Human monoclonal antibodies to HIV-1: cross-reactions with gag and env products.

Human monoclonal antibodies were raised by in vitro immunization of normal human spleen cells with denatured HIV-1 and subsequent fusion to an Epstein-Barr virus (EBV) transformed cell line. Three monoclonals reacted with both gag-encoded p24 core protein and env-encoded gp41 transmembrane glycoprotein. The cross-reaction was confirmed by reactivity with p24- and gp41-derived recombinant peptides. The peptides share two amino acid sequences which may be the basis for the cross-reaction. Attempted epitope mapping using synthetic peptides was unsuccessful due to non-specific reactivity with the short linear peptides.     

Epitope mapping of horseradish peroxidase with use of monoclonal antibodies.

Nine mouse monoclonal antibodies (MAbs) to horseradish peroxidase (HRP) were used to develop an epitope map of the enzyme. The results of a competitive binding assay indicated three distinct patterns of reactivity. Two groups of MAbs (I and III) recognized epitopes located in separate antigenic regions on the molecule; another (II) bound to sites that overlapped with epitopes in either region I or III. Further definition of these regions was obtained by analyzing the MAbs for their binding to isolated heme, other peroxidases and heme-containing proteins, and to denatured and apo-HRP. None of the group I MAbs bound heme, suggesting that this region was removed from the active site of the enzyme. All of the group II and III MAbs bound heme as well as the other peroxidases and heme-containing proteins, indicating that they recognized heme-associated epitopes at or near the active site. Only one MAb (2A2) in groups II and III bound to apo-HRP but not to denatured HRP; it was also the only MAb in the entire panel that inhibited the catalytic activity of HRP. This suggests that the epitope recognized by 2A2 involves both the heme moiety and a conformationally dependent protein determinant near the active site.     

Epitope mapping of anti-breast and anti-ovarian mucin monoclonal antibodies.

Anti-breast cancer antibodies (BC2, HMPV and 4B6) and an anti-ovarian cancer antibody (OM1) were found to react with mucins--indeed with the protein core encoded by the MUC1 gene. This gene contains a VNTR (variable number of tandem repeats) encoding a 60 bp (= 20 amino acids) repeat sequence and within this amino acid sequence SAPDTRPAP was predicted, by hydrophilicity analysis, to be the immunogenic peptide sequence. The four antibodies were shown to react with MUC1 VNTR encoded peptides in direct binding and inhibition studies. The precise reactivity of the 4 mAbs was mapped using ELISA in both solid and liquid phase, and demonstrated the epitopes to be: APDTR (BC2 and HMPV), PDTR (4B6) and DTRPA (OM1). By using the pepscan method, the epitopes were shorter (PDTR, DTR and DTRP). However when these short peptides (except DTR) were synthesized they did not react; flanking amino acids are needed for the epitopes. Clearly several different methods should be used to define the reactive epitope. Within (S)APDTR, major amino acid substitutions could be made--even of three to four amino acids without altering antibody binding, provided that P and R were not substituted. It was of interest that an anti-ovarian cancer antibody gave similar anti-peptide reactions to the anti-breast cancer antibodies; apparently MUC1 peptides in ovarian cancer are the same as in breast cancer.     

Epitope mapping of dengue 1 virus E glycoprotein using monoclonal antibodies

Summary Ten monoclonal antibodies (MAbs) were raised against dengue 1 (DEN 1, Hawaii) virus E glycoprotein. Specificity of the MAbs was tested by ELISA and immunofluoresence. Eight were DEN 1 type-specific, one was DEN group-reactive (DGR) and one was flavivirus cross-reactive (FCR). Two of these type specific MAbs exhibited haemagglutination-inhibition (HI) and neutralized (N) DEN 1 virus in vivo (HS). These two MAbs showed 100% protection against a challenge of 100 LD₅₀ of DEN 1 virus in adult Swiss albino mice. The remaining six MAbs were HI negative, N negative and non-protective against challenge (NHS). Of these only three were reactive in the CF test. The DGR, FCR and one of the NHS MAbs (NHS-3) did not react with DEN 1 virus grown in Vero cells, whereas they reacted with DEN 1 virus grown in LLC-MK2 and C6/36 cells in immunofluorescence, probably indicating a difference in the synthesis/processing of viral proteins in these different cell lines. An epitope map of the E gp was drawn using a computer programme based on the additivity index values. The epitope map delineated five domains, a) S-I representing type-specific, HI positive, N positive and protecting MAbs. b) S-II representing type-specific, HI negative, N negative MAbs. c) S-III representing type-specific HI/N negative MAb, but distinct from S-II. d) DGR representing HI/N negative DEN group reactive MAb. e) FCR representing HI/N negative flavivirus cross-reactive MAb. Epitope analysis of a number of different DEN 1 strains isolated in India over a period of 30 years showed that the domains S-II and S-III which react with HI negative, DEN-1 specific MAbs were variable. The DGR domain and the S-I domains were conserved.     

Fine mapping of HIV-1 Nef-epitopes by monoclonal antibodies

Summary A panel of newly isolated murine monoclonal antibodies is described which are specific for the Nef protein of the human immunodeficiency virus type 1 (HIV-1). Epitope mapping using recombinant Nef-related proteins, synthetic peptides and lipopeptides showed 3 independent antigenic determinants located within the regions of amino acids 83–93, 175–190 and 86–166 of the Nef protein. None of the monoclonal antibodies reacted with recombinant Nef proteins of HIV-2.     

Epitope mapping of four monoclonal antibodies specific for the human RhD antigen.

RhD is a highly immunogenic erythrocyte membrane protein, implicated in hemolytic disease of the newborn and other hemolytic disorders. Anti-RhD antibodies are used in the treatment of such disease states. Six mutant forms of recombinant RhD were stably expressed in K562 cells, and these cells were used to investigate epitope specificities of four anti-RhD monoclonal antibodies (mAbs). Amino acid substitutions were made in the exofacial loops of RhD to the corresponding residues found in the related RhCE polypeptide; M169L/M170R and I172F in the third loop, F223V and E233Q in the fourth loop, and D350H and G353W/A354N in the sixth loop. Each mAb was found to have a unique fine specificity and recognized multiple distant sites within RhD. The mAbs also differed in how they recognized individual amino acids in the exofacial loops of RhD.     

Epitope mapping of monoclonal antibodies against N-domain of carcinoembryonic antigen

Monoclonal antibodies (MoAbs) against N-domain of carcinoembryonic antigen (CEA), C249, K348, K1338, and K1444, that inhibit CEA-mediated cell adhesion, did not crossreact with nonspecific cross-reacting antigen (NCA). To determine amino acid sequences involved in the adhesion, epitopes of the MoAbs were mapped with recombinant NCAs carrying CEA-NCA chimeric N-domain. The data showed that the epitopes of C249, K1338, K1444 are located within the regions 1-32, 1-32, and 33-59 of CEA, respectively, and that two discrete regions 1-32 and 60-93 may be related to the epitope of K348. Comparison of amino acid sequences between CEA and NCA suggested that four residues (21, 27-29), eight residues (21, 27-29, 66, 78, 79, 89), and three residues (43, 44, 46) are important for recognition by C249 (or K1338), K348, and K1444, respectively. These residues seem to participate in the cell adhesion mediated by CEA.     

Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies

Summary Monoclonal antibodies with differing specificity were prepared against the Josiah strain of the Lassa virus. All monoclonal antibodies were characterized by subclass determination and the immunofluorescence test against Lassa, LCM (WE & Arm strain), Junin, Machupo, and other arenavirus antigens. In radioimmune precipitation tests using purified Lassa virus antigen all monoclonal antibodies precipitated a single band of 60 kd, specific for the viral nucleoprotein (p 60). Three domains (A, B, C) were identified on the surface of the Lassa virus nucleoprotein using an ELISA-inhibition test. All domains carried different Lassa virus specific epitopes. In addition, the A-domain carried a group specific epitope present within the arenavirus family as a whole as shown by cross-reaction in immunofluorescence tests. The B-domain only carries Lassa virus specific epitopes, whereas the C-domain has a type specific and a subgroup specific (Lassa, LCM) epitope.     

Epitope mapping of recombinant human gamma interferon using monoclonal antibodies

Five monoclonal antibodies (MAbs B22, B27, 3-6, 32 and 35) specific for human recombinant IFN-gamma were characterized. These MAbs were used to set up quantitative sandwich ELISAs which allowed the detection of 1.25 ng/ml of IFN-gamma when diluted in normal human serum. Epitope mapping of the IFN-gamma molecule using these MAbs demonstrated that antibodies 3-6 and 32 which did not inhibit the biological activity of IFN-gamma recognized an epitope localized on the 15 C-terminal amino acids, suggesting that this portion of the molecule was not implicated in the biological activity of IFN-gamma. Sandwich ELISAs were performed using various pairs of MAbs. The level of reactivity obtained when antibodies B22 and B27 were used simultaneously as catcher and tracer was similar to the result obtained with two antibodies recognizing different epitopes. These results confirm that the IFN-gamma molecule is a dimer in solution and indicate that the two sites of the IFN-gamma dimeric molecule which are associated with the biological activity (epitope B22/B27) are fully exposed. In contrast, the C-terminus is only partially accessible to the antibodies 3-6/32, suggesting that the dimerization of IFN-gamma molecule results in the interaction of regions of the monomers that are homologous and adjacent to the C-terminus.     

Epitope mapping of major insect allergens (chironomid hemoglobins) with monoclonal antibodies

Monoclonal antibodies (m ABs) were raised against the physically and chemically well-defined insect allergen, component CTT III from Chironomus thummi thummi (CTT) hemoglobin. With these m ABs, we observed epitopes present in as many different hemoglobin (Hb) molecules from the widespread insect family Chironomidae as possible. These Hbs were identified as important allergens in previous studies. Twenty m ABs from seven different clones were tested for specificity against the CTT Hbs III, IIIa, IV, and I to X, as well as against Hbs from 14 other chironomid species; m ABs 1 to 3 were found to be specific for an epitope that was expressed on the CTT III component as well as in Hbs from three other species. m ABs 5 to 7 and 15 recognized an epitope expressed in CTT III, CTT IIIa, CTT IV, and in Hbs from 13 other species. m ABs 8 to 14 and 16 to 20 demonstrated heteroclitic behavior, that is, they bound CTT IIIa better than the immunogen CTT III. Reactivity of m AB 6 with a clinically relevant epitope was demonstrated by up to 15% inhibition of antibody binding by human IgE from sensitized patients. Our results demonstrate that clinically relevant allergenic epitopes can be localized on different molecules with m ABs. This could be important in the development of immunotherapy protocols with highly purified or fragmented allergens.     

Epitope mapping of monoclonal antibodies against Bordetella pertussis adenylate cyclase toxin

Adenylate cyclase (AC) toxin from Bordetella pertussis is a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.     

Epitope mapping of human herpesvirus-7 gp65 using monoclonal antibodies

Summary.  Human herpesvirus (HHV)-7 encodes a unique 65-kDa heparin- binding glycoprotein, designated gp65. This molecule is thought to play a role in virus attachment and entry. To obtain reagents to map the structure and function of HHV-7 gp65, we produced monoclonal antibodies to this molecule. Ten monoclonal antibodies reacting with gp65 on ELISA were subdivided in four groups on the basis of their isotype and differential reactivity with (i) native versus denatured forms of gp65, and (ii) mature (virion-associated) versus immature (cell-associated) forms of the molecule. We were able to map the binding epitopes for eight of these ten antibodies, and these were found to cluster to one site on gp65 (amino acids 239–278); within this region, the antibodies reacted with at least three distinct domains (244–251, 255–262, 263–278). The reasons for the apparent immunodominance of this region are uncertain. Taken together, this panel of antibodies constitutes an extensive and well-characterized set of HHV-7 specific antibodies that may have utility for future analyses of the structure/function of gp65, and for studies on the virus life cycle. Accepted April 9, 2001 Received January 19, 2001     

Epitope mapping of the influenza A virus RNA polymerase PA using monoclonal antibodies

Summary.  To obtain reagents to functionally map the PA protein, we produced monoclonal antibodies specific to this protein. Twenty-two monoclonal antibodies reacting with PA protein in ELISA were divided into 10 groups on the basis of competitive binding patterns to this protein. Of these, seventeen monoclonal antibodies bound to PA polypeptide spanning amino acids 101–400 and three bound to that of amino acids 518–600, while the other two did not react with any PA polypeptides tested with the exception of full-length PA. Among these monoclonal antibodies, only five reacted with PA in A/PR/8/34 virus-infected cells in indirect immunofluorescence assay. Thus, we obtained monoclonal antibodies that recognize at least 10 distinct regions of the PA molecule. These monoclonal antibodies should be useful in dissecting functions of the PA protein. Received September 6, 1999/Accepted January 5, 2000     

Epitope diversity of angiotensin II analysed with monoclonal antibodies.

The antigenic heterogeneity of angiotensin II (AII) was studied with monoclonal antibodies. Twelve antibodies were produced and characterized. Association constants for AII varied from 1.2 X 10(8) to 1.1 X 10(10) M-1. The fine specificity of the Mab was studied by immunoenzymoassay using solid-phase AII. Using AII analogues in binding inhibition experiments, three groups of specificity could be characterized: (1) five antibodies reacted only with peptides in which phenylalanine is the carboxy terminal aminoacid; for two of these antibodies, tyrosine4 is closely associated with the binding site, since iodine labelling suppresses reactivity; (2) two antibodies also required phenylalanine in position 8, but, in addition, reacted with AI, a decapeptide in which phenylalanine is not terminal; (3) five antibodies reacted with analogues in which phenylalanine had been substituted for another amino acid. In addition, studies in which binding of a biotinylated Mab to solid-phase AII was analysed in the presence of various unlabelled Mab suggest further antigenic heterogeneity of AII.     

Epitope mapping of monoclonal antibodies raised to recombinant Mengo 3D polymerase

The cDNA coding sequence of the RNA-dependent RNA polymerase (3D^pol) of Mengovirus was cloned and expressed in a bacterial system. Eleven monoclonal antibodies were raised against the recombinant Mengo 3D^pol (rM3D). All of them recognized the recombinant and the viral-induced form of the protein. The panel of monoclonals belonged to the IgG1 and IgG2a isotypes and were mapped to four different epitopes in the 3D molecule by competition assays. All monoclonals recognized Mengo 3D^pol in western blots and cross-reacted with the homologous polymerases of seven other cardioviruses but failed to react with 3D^pol from poliovirus type 1 and 3 or rhinovirus type 14 and 16.     

Detection and subtyping of HIV-1 isolates with a panel of characterized monoclonal antibodies to HIV p24gag

A panel of monoclonal antibodies (Mabs) was used to analyze the number and localization of B-cell epitopes on human immunodeficiency virus (HIV) p24gag and the variability of these epitopes in sequential HIV isolates and in isolates from different geographical origin. The specificity of these Mabs was demonstrated by immunoblotting and radioimmunoprecipitation assays. Cross-inhibition experiments indicated the presence of at least five different epitopes on p24. Analysis with p24 recombinant products revealed that three of the Mabs to p24 were directed to epitopes localized on the C-terminal part. Four other Mabs were directed to epitopes localized on the N-terminal half of the protein. Anti-p24 Mabs were used to develop HIV p24 antigen-capture assays. Application of these assays in HIV isolation resulted in more efficient recovery of HIV. Serotyping of HIV-1 isolates with five anti-p24 Mabs demonstrated that 55/65 isolates recovered from Dutch and Belgian individuals, but only 4/9 HIV-1 African isolates, were recognized by all five Mabs. Five of nine Central African HIV-1 isolates were not reactive with at least one of these Mabs. The variability of p24 appeared to be predominantly localized on the N-terminal part of the protein. Lack of expression of antigenic determinants on p24 was shown to be independent of culture conditions. Moreover, an infectious molecular clone was shown to have the same serotype as the corresponding HIV isolate. The serotype of sequential isolates obtained from 17 individuals over a 1 1/2- to 2 1/2-year period did not change, suggesting a limited in vivo p24 variation over time.     

Epitope mapping of monoclonal antibodies capable of neutralizing cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli

Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic Escherichia coli belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation. Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin. Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity. To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies. Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains. All MAbs showed reactivity to CNF1 by Western and dot blot analyses. However, only 7 of the 16 MAbs exhibited cross-reactivity with CNF2. Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity). Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a [IgG2a]), BF8 (IgA), and NG8 (IgG2a), were more precisely defined. MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain. MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164. Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2. MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain. Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.     

Epitope mapping of apamin by means of monoclonal antibodies raised against free or carrier-coupled peptide.

A panel of 20 monoclonal antibodies raised against the bee-venom peptide apamin (18 residues, 2 disulfide bridges) was prepared. Nine monoclonal antibodies (mAb) were obtained from a mouse immunized with free apamin and 11 from a mouse immunized with a mixture of free and carrier-coupled peptide. Using a panel of 11 synthetic apamin analogs, we examined the fine antigenic specificity of each antibody. The mAb generated against free apamin preferentially bound to the central part of the peptide and less frequently recognized the N- and C-terminal regions. However, monoclonal antibodies obtained by immunization with carrier-bound apamin showed a broader range of specificities, consistent with the possibility of the entire surface of this small antigen becoming immunogenic upon coupling to the carrier.     

Epitope mapping of anti-proteinase 3 and anti-myeloperoxidase antibodies.

Anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) autoantibodies are present in many patients with Wegener's granulomatosis (WG) and microscopic polyarteritis. The aim of this study was to determine whether these antibodies bound to linear peptide sequences on their target antigens. If common linear epitopes were demonstrated, then these could be manufactured and used in diagnostic ELISAs for anti-PR3 and anti-MPO antibodies. In addition, any homology between these epitopes and bacterial or viral sequences might implicate those microorganisms in the development of these antibodies and the pathogenesis of the associated diseases. The presence of linear epitopes on PR3 and MPO was suggested by the binding of the corresponding autoantibodies to these proteins after they had been reduced with beta-mercaptoethanol (beta-ME) and denatured with SDS or boiling, and digested with proteases. Four of the 22 sera with anti-PR3 antibodies bound to PR3 in Western blots after treatment with SDS, beta-ME and boiling for 5 min. Thermal denaturation reduced the amount of binding more than other forms of denaturation. One serum with anti-PR3 antibodies bound to Lys-C and Glu-C-digested PR3 in dot blots. Linear epitopes could not be further defined by their binding in an ELISA using overlapping peptides corresponding to the PR3 molecule because of non-specific binding. Three of the five sera with anti-MPO antibodies bound to MPO in Western blots after treatment with SDS, beta-ME and boiling for 5 min. One serum with anti-MPO antibodies bound to Lys-C and Glu-C-digested MPO in dot blots. Again, linear epitopes could not be further defined using an ELISA with overlapping peptides because of non-specific binding. Some anti-PR3 and anti-MPO antibodies are likely to recognize linear epitopes, but these cannot be defined by use of a PIN ELISA system.     

Epitope mapping of anti-rat CD53 monoclonal antibodies. Implications for the membrane orientation of the Transmembrane 4 Superfamily

CD53 is a pan-leukocyte glycoprotein which is a member of the recently described Transmembrane 4 Superfamily (TM4SF) of membrane proteins that are predicted to span the lipid bilayer four times. The major hydrophilic region of murine CD53 was expressed as a glutathione-S-transferase fusion protein, and the epitopes of four mouse anti-rat CD53 monoclonal antibodies (mAb) (OX-44, 2D1, 6E2 and 7D2) were mapped to this region using mouse/rat chimeric fusion proteins. The epitopes of OX-44,6E2 and 7D2 are restored by the substitution of a single isoleucine residue for threonine at position 154 in the mouse protein. The 2D 1 epitope is non-linear and appears to require the juxtaposition of isoleucine at position 154 with one or more of the amino acids arginine (132), methionine (133) and serine (140). All of these epitopes are shown to be sensitive to reduction, thus indicating the importance of disulfide bonding in the correct folding of the CD53 hydrophilic domain. Moreover, as these four mAb recognize CD53 at the cell surface, the data provide direct molecular evidence for the proposed membrane orientation of the TM4SF.