peptides from loops 5 and 8 were expressed in the pGEX-2T vector. Immunoblot assay of fusion peptides of loops 5 and 8 confirmed that 5F2 recognized an epitope within residues 338 to 354 of loop 8; 6G3 and the remaining MAbs recognized an epitope within residues 213 to 229 of loop 5. These studies indicate that nontypeable H. influenzae contains bactericidal epitopes which have been mapped to two different surface-exposed loops of the P2 molecule. These potentially protective epitopes are strain specific and abundantly expressed on the surface of the intact bacterium.     

Characterization of three novel monoclonal antibodies against hepatitis C virus core protein

Three novel monoclonal antibodies (MAbs) were established against a recombinant hepatitis C virus (HCV) core protein derived from cloned genotype 1b HCV cDNA. MAbs C7-50 and C8-59 recognize a conserved linear epitope represented by amino acid residues 21 to 40 of the nucleocapsid protein. MAb C8-48 is directed against a strain-specific conformational epitope located within the first 82 amino acids. A sensitive two-site MAb-based immunoradiometric assay was established using antibodies directed against distinct epitopes on the nucleocapsid protein. Processed 21 kDa core protein was detected by immunoblotting in human hepatocellular carcinoma cell lines and primary adult rat hepatocytes transfected with a cytomegalovirus promoter-driven expression construct. Immunofluorescence microscopy studies revealed a granular and vesicular cytoplasmic staining pattern. MAb C7-50 was used successfully to detect HCV core antigen in chronically infected chimpanzee liver tissue. These MAbs represent important reagents for the study of HCV biology and for the development of immunodiagnostic assays. © 1996 Wiley-Liss, Inc.     

Neutralizing human monoclonal antibodies to hepatitis A virus recovered by phage display

Four human monoclonal antibodies (MAbs) to hepatitis A virus (HAV) were isolated from a phage-displayed antibody library constructed from the peripheral blood lymphocytes (PBLs) of HAV-immune donors. The four MAbs showed differences in their affinity: two (HA6, HA9) of them were dominant after four rounds of panning, and showed higher affinity than the other two (HA1, HA12). All four MAbs showed HAV-neutralizing activity in radioimmunofocus inhibition assay and their neutralizing activity was positively correlated with their affinities. Analysis of their epitope specificity by cross-competition binding assays suggested that HA6 and HA9 recognize extensively overlapping epitopes, which overlap with those of HA1 and HA12, although HA1 and HA12 recognize distinct epitopes. In addition, competition assays with known neutralizing murine MAbs suggested that the epitopes of four human MAbs extensively overlap with those of B5B3 and K34C8 which are distinct but reside within the single, immunodominant neutralization site on the HAV capsid. The human MAbs (HA6 and HA9) with highest affinity may be useful in the immunoprophylaxis of HAV infection.     

Antigenic characterization and analysis of the human immune response to outer membrane protein E of Branhamella catarrhalis.

Outer membrane protein E (OMP E) is a 50-kDa major OMP of Branhamella catarrhalis. Polyclonal antisera and four monoclonal antibodies (MAbs) to OMP E were generated to study its antigenic structure. All antibodies recognized epitopes in all 19 B. catarrhalis strains tested by immunoblot assays. By flow cytometry, it was determined that MAbs 1B3 and 9G10d recognized epitopes which are expressed on the surface of the intact bacterium, while MAbs IC11 and 7C10 recognized epitopes which were buried within the outer membrane. A competitive enzyme-linked immunosorbent assay showed that MAbs 1B3 and 9G10d recognize the same or closely related epitopes. Proteinase K treatment of whole bacterial cells revealed that MAbs 1B3 and 9G10d recognize a surface-exposed epitope located in the 17-kDa region towards the amino terminus of OMP E. The human serum and mucosal antibody responses to OMP E in adults with chronic bronchitis were studied. A majority of these patients had immunoglobulin A to OMP E in sputum supernatants. None of ten adults who experienced lower respiratory tract infections due to B. catarrhalis demonstrated a clear-cut rise in antibody titer to OMP E in serum or sputum supernatant. This study has demonstrated that OMP E has at least one surface-exposed epitope which is highly conserved among strains of B. catarrhalis and which is located in the amino-terminal 184 amino acids of the molecule.     

Monoclonal antibodies that distinguish antigenic variants of canine parvovirus

Canine parvovirus (CPV) is classified as a member of the feline parvovirus (FPV) subgroup. CPV isolates are divided into three antigenic types: CPV type 2 (CPV-2), CPV-2a, and CPV-2b. Recently, new antigenic types of CPV were isolated from Vietnamese leopard cats and designated CPV-2c(a) or CPV-2c(b). CPV-2c viruses were distinguished from the other antigenic types of the FPV subgroup by the absence of reactivity with several monoclonal antibodies (MAbs). To characterize the antigenicity of CPV-2c, a panel of MAbs against CPV-2c was generated and epitopes recognized by these MAbs were examined by selection of escape mutants. Four MAbs were established and classified into three groups on the basis of their reactivities: MAbs which recognize CPV-2a, CPV-2b, and CPV-2c (MAbs 2G5 and 20G4); an MAb which reacts with only CPV-2b and CPV-2c(b) (MAb 21C3); and an MAb which recognizes all types of the FPV subgroup viruses (MAb 19D7). The reactivity of MAb 20G4 with CPV-2c was higher than its reactivities with CPV-2a and CPV-2b. These types of specificities of MAbs have not been reported previously. A mapping study by analysis of neutralization-resistant mutants showed that epitopes recognized by MAbs 21C3 and 19D7 belonged to antigenic site A. Substitution of the residues in site B and the other antigenic site influenced the epitope recognized by MAb 2G5. It was suggested that the epitope recognized by MAb 20G4 was related to antigenic site B. These MAbs are expected to be useful for the detection and classification of FPV subgroup isolates.     

Antigenic analysis monoclonal antibodies against different epitopes of σB protein of Muscovy duck reovirus

σB is one of the major structural proteins of Muscovy duck reovirus (DRV), which is able to induce protective immune response in target birds. Four anti-DRV σB MAbs were identified belong to two distinct epitopes, designated A (1E5, 4E3, and 5D8) and B (2F7) (Liu et al., 2010). To understand antigenic determinants of the σB protein, a set of 20 (P1-P20), partially overlapping and consecutive peptides spanning σB were expressed and then screened by MAbs. With Western blot and enzyme-linked immunosorbent assay (ELISA), two minimal units of the linear epitopes, 19YIRAPACWD27 (epitope B) and 65TDGVCFPHHK74 (epitope A), were identified within N-terminal region of the σB protein. The epitope B was highly conserved among DRV and avian reovirus (ARV) strains through sequence alignment analysis. Immunofluorescence assays (IFA) and ELISA, confirmed that epitope B is a broad group-specific epitope among DRV and ARV. Epitope A could only react with chicken embyonated fibroblast cells (CEF) infected with DRV, but not ARV. However, both peptides have good immunogenicity and could induce antibodies against DRV in BALB/c mice. This report documents the first identification of σB epitopes in the precise locations. The two probes would be useful in the development of discriminating diagnostic kits for DRV and ARV infection.     

Three epitopes located on the coat protein amino terminus of viruses in the bean common mosaic potyvirus subgroup

Summary.  Twenty-seven of 29 strains of viruses in the bean common mosaic virus (BCMV) subgroup of legume-infecting potyviruses reacted strongly with one or more of the monoclonal antibodies (MAbs) which are known to be specific for epitopes located along the 50 amino acids which constitute the N-terminal end of the viral coat protein. Approximately one half of the virus strains reacted with the N-terminal epitope specific (NTES) MAb 4G12 which is specific for epitope E/B4, while the other half reacted with NTES MAbs 4 A1 or 4F9 which are specific for epitope E/B3. All but two strains contained at least one of these epitopes while no strain contained both. Competitive assays using five sequential, non-overlapping, synthetic, 10mer peptides indicated that the amino acids critical for epitope E/B3 reaction were located at positions 5, 7, and 10 from the N-terminal end of the coat protein. By deduction we postulate that the amino acids critical for epitope E/B4 are located at positions 10, 16, and 17. Because epitope E/B3 requires isoleucine at position 10 for expression whereas epitope E/B4 requires valine to be expressed, no one strain can express both epitopes. Two viruses in our tests (azuki mosaic and Dendrobium mosaic viruses) had deletions in this portion of their sequence explaining their failure to react MAbs specific for either epitope. The critical amino acids for a third epitope, E/B3A, were located at positions 16 and 17. We found no correlation between any of the three N-terminal epitopes defined in this study and the presence or absence of any biological property that we could accurately measure: i.e., symptomatology, host range, or pathotype. However, when coat protein sequences were aligned according to epitope type E/B3 or E/B4, we found that sequences within groups had high levels of identity while between group identities were low. We also found that sequences in the 3′-end non-coding region exhibited similar relationships within and between epitope groups. Two strains of BCMV (NL-4 and RU-1) were found to possess coat protein sequences typical of epitope E/B4 but 3′-NCR sequences typical of epitope E/B3. These data suggest that both strains may be the result of natural recombinants between the two epitope groups. Received May 18, 1998 Accepted October 26, 1998     

Epitope mapping and functional analysis of sigma A and sigma NS proteins of avian reovirus

We have previously shown that avian reovirus (ARV) sigmaA and sigmaNS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on sigmaA (I and II) and three epitopes (A, B, and C) on sigmaNS. To further define the location of epitopes on sigmaA and sigmaNS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of sigmaA and sigmaNS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on sigmaA was located at amino acid residues 340QWVMAGLVSAA350 and epitope B on sigmaNS at amino acid residues 180MLDMVDGRP188. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of sigmaA and sigmaNS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete sigmaNS but not with truncated sigmaNS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on sigmaNS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on sigmaA was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the sigmaNS with MAb 1F9. The sigmaNS of ARV with ssRNA-binding activity could be blocked by monoclonal antibody 1F9. The epitope B on sigmaNS is required for ssRNA binding because its deletion fully abolished the ssRNA binding activity of sigmaNS.     

Mapping of three antigenic sites on the haemagglutinin-neuraminidase protein of Newcastle disease virus

Nine neutralizing monoclonal antibodies (MAbs), each of which react with the haemagglutinin-neuraminidase (HN) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV), have been used in competitive binding assays to delineate three non-overlapping antigenic sites A, B and C. Epitopes within these sites have been identified on the basis of cross-reactivity of MAb-resistant mutants against the panel of MAbs, determined by plaque assays and Western blotting. Site A contains three non-overlapping epitopes (A1, A2 and A3). A1 is the only linear epitope; all remaining epitopes are conformational. MAbs which react with epitopes A2 and A3 inhibit neuraminidase activity (NA) when assayed with neuraminlactose. Site B contains three partially overlapping epitopes (B1, B2 and B3) and site C is represented by a single epitope (C1). HN gene sequence analysis of MAb-resistant mutants showed that they each had only single amino acid substitutions which range from amino acid residues 347-460 for site A, 284-325 for site B, and at 481 for the C1 epitope. The apparent molecular mass of the HN glycoprotein of one mutant was increased from 72 to 75 kDa. This correlates well with the creation of an additional potential glycosylation site in this mutant from Asn-Ser-Pro(325) to Asn-Ser-Ser(325).     

An analysis of the properties of monoclonal antibodies directed to epitopes on influenza virus hemagglutinin

Summary Monoclonal antibodies (MAbs) specific for the hemagglutinin (HA) of the H3 subtype of influenza A virus were grouped according to their inability to bind to particular MAb-selected neutralization escape mutants of the virus having an amino acid substitution in one of the five postulated antigenic sites on the molecule. Additional residues critical to the binding of the MAbs were deduced from their patterns of reactivity with a panel of field strains and receptor mutants of the H3 subtype. The relationship of these residues to the actual epitopes recognized by the MAbs was inferred from their location on the three-dimensional structure of the HA molecule. In this way it was generally possible to identify a number of residues that are critical to the integrity of the epitope recognized by each of the MAbs examined. It was found that: (1) Several of these epitopes appear to be discontinuous and some may depend on residues contributed by more than one monomer. For example, residue 205, in the interface between monomers of the HA, was found to affect the integrity of the epitopes for several MAbs, possibly by stabilizing the conformation of residues around the receptor-binding pocket and/or in site B on the adjacent monomer. The activity of these particular MAbs was greatly decreased if the virus was exposed to pH 5. (2) All the MAbs tested neutralized viral infectivity and inhibited hemagglutination, although the single MAb directed to site C, which is the most distant from the receptor-binding site, was the least efficient. (3) Hemagglutination inhibition, and particularly neutralization tests, were more discriminating than ELISA in discerning subtle differences between the corresponding epitopes recognized by MAbs on different field strains. (4) Efficiency of neutralization of infectivity did not correlate consistently with hemagglutination inhibiting efficiency; MAbs postulated to bind to epitopes close to the receptor-binding pocket were very efficient at inhibiting hemagglutination, whereas neutralization efficiency tended to be more influenced by the affinity of binding of the MAb. (5) A MAb binding to any particular epitope could affect the binding of a second MAb directed to an epitope within the same or even a different antigenic site. The observed effect was most commonly inhibition of binding, which was not always reciprocal; enhancement of binding was also observed with certain combinations of MAbs. The relative affinity of the MAbs, in addition to steric constraints, were shown to be important factors in the ability to compete for interaction with HA.     

Characterization of adenovirus hexons by their epitope composition

Summary For the investigation of epitope composition of different adenovirus hexon types sixty-one mouse ascitic fluids containing monoclonal antibodies (MAbs) developed in three different panels were used. The distinction and marking of the different epitopes recognized by the MAbs were carried out by the determination of the composite cross-reactivity pattern, the titer and the correlation coefficient of all the 61 MAbs with 21 different hexon types representing all the six human subgenera, as well as different bovine and simian adenoviruses. The distinct epitopes were marked by two numbers refering the homologous hexon type to which the MAbs were directed and the serial number of the epitope specified by the different members of the given panel of the MAbs. The three panels of MAbs recognized 22 epitopes on the 21 hexon types among them a genus and three type specific ones and 18 different bi- and multilateral intertype (IT) specific epitopes that grouped adenoviruses within the genus, independently from the subgenus they belong to. Considering that the type specific epitope could be present only on the homologous hexon type, the largest number of the different epitopes distinguishable by the MAbs used could be 20 on the homologous hexon and 19 on the heterologous ones. It was found that the total number of IT specific epitopes on the hexons varied between 2 and 18. The distribution of the distinct specific epitopes on the different hexon types was different, as expected. The antigenic structure of the individual hexon types were characterized by the determination of their IT specific epitope spectrum. By pairwise analysis ten human hexon types formed three epitope clusters (types 4 and 19; types 8, 9, 9/13 and 10; as well as all types of subgenus C) showing identical epitope spectra. No clustering was found with human type 7, 12, 13, 18, 26, 27, 35 and 41, as well as with bovine and simian adenovirus hexons studied. However, they displayed a closer or looser antigenic relationship among each other and to members of the epitope clusters. The degree of antigenic relationship could be expressed by the similarity/dissimilarity percentage calculated from the number of the identical and different epitopes present on any two given hexon types.     

Epitopes on the S1 subunit of pertussis toxin recognized by monoclonal antibodies.

To identify the neutralizing epitopes on the S1 subunit (A promoter) of pertussis toxin, we characterized anti-S1 monoclonal antibodies (MAbs) X2X5, 3CX4, and 6FX1. We confirmed by immunoblot analysis that these MAbs bind to the S1 subunit and not to the B oligomer of pertussis toxin and that they recognize different epitopes by a competitive binding enzyme-linked immunosorbent assay. These MAbs had differential abilities to neutralize the lymphocytosis-promoting factor activity of pertussis toxin in mice: 3CX4 and 6FX1 had partial neutralizing abilities, while MAb X2X5 had none. With these MAbs, the epitopes on the S1 subunit were examined by using trypsinized S1 peptides, recombinant truncated S1 molecules, and synthetic peptides. The non-neutralizing MAb X2X5 bound in immunoblots to tryptic peptides of various sizes as small as 1.5 kilodaltons; the neutralizing MAbs 3CX4 and 6FX1 bound only to a 24-kilodalton tryptic peptide band. Immunoblot studies with recombinant truncated S1 molecules demonstrated that amino acid residues 7 to 14 and 15 to 26 play an important role in the binding of neutralizing MAbs and the non-neutralizing MAb, respectively. The binding of these MAbs was not dependent upon the presence of C-terminal amino acid residues 188 to 234. To further define B-cell epitopes, the binding of the MAbs we tested to synthetic peptides representing the entire S1 subunit were examined. Neutralizing MAbs 3CX4 and 6FX1 bound to none of these peptides, further suggesting that these MAbs recognize conformational epitopes. The non-neutralizing MAb X2X5 bound to peptides 11 to 26 and 16 to 30, demonstrating that the major antigenic determinant recognized by this MAb is a linear epitope located within residues 16 to 26.     

Monoclonal antibodies against human basic fibroblast growth factor

Recombinant human basic fibroblast growth factor (hbFGF) was used as an antigen to develop, by a somatic cell fusion technique, four monoclonal antibodies (MAbs), that recognize the complete and amino-terminal truncated form of hbFGF. Isotype identification showed that MAbs designated MAb12 and MAb98 were IgG1; and those designated MAb52 and MAb78 were IgG2b. All these MAbs bound the complete form of hbFGF produced in E.coli. Competition with synthetic polypeptides, a replication of 1-9 aa and of 141-146 aa of hbFGF, and truncated forms of hbFGF by 13 and 40 amino acid residues in its amino-terminal produced in E. coli by recombinant technique, revealed at least two epitopes recognized by the four IgG type MAbs. MAb12 and MAb78 recognized the epitope located within the first 9 amino acid residues at the amino terminal of the complete hbFGF. MAb52 and MAb98 recognized the one located between the amino acid residue no. 14 and 40. None of MAbs bound bovine acidic FGF (aFGF). Using MAb52 or MAb98 and MAb78, a two-site EIA has been developed. This EIA is sensitive enough to detect 0.5 ng/ml of hbFGF. Furthermore, MAb78 was used as a ligand for affinity chromatography to purify hbFGF mutein CS4, which binds weakly to a heparin affinity column.     

Monoclonal antibodies to cyanogen bromide fragments of glycophorin A

Eleven mouse monoclonal antibodies directed against epitopes on CNBr peptides of the major sialoglycoconjugate of the human red blood cell, glycophorin A, have been produced by hybridomas derived from P3-X63-Ag8.653 myeloma cells and spleen cells from BALB/c mice immunized with purified glycophorin. The monoclonal antibodies could be divided into four groups according to their reactivities with CNBr peptides in a direct ELISA assay: one antibody (6B5) that binds solely to the aminoterminal octapeptide (CNBr3); two antibodies (8F10 and 9C3) that bind to CNBrl (residues 9-81); two antibodies (3D2 and 4C6) that are reactive with CNBr2, The C-terminal portion of the molecule (residues 82-131); six antibodies (1B4, 4C3, 4E7, 7B10, 7C11 and 9D6) which are cross-reactive with an epitope on both CNBr1 and CNBR3 glycopeptides. This cross-reactive epitope(s) appears to involve both carbohydrate and protein residues.     

Reactivity with monoclonal antibodies of viruses from an episode of foot-and-mouth disease

A panel of 12 monoclonal antibodies (MAbs) raised against foot-and-mouth disease virus (FMDV) of serotype C1 (FMDV C-S8c1) and 11 MAbs raised against other FMDVs have been used to evaluate the reactivity of 14 isolates of FMDV of serotype C1 (series FMDV C-S), 12 of them from one disease episode (Spain 1979-1982). The assays used were immunoelectrotransfer blot, immunodot and neutralization of infectivity. None of the isolates could be clearly distinguished by its reactivity with 6 non-neutralizing and 2 neutralizing MAbs raised against FMDV C-S8c1. In contrast, the isolates were distinguished in two groups by a 10(2)-fold difference in their reactivity with 6 neutralizing MAbs. The reactivity of MAbs with synthetic peptides indicated that conserved and non-conserved epitopes recognised respectively by neutralizing MAbs 4G3 and SD6 are localized in the immunogenic region (amino acids 138-156) of VP1. Thus, epidemiologically related FMDVs differ in at least one epitope critical for virus neutralization.     

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.     

Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120

Recombinant gp120 of the HIV-1(IIIB) isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120(LAI). Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A,1570C,1570D,1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V(H)3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies.     

Antigenic organization of the N-terminal part of the polymorphic outer membrane proteins 90, 91A,and 91B of Chlamydophila abortus

A series of overlapping recombinant antigens, 61 to 74 residues in length, representing polymorphic outer membrane protein 90 (POMP90) of Chlamydophila abortus and two recombinant peptides spanning gene fragment p91Bf99 of POMP91B were assessed by immunoblotting to determine the antigen-binding sites of 20 monoclonal antibodies to POMP90, -91A, and -91B. The epitopes were further restricted by scanning 52 overlapping synthetic 12-mer peptides representing the N-terminal part of POMP90, and the 12-mer epitopes were then analyzed by using hexapeptides to the resolution of a single amino acid. Ten epitopes were defined: 1, TSEEFQVKETSSGT; 2, SGAIYTCEGNVCISYAGKDSPL; 3, SLVFHKNCSTAE; 4, AIYADKLTIVSGGPTLFS; 5, SPKGGAISIKDS; 6, ITFDGNKIIKTS; 7, LRAKDGFGIFFY; 7a, DGFGIF; 7b, GIFFYD; 8, IFFYDPITGGGS; 8a, FFYDPIT; 9, GKIVFSGE; and 10, DLGTTL. The 20-mer peptide LRAKDGFGIFFYDPITGGGS was a major epitope that was recognized by seven antibodies. Epitopes 7 to 10 were conserved in reference strains of the former species C. psittaci, whereas the strong antigenic peptides FYDPIT and IVFSGE were conserved among members of the genus CHLAMYDOPHILA: Epitopes 3 to 8 were located within the best-scoring beta-helical wrap (residues 148 to 293) predicted for POMP91B by the program BETAWRAP. Other studies have suggested an association of the POMPs with type V secretory autotransporter proteins. The results presented in this study provide some evidence for a passenger domain that is folded as a beta-helix pyramid with compact antigenic organization.