Unique epitope of bovine immunodeficiency virus gag protein spans the cleavage site between p16(MA) and p2L

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are closely related bovine lentiviruses that are difficult to distinguish by presently available diagnostic methods. Recently, in our laboratory, a monoclonal antibody (MAb; MAb 10H1) against the BIV Gag protein identified a differential epitope, located at the 6.4-kDa N terminus of a 29-kDa Gag capsid protein, which was absent in JDV. To define the essential amino acids of the epitope, a series of primers within the 163 bp of DNA corresponding to the 6.4-kDa protein were designed. The full-length 163-bp DNA fragment and the smaller DNA fragments with deletions were amplified by PCR and then cloned into pQE32 vectors for protein expression studies. The expressed proteins were analyzed with MAb 10H1 by Western blotting. The differential epitope has been narrowed to a 26-amino-acid region (R121 to R146), which includes 6 residues of p16(MA) (where MA represents the matrix protein) and 20 residues of p2L. A synthetic peptide corresponding to the putative 26-amino-acid epitope blocked MAb 10H1 binding to the expressed peptide. These experiments revealed that the epitope spans the cleavage site between p16(MA) and p2L and presumably will be valuable in distinguishing the two viruses.     

Differentiation of Two Bovine Lentiviruses by a Monoclonal Antibody on the Basis of Epitope Specificity

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically. A recombinant fusion protein containing the capsid protein of BIV expressed in Escherichia coli was used to immunize mice and produce monoclonal antibodies. Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further. Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein. The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses. However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV. Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein. This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.     

Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically. A recombinant fusion protein containing the capsid protein of BIV expressed in Escherichia coli was used to immunize mice and produce monoclonal antibodies. Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further. Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein. The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses. However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV. Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein. This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.     

Recombinant Jembrana disease virus gag proteins identify several different antigenic domains but do not facilitate serological differentiation of JDV and nonpathogenic bovine lentiviruses

In Indonesia, it is suspected that there are two bovine lentiviruses circulating in the cattle population: a pathogenic Jembrana disease virus (JDV), and a nonpathogenic bovine immunodeficiency-like virus (BIV). Both viruses cross-react antigenically and cannot be differentiated by current serological tests using JDV antigens. To identify possible type-specific epitopes, a series of recombinant protein constructs including the matrix, capsid and nucleocapsid proteins were produced from JDV gag and the expressed proteins were tested by Western blot using JDV and BIV hyperimmune sera. JDV matrix and truncated capsid proteins were recognised by both JDV and BIV hyperimmune sera indicating that there were multiple cross-reactive epitopes present in JDV gag. At least three epitopic regions were identified in these constructs, including the major homology region, by monoclonal antibody binding studies. JDV nucleocapsid recombinant protein was not recognised by either JDV or BIV hyperimmune sera and none of the recombinant gag proteins were able to differentiate between JDV positive sera from Jembrana disease endemic and Jembrana disease-free areas. Additionally, a 40 amino acid recombinant subunit protein encompassing the region recently found to contain an epitope unique to BIV [Zheng, L., Zhang, S., Wood, C., Kapil, S., Wilcox, G.E., Loughin, T.A., Minocha, H.C., 2001. Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity. Clin. Diagn. Lab. Immunol. 8, 283-287] was tested but was not recognised by either JDV positive sera from Jembrana disease-endemic or Jembrana disease-free areas.     

Identification of Residues within the Herpes Simplex Virus Type 1 Origin-Binding Protein That Contribute to Sequence-Specific DNA Binding

Gene UL9 of herpes simplex virus type 1 encodes an 851-amino-acid protein which is essential for viral DNA synthesis and functions as a sequence-specific origin-binding protein and DNA helicase. We generated monoclonal antibodies against purified UL9 protein and identified one such antibody (MAb 13924) that can block the interaction of the UL9 C-terminal DNA-binding domain (amino acids 534–851) with its recognition sequence. MAb 13924 interacted with immobilized peptides containing residues 780–786 of UL9. Although the corresponding region of the homologous protein encoded by varicella-zoster virus differs at only a single position it was not recognized by MAb 13924. Site-directed mutagenesis experiments confirmed that residues within this region contribute to the epitope recognized by MAb 13924 and may be involved in sequence-specific DNA binding. In addition, all eight lysine residues within the DNA-binding domain were separately changed to alanine and the DNA-binding properties of the mutated proteins were examined. The results showed that lysine residues that are located close to the peptide recognized by MAb 13924 or lie within the region of the DNA-binding domain most highly conserved among homologous alphaherpesvirus proteins play a role in sequence-specific DNA binding. Moreover, alteration of a lysine residue 18 amino acids from the recognized peptide prevented the interaction of MAb 13924 with the UL9 C-terminal DNA-binding domain. Three helical segments are predicted to occur within the region containing mutations that affect sequence-specific binding and interaction with MAb 13924.     

Tat protein expression in MDBK cells does not confer susceptibility to bovine immunodeficiency virus

Summary.  The ability of BIV strain R29 to infect bovine cell lines in the presence or absence of a functional lentiviral Tat protein is described. Jembrana disease virus (JDV) Tat protein was stably expressed in MDBK cells. No viral replication could be detected in this cell line after infection with BIV R29. Transfection of MDBK cells and MDBK Tat expressing cells with BIV R29 proviral DNA established that BIV R29 could not replicate in MDBK cells. Whether viral entry into MDBK cells is also a block to BIV R29 infection of MDBK cells has yet to be established. Received August 8, 2001 Accepted October 18, 2001     

Prior bovine immunodeficiency virus infection does not inhibit subsequent superinfection by the acutely pathogenic Jembrana disease virus

In cattle the interaction between the two genetically and antigenically related bovine lentiviruses, the acutely pathogenic Jembrana disease virus (JDV) and the non-pathogenic Bovine immunodeficiency virus (BIV) has not been reported although both JDV and a BIV-like virus have been reported in the Bali cattle (Bos javanicus) population in Indonesia. The outcome of infection of Bali cattle with the R29 strain of BIV prior to superinfection 42 days later with JDV_TAB/87 was determined. All BIV-inoculated cattle were successfully infected and developed an antibody response to the TM and CA proteins. BIV infection did not prevent subsequent infection with JDV or ameliorate the clinical signs of Jembrana disease in the infected cattle. It did, however, modify the dynamics of the JDV infection with an earlier onset and end of the acute disease process, and a reduction in the duration of viremia that exceeded 10⁶ genome copies/ml of plasma.     

Development of a Western Blot Assay for Detection of Bovine Immunodeficiency-Like Virus Using Capsid and Transmembrane Envelope Proteins Expressed from Recombinant Baculovirus

A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.     

Epitope Detection in the Envelope of Intracellular Naked Orthopox Viruses and Identification of Encoding Genes

Monoclonal antibodies (MAbs) were generated against vaccinia virus, cowpox virus KR2 Brighton, monkeypox virus Copenhagen, or ectromelia virus. Pairwise epitope specificity studies by competition ELISAs identified 23 distinct antigenic sites in 19 different orthopox virus strains. Six epitopes were completely independent of each other, and 17 closely related antigenic sites formed three separate epitope complexes. As shown by immunogold electron microscopy (ELMI), all MAbs reacted with epitopes in the envelope of intracellular naked virus, 16 MAbs recognized proteins of 32, 30, 16 or 14 kDa in Western blotting (WB), and 9 MAbs neutralized virus infectivity. In rabbitpox virus (RPV) 18 epitopes were detected. A λgt11 expression library of RPV DNA was screened with the corresponding 18 MAbs. Fourteen recombinant bacteriophage clones (ph) were isolated. Cross-hybridizations of phage and RPV DNA demonstrated a reaction with the HindIII A, HindIII D, or HindIII H fragments, respectively. DNA of ph3D was related to the A25L gene, which corresponds to the A-type inclusion body gene of cowpox virus. Two phage clones contained sequences of the 14-kDa fusion protein gene (A27L gene). Ph1A contained nearly the entire 14-kDa gene encoding 4 neutralizing (neutr) and 2 nonneutr epitopes. Ph5, expressing only half of this gene product, encoded 1 nonneutr epitope. The fusion protein of vaccinia virus MVA was isolated by immune-affinity chromatography with a neutr. catching MAb. The protein formed hollow rods (ELMI) and the 6 antigenic sites that were present were identical to those expressed by Escherichia coli infected with ph1A. WB detection with a polyclonal hyperimmune serum detected protein bands of 54, 32, 30, 16, and 14 kDa. The catching MAb bound only to a 16-kDa band. The purified fusion protein induced neutralizing antibodies in mice and rabbits.     

Development of a Western blot assay for detection of bovine immunodeficiency-like virus using capsid and transmembrane envelope proteins expressed from recombinant baculovirus

A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.     

A novel T‐cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice

We used a newly generated T‐cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10‐mer) or SSVWNATTA (9‐mer) in complex with class I MHC antigen H‐2D^b, were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8⁺ T cells can detect 10‐ and 9‐mer length variants similarly. This result varies from the idea that a given T‐cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10‐mer was presented on the surface of uninfected and infected CD8α⁺CD11c⁺ dendritic cells, which suggests the use of direct and cross‐presentation pathways. In contrast, CD11b⁺CD11c^? cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell‐type specific mechanisms of antigen presentation in vivo.     

Localization of tick-borne encephalitis virus protein E antigenic determinant recognized by antihemagglutinating monoclonal antibodies using a phage-display peptide library

Bacteriophages bearing peptides reacting with antihemagglutinating monoclonal antibodies (MAb) 10H10 to tick-borne encephalitis (TBE) virus protein E were selected from a phage-display peptide library by affinity selection and enzyme immunoassay. The library contained randomized peptides that are 6 amino acids long, fused with protein pIII and exposed on the surface of the bacteriophage. No significant homology between the sequences of selected peptides and TBE virus protein E was detected. Computer software was created to locate the conformation epitopes on the surface of protein E. Amino acids R73, C74, T76, M77, N103, C105, L107, and S112 were found to form a discontinuous epitope recognized by MAb 10H10. These amino acids are remote in the protein sequence but close in the tertiary structure and form a whole epitope located in the structural domain II of protein E. Presumably the localized amino acids bind to the cellular receptor for TBE virus.     

Definition of a divergent epitope that allows differential detection of early protein p41 from human herpesvirus 6 variants A and B

The human herpesvirus 6 (HHV-6) early protein, p41, encoded by the U27 gene has been detected in oligodendrocytes of multiple sclerosis (MS) patients by using a monoclonal antibody (MAb to p41/38). We here report the antigenic epitope of HHV-6 p41 recognized by this MAb. First, we established that the MAb to p41/38 recognizes a nuclear antigen in HHV-6A strain GS-infected cells but not in HHV-6B strain Z29-infected cells. Secondly, we compared the reactivity of the MAb to p41/38 to that of another p41-specific MAb (MAb to p41) on immunoblots with purified p41-glutathione S-transferase fusion protein from strains GS and Z29 and GS- and Z29-infected-cell lysates. The two MAbs were tested in an enzyme-linked immunosorbent assay against a panel of synthetic peptides covering the amino acid substitutions between the GS- and Z29-derived p41 proteins, as determined by DNA sequencing of our cloned isolates of the U27 gene. The MAb to p41/38 reacted specifically with a peptide comprising p41 residues 321 to 340 from strain GS. The critical residue in this peptide was serine 328, as the substitution S328N in the Z29 strain rendered the corresponding peptide nonreactive. The p41 S328 marker was present in three of three HHV-6A strains, while four of four sequenced p41 genes from HHV-6B strains had N328. Our findings are of value for the interpretation of previous findings of p41 expression in brains of MS patients and may allow a more detailed analysis of the role of HHV-6 variants in other disorders.     

T cell receptor and peptide-contacting residues in the HLA-DR17(3) β1 chain

Previously, we have proposed that the β1 residues 9–13, 26, 28 and 86 in HLA-DR17, the most common subtype of DR3, might be critical for the binding of an immunodominant, mycobacterial epitope (peptide 3–13 of the 65-kDa heat shock protein). In order to examine directly (i) which DR17 residues are involved in peptide binding, (ii) whether the same or other DR17 residues are involved in the binding of different peptides, and (iii) whether subtle differences in the mode of peptide binding can influence T cell stimulation, we have now systematically mutated 15 highly polymorphic DR17β1 residues, located in the proposed peptide binding groove of DR17, and examined the effect thereof on binding and presentation of two peptides, hsp65 p3–13 and p56–65 of the 30/31-kDa secreted mycobacterial protein. Mutations in residues 28 (D → H) and 86 (V → G) completely eliminated binding of p3–13 and significantly reduced binding of p56–65. A mutation in residue 26 (Y → F) decreased binding of p3–13 but did not affect binding of p56–65. Substitutions of amino acid residues 28, 67, 71 and 86 in the DR17β1 chain abrogated peptide-specific stimulation of both the p3–13- and the p56–65-specific T cell clones, while specific stimulation by only one peptide was eliminated by substitution at positions 26 and 74 (p3–13) and by substitution of residues 11 and 37 (p56–65). The observation that substitution of several other peptide-contacting DR17β1 chain residues does not significantly affect peptide binding but does affect T cell stimulation, suggests that these substitutions alter the conformation of the bound peptide.     

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.     

Drug-associated changes in amino acid residues in Gag p2, p7(NC), and p6(Gag)/p6(Pol) in human immunodeficiency virus type 1 (HIV-1) display a dominant effect on replicative fitness and drug response

Regions of HIV-1 gag between p2 and p6^Gag/p6^Pol, in addition to protease (PR), develop genetic diversity in HIV-1 infected individuals who fail to suppress virus replication by combination protease inhibitor (PI) therapy. To elucidate functional consequences for viral replication and PI susceptibility by changes in Gag that evolve in vivo during PI therapy, a panel of recombinant viruses was constructed. Residues in Gag p2/p7^NC cleavage site and p7^NC, combined with residues in the flap of PR, defined novel fitness determinants that restored replicative capacity to the posttherapy virus. Multiple determinants in Gag have a dominant effect on PR phenotype and increase susceptibility to inhibitors of drug-resistant or drug-sensitive PR genes. Gag determinants of drug sensitivity and replication alter the fitness landscape of the virus, and viral replicative capacity can be independent of drug sensitivity. The functional linkage between Gag and PR provides targets for novel therapeutics to inhibit drug-resistant viruses.     

Folding of the rabbit hemorrhagic disease virus capsid protein and delineation of N-terminal domains dispensable for assembly

Summary.  Rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV) are caliciviruses that produce severe symptoms and are lethal to rabbits and hares. The folding of the capsid protein was studied by determination of the antigenic pattern of chimeric capsid proteins, composed of regions from RHDV and EBHSV capsid proteins. The anti-RHDV monoclonal antibody (MAb) E3, which is known to bind an external conformational epitope, recognized the RHDV C-terminal region. The anti-RHDV MAb A47, which binds a buried epitope, recognized the RHDV N-terminal region. Using a pGEX expression library, we more precisely mapped the MAb A47 epitope on a 31 residues length peptide, between residue 129 and 160 of the VP60, confirming its location in the N-terminal part of the protein. These results demonstrate that the C-terminal part of the protein is accessible to the exterior whereas the N-terminal domain of the protein constitutes the internal shell domain of the particle. With the aim of using virus-like particles (VLPs) of RHDV as epitope carriers or DNA transfer vectors, we produced in the baculovirus system three proteins, ΔN1, ΔN2 and ΔN3, truncated at the N terminus. The ΔN1 protein assembled into VLPs, demonstrating that the first 42 amino acid residues are not essential for capsid assembly. In contrast, ΔN2, from which the first 75 residues were missing, was unable to form VLPs. The small particles obtained with the ΔN3 protein lacking residues 31 to 93, located in the immunodominant region of the RHDV capsid protein, indicate that up to 62 amino acid residues can be eliminated without preventing assembly. Received September 6, 2001; accepted March 13, 2002 Published online June 21, 2002     

Characterization of two epitopes on insulin using monoclonal antibodies

Three monoclonal antibodies (MAb), 21 (IgG1), I10 (IgG1) and H38 (IgG2b), to insulin have been tested for cross-reactivity with 11 species variants of insulin and three of proinsulin. Correlations of differences of reactivities between the MAb and the species variants of insulin with the respective amino acid sequences of the latter have permitted the identification of two epitopes recognized by the MAb which encompass the regions in the A- and B-chains of insulin subject to frequent evolutionary amino acid substitutions. MAb 21 and H38 are directed to an epitope which includes residues B27-30 and A1 or A4 and can discriminate between human and pig insulins which differ only at B30. MAb 21 reacts with human (B30 thr) but not with pig (B30 ala) insulins, whereas MAb H38 exhibits a reciprocal specificity. Neither MAb 21 nor MAb H38 react with human or pig proinsulins respectively indicating that the presence of the C-peptide joining A1 to B30 masks the epitope. MAb 21 reacts with human insulin 125I-labeled at tyr A14 but not B26 suggesting that incorporation of the I atom at B26 also masks the epitope. MAb I10 is directed to an epitope which includes A8-10 and A4 or B3 with a specificity for the human A8-10 sequence. MAb I10 reacts with human proinsulin and human insulin 125I-labeled at either tyr A14 or B26.     

A novel monoclonal antibody against FbaA can inhibit the binding of the complement regulatory protein factor H to group A streptococcus

Some microbial pathogens utilize human complement regulatory proteins, such as factor H (FH) and factor H-like protein 1 (FHL-1), for immune evasion. FbaA is an FHL-1 and FH binding protein expressed on the surface of group A streptococcus (GAS), a common agent of pharyngeal, skin, and soft tissue infections. In this study, we prepared monoclonal antibodies (MAbs) against FbaA, assayed them for specificity, and located their binding domains in FbaA. We found an MAb called FbaA MAb2, which demonstrated the highest affinity to GAS among all of the MAbs. Based on the binding with component peptides, the detected epitope, which was specific for FbaA MAb2, was the amino acid residues 95 to 118 of FbaA; on the other hand, it did not bind with the truncated protein of the internally deleted residues of the segment from 95 to 118 of FbaA. Furthermore, the predominant amino acids specific for FbaA MAb2 screened by phage display epitope library were I, T, P, D, and L, corresponding to the amino acid residues 101, 103, 105, 106, and 110 of FbaA, respectively. The binding location of FbaA with FH and FHL-1 was a 16-amino-acid region corresponding to amino acid residues 97 to 112 of FbaA, which overlapped the FbaA MAb2 binding domain, as confirmed by competitive inhibition enzyme-linked immunosorbent assay and immunofluorescence microscopy. Based on the results of the invasion assay, FbaA MAb2 can inhibit the binding of FH to GAS.     

Cloning and sequencing of the genes coding for the 10- and 60-kDa heat shock proteins from Pseudomonas aeruginosa and mapping of a species-specific epitope.

A genomic library of Pseudomonas aeruginosa DNA was screened with a monoclonal antibody (MAb 2528) specific for the P. aeruginosa 60-kDa heat shock protein. A positive clone, pAS-1, was isolated. The gene coding for P. aeruginosa chaperonin (hsp60) was localized to a 2-kb EcoRI fragment subcloned in pAS-2. A sequence analysis of pAS-2 and parts of pAS-1 identified two open reading frames that encoded proteins with calculated molecular masses of 10 and 57 kDa. In amino acid sequence comparison studies the sequences of these proteins, which were designated GroES and GroEL, exhibited up to 78% homology with known prokaryotic sequences of 10- and 60-kDa heat shock proteins (hsp10 and hsp60). In order to map the epitope recognized by MAb 2528, a series of GroEL nested carboxy-terminal deletion clones were tested with MAb 2528. We identified the clone with the shortest insertion that was still recognized by MAb 2528 and the clone with the largest insertion that was not recognized by MAb 2528. The 3' ends of the insertions were determined by sequencing and were found to delimit a region that encoded 25 amino acid residues. Synthetic oligonucleotides that coded for peptides possibly resembling the epitope within this region were ligated into expression vector pGEX-3X, and fusion proteins expressed by these clones were tested for reactivity with MAb 2528. By using this method we determined that the decapeptide QADIEARVLQ (positions 339 to 348 on GroEL) was responsible for the binding of P. aeruginosa-specific MAb 2528.