Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody.

A human monoclonal antibody designated 15e is reactive with the envelope glycoprotein (gp120) of multiple isolates of human immunodeficiency virus type 1 (HIV-1). Antibody 15e also neutralizes HIV-1 with broad specificity and blocks gp120 binding to CD4. Characterization of the 15e epitope shows that it is conformation dependent and is distinct from previously recognized functional domains of gp120, suggesting that this epitope represents a novel site important for HIV-1 neutralization and CD4 binding. These findings have implications for the development of a vaccine for AIDS.     

Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope.

A monoclonal antibody was produced to the exterior envelope glycoprotein (gp120) of the human T-cell lymphotropic virus (HTLV)-IIIB isolate of the human immunodeficiency virus (HIV). This antibody binds to gp120 of HTLV-IIIB and lymphadenopathy-associated virus type 1 (LAV-1) and to the surface of HTLV-IIIB- and LAV-1-infected cells, neutralizes infection by cell-free virus, and prevents fusion of virus-infected cells. In contrast, it does not bind, or weakly binds, the envelope of four heterologous HIV isolates and does not neutralize heterologous isolates HTLV-IIIRF and HTLV-IIIMN. The antibody-binding site was mapped to a 24-amino-acid segment, using recombinant and synthetic segments of HTLV-IIIB gp120. This site is within a segment of amino acid variability known to contain the major neutralizing epitopes (S. D. Putney, T. J. Matthews, W. G. Robey, D. L. Lynn, M. Robert-Guroff, W. T. Mueller, A. J. Langlois, J. Ghrayeb, S. R. Petteway, K. J. Weinhold, P. J. Fischinger, F. Wong-Staal, R. C. Gallo, and D. P. Bolognesi, Science 234:1392-1395, 1986). These results localize an epitope of HIV type-specific neutralization and suggest that neutralizing antibodies may be effective in controlling cell-associated, as well as cell-free, virus infection.     

Characterization and epitope mapping of a human monoclonal antibody reactive with the envelope glycoprotein of human immunodeficiency virus

A human monoclonal antibody (IgG2, lambda), 1B8.env, was produced, reactive with the envelope glycoprotein of human immunodeficiency virus (HIV). The antibody specifically stains cells infected with HIV, as assessed by indirect immunofluorescence analysis and reacts with determinants displayed on the surface of infected cells. In Western blot analysis, the antibody reacts with bands of 160 and 41 kD, consistent with the precursor and transmembrane forms of the HIV envelope glycoprotein. The antibody also reacts specifically in immunofluorescence and Western blot analysis with cells infected with the recombinant vaccinia virus VSC-25, which contains the envelope gene of HIV. With the lambda gt11 expression vector, the epitope recognized by 1B8.env was mapped to a region of 11 amino acids in the coding region of gp41. This domain is highly conserved between several otherwise highly variable HIV isolates. In addition, this epitope appears to be recognized by the vast majority of HIV seropositive individuals. Although antibody IB8.env does not neutralize HIV virion infectivity or virally mediated cell fusion, the results presented here demonstrate the feasibility of generating and characterizing human monoclonal antibodies to HIV with these techniques. Additional antibodies produced in this manner will help to further characterize the humoral response to HIV infection, define biologically significant determinants on HIV proteins, and may be useful in clinical applications.     

Characterization of a monoclonal antibody reactive with rabbit T lymphocytes and neutrophils

An IgG1 monoclonal antibody, termed ACM-1, has been shown to react with rabbit T cells, but not Ig+ cells or macrophages. This antibody appears to recognize the same epitope as the previously described 9AE10 antibody and, together with 9AE10, has been used to obtain highly pure and fully functional T- and B-cell populations. However, the relevant epitope does not appear to be homologous to rodent Thy-1 since quantitative absorptions failed to show reactivity with rabbit brain. Furthermore, attempts to obtain in vivo T-cell depletion resulted in larger decreases in white blood cells than would be expected for simple T-cell removal. In vitro assays on enriched neutrophil preparations revealed that 80-95% of these cells were reactive with ACM-1 and 9AE10. Thus, it appears that in the rabbit, T cells and neutrophils share a major epitope.     

Homologies between paraflagellar rod proteins from trypanosomes and euglenoids revealed by a monoclonal antibody

A Nonidet P 40 insoluble fraction was isolated from Trypanosoma brucei and was used to raise a monoclonal antibody (5E9). The antigen was localized by indirect immunofluorescence in the flagellum of T. brucei and of two species of euglenoids, Euglena gracilis and Distigma proteus. In immunoblot analysis, 5E9 appeared to bind to paraflagellar rod proteins PFR1 and PFR2 of T. brucei (72000 and 75000 mol. wt.) and of E. gracilis (67000 and 76000 mol. wt.). The presence of a common epitope in paraflagellar rod proteins from species of trypanosomes and euglenoids shows that despite distinct structures of the rods some identical domain exists in the proteins that could be involved in their supramolecular assembly into a similar organelle. The antigenic determinant defined by 5E9 was also shown to be present in a 87000 molecular weight polypeptide located in the proximal part of the flagellum of Crithidia oncopelti in which a paraflagellar rod is not detectable at the ultrastructural level.     

Fine mapping of a foot-and-mouth disease virus epitope recognized by serotype-independent monoclonal antibody 4B2

VP2 is a structural protein of the foot-and-mouth disease virus (FMDV). In this study, a FMDV serotype-in-dependent monoclonal antibody (MAb), 4B2, was generated. By screening a phage-displayed random 12-peptide library, we found positive phages displaying the consensus motif ETTXLE (X is any amino acid (aa)), which is highly homologous to ⁶ETTLLE¹¹ at the N-terminus of the VP2 protein. Subsequently, a series of GST-fusion proteins expressing a truncated N-terminus of VP2 were examined by western blot analysis using the MAb 4B2. The results indicated that the motif ⁶ETTLLE¹¹ of VP2 may be the minimal requirement of the epitope recognized by 4B2. Moreover, a 12-aa peptide ²KKTEETTLLEDR¹³ was shown to be the minimal unit of the epitope with maximal binding activity to 4B2. Alanine-scanning analysis demonstrated thatThr⁷, Thr⁸, and Leu¹⁰ are the functional residues of the 4B2 epitope Glu⁶ and Leu⁹ are required residues, and Glu¹¹ plays a crucial role in the binding of MAb 4B2. The fine mapping of the epitope indicated that MAb 4B2 has the potential to be used in FMDV diagnosis.     

Visualization of cardiac ventricular myosin heavy chain homodimers and heterodimers by monoclonal antibody epitope mapping

Two mAbs, one specific for cardiac alpha-myosin heavy chains (MHC) and the other specific for cardiac beta-MHC, were used to investigate the heavy-chain dimeric organization of rat cardiac ventricular myosin. Epitopes of the two mAbs were mapped on the myosin molecule by electron microscopy of rotary shadowed mAb-myosin complexes. mAbs were clearly identifiable by the different locations of their binding sites on the myosin rod. Thus, myosin molecules could be directly discriminated according to their alpha-or beta-MHC content. alpha alpha-MHC and beta beta-MHC homodimers were visualized in complexes consisting of two molecules of the same mAb bound to one myosin molecule. By simultaneously using the alpha-MHC-specific mAb and the beta-MHC- specific mAb, alpha beta-MHC heterodimers were visualized in complexes formed by one molecule of each of the two mAbs bound to one myosin molecule. Proportions of alpha alpha-and beta beta-MHC homodimers and alpha beta-MHC heterodimers were estimated from quantifications of mAb- myosin complexes and compared with the proportions given by electrophoreses under nondenaturing conditions. This visualization of cardiac myosin molecules clearly demonstrates the arrangement of alpha- and beta-MHC in alpha alpha-MHC homodimers, beta beta-MHC homodimers, and alpha beta-MHC heterodimers, as initially proposed by Hoh, J. F. Y., G. P. S. Yeoh, M. A. W. Thomas, and L. Higginbottom (1979).     

Saturation-transfer difference NMR studies for the epitope mapping of a carbohydrate-mimetic peptide recognized by an anti-carbohydrate antibody

Saturation-transfer difference NMR spectroscopy (STD-NMR) experiments have been performed to analyze the topography or epitope of the octapeptide MDWNMHAA recognized by the anti-carbohydrate antibody SYA/J6 in solution; the antibody is directed against the Shigella flexneri Y O-antigen polysaccharide. The results permit a valuable comparison of solution versus crystal-structure data, and provide insight for the design of the next-generation binding ligands.     

Fine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzyme

Angiotensin I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) is a key enzyme in cardiovascular pathophysiology. A wide spectrum of monoclonal antibodies to different epitopes on the N and C domains of human ACE has been used to study different aspects of ACE biology. In this study we characterized the monoclonal antibody (mAb) 5F1, developed against the N domain of human ACE, which recognizes both the catalytically active and the denatured forms of ACE. The epitope for mAb 5F1 was defined using species cross-reactivity, synthetic peptide (PepScan technology) and phage display library screening, Western blotting, site-directed mutagenesis, and protein modeling. The epitope for mAb 5F1 shows no overlap with the epitopes of seven other mAbs to the N domain described previously and is localized on the other side of the N domain globule. The binding of mAb 5F1 to ACE is carbohydrate-dependent and increased significantly as a result of altered glycosylation after treatment with alpha-glucosidase-1 inhibitor, N-butyldeoxynojirimycin (NB-DNJ), or neuraminidase. Out of 17 species tested, mAb 5F1 showed strict primate ACE specificity. In addition, mAb 5F1 recognized human ACE in Western blots and on paraffin-embedded sections. The sequential part of the epitope for mAb 5F1 is created by the N-terminal part of the N domain, between residues 1 and 141. A conformational region of the epitope was also identified, including the residues around the glycan attached to Asn117, which explains the sensitivity to changes in glycosylation state, and another stretch localized around the motif 454TPPSRYN460. Site-directed mutagensis and inhibition assays revealed that mAb 5F1 inhibits ACE activity at high concentrations due to binding of residues on both sides of the active site cleft, thus supporting a hinge-bending mechanism for substrate binding of ACE.     

Anti-idiotype-mediated epitope spreading and diminished phagocytosis by a human monoclonal antibody recognizing late-stage apoptotic cells

Apoptotic cells are considered an important auto-antigenic source in diseases such as systemic lupus erythematosus (SLE). A human monoclonal antibody demonstrating exquisite specificity towards late-stage apoptotic cells was generated from an SLE patient. Polyreactive recognition of ribonucleoproteins Ro52 and Ro60 was observed. The antibody significantly diminished the phagocytosis of apoptotic cells and a concomitant decrease in transforming growth factor-beta (TGF-beta) secretion was observed. Light and heavy chain sequencing revealed the antibody to be in essentially germline configuration. Elicited anti-idiotypic antibodies bound distinct self-antigens and showed augmented reactivity towards apoptotic cells as well. Thus, near-germline encoded antibodies recognizing antigens externalized during the process of apoptosis can mediate a variety of potentially pathogenic effects; decreases in the phagocytic uptake of dying cells would constitute a disease-perpetuating event and stimulation of the idiotypic network could lead to intermolecular epitope spreading, increasing the range of molecular targets..     

Topology of MDR1-P-glycoprotein as indicated by epitope mapping of monoclonal antibodies to human MDR cells

The MDR1-P-glycoprotein binding sites of three different murine monoclonal antibodies (MM4.17, MM6.15 and MC57), directed towards living, intact human multidrug-resistant cells were investigated in order to study P-glycoprotein topology. By using synthetic peptide scanning, we demonstrated that well-defined regions localized on the predicted first, fourth and sixth extracellular loops are external. On the basis of the structure of MM6.15 epitope, which is distributed on the above three different extracellular loops (and thus is discontinuous), P-glycoprotein molecules result to be differently organized in the lipid bilayer. Moreover, the outcome of the MC57 and MM4.17 epitopes localization experiments, obtained through the use of phage-displayed peptide libraries, represent an additional challenge to the classical 12-transmembrane domain model of P-glycoprotein, since they agree with the novel topography of the molecule (10-transmembrane domain), which was recently proposed on the basis of biochemical and expression studies.     

Characterization of a discontinuous human immunodeficiency virus type 1 gp120 epitope recognized by a broadly reactive neutralizing human monoclonal antibody.

While one hypervariable, linear neutralizing determinant on the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein has been well characterized, little is known about the conserved, discontinuous gp120 epitopes recognized by neutralizing antibodies in infected individuals. Here, the epitope recognized by a broadly reactive neutralizing monoclonal antibody (F105) derived from an HIV-1-infected patient was characterized by examining the effects of changes in conserved gp120 amino acids on antibody reactivity. The F105 epitope was disrupted by changes in gp120 amino acids 256 and 257, 368 to 370, 421, and 470 to 484, which is consistent with the discontinuous nature of the epitope. Three of these regions are proximal to those previously shown to be important for CD4 binding, which is consistent with the ability of the F105 antibody to block gp120-CD4 interaction. Since F105 recognition was more sensitive to amino acid changes in each of the four identified gp120 regions than was envelope glycoprotein function, replication-competent mutant viruses that escaped neutralization by the F105 antibody were identified. These studies identify a conserved, functional HIV-1 gp120 epitope that is immunogenic in man and may serve as a target for therapeutic or prophylactic intervention.     

Lysis of cells infected with HIV-1 by human lymphocytes targeted with monoclonal antibody heteroconjugates

The present study was undertaken to determine whether human PBL can be specifically focused to lyse cells infected with HIV-1 by mAb heteroconjugates that can bridge target and effector cells. A mAb directed against the central portion of HIV-1 glycoprotein gp110 was chemically cross-linked to a mAb directed against the CD3/TCR complex or to a mAb directed against the CD16 Fc gamma-R expressed on large granular lymphocytes (LGL). HIV-1-infected cells, but not uninfected cells, were found to be lysed to a greater extent by PBL in the presence of the gp110 X CD3 or the gp110 X CD16 antibody heteroconjugate than in the presence of the single antibodies or a mixture of the mAb comprising the heteroconjugates. Pretreatment of PBL with anti-CD3 or IL-2 augments their ability to lyse HIV-1-infected cells in the presence of the heteroconjugates. Lysis by anti-CD3-activated PBL in the presence of the gp110 X CD3 heteroconjugate was found to be mediated by CD8+-enriched T cells, whereas lysis by IL-2-treated PBL in the presence of the gp110 X CD16 heteroconjugate is mediated by PBL enriched for CD16+ cells, which are primarily LGL. Furthermore, PBL from asymptomatic, HIV-1-infected seropositive donors were found to be functional in lysing HIV-1-infected cells in the presence of the antibody heteroconjugates. Such antibody heteroconjugates, which can target T cells or LGL to lyse HIV-1-infected cells, may be of prophylactic or therapeutic value in HIV-1-infected individuals.     

Molecular mapping of a new public HLA class I epitope shared by all HLA-B and HLA-C antigens and defined by a monoclonal antibody

It has previously been shown that a mouse monoclonal antibody, designated 4E, reacts with an epitope common to all HLA-B and -C antigens and those of the HLA-Aw19 cross-reactive group, namely, HLA-A29,-A30, -A31, -A32, -Aw33, and -Aw74. In order to pinpoint the amino acid residues which comprise the public specificity recognized by 4E, an HLA-A29 cDNA clone was isolated and its predicted amino acid sequence compared with those of other clonedHLA class I genes. The isolated HLA-A29 cDNA corresponded to the rarer of the twoA29 variant alleles,A29.1. Two amino acid residues of HLA-A29.1, gln-144 and arg-151, were found in all 24HLA-B andHLA-C alleles examined but were present in only one of 15HLA-A alleles for which sequence data are available. Importantly, this exceptional allele wasHLA-A32, another member of the HLA-Aw19 cross-reactive group. Gln-144 and arg-151 should be capable of jointly contributing to the binding site for 4E, as they are situated in successive alpha-helical subregions and are predicted to be juxtaposed in the three-dimensional HLA molecule. Four other residues in the first or second external domains of HLA-A29.1 (thr-9, leu-62, gln-63, and his-102) were unique among theHLA-A alleles, but none of these was found in corresponding positions ofHLA-B or-C alleles and thus failed to correlate with presence or absence of the 4E determinant. These observations are consistent with the notion that gln-144 and arg-151 define a determinant common to HLA-B, HLA-C, and the HLA-Awl9 cross-reactive group and the binding site of the monoclonal antibody 4E.     

Fine mapping of an epitope recognized by an invasion-inhibitory monoclonal antibody on the malaria vaccine candidate apical membrane antigen 1

Antibodies that inhibit red blood cell invasion by the Plasmodium merozoite block the erythrocytic cycle responsible for clinical malaria. The invasion-inhibitory monoclonal antibody (mAb) 4G2 recognizes a conserved epitope in the ectodomain of the essential Plasmodium falciparum microneme protein and vaccine candidate, apical membrane antigen 1 (PfAMA1). Here we demonstrate that purified Fab fragments of 4G2 inhibit invasion markedly more efficiently than the intact mAb, suggesting that the invasion-inhibitory activity of this mAb is not due solely to steric effects and that the epitope lies within a functionally critical region of the molecule. We have taken advantage of a synthetic gene encoding a modified form of PfAMA1, and existing x-ray crystal structure data, to fully characterize this epitope. We first validate the gene by demonstrating that it fully complements the function of the authentic gene in P. falciparum.We then use it to identify a group of residues within the previously described domain II loop of PfAMA1 that are critical for recognition by mAb 4G2 and demonstrate that the epitope lies exclusively within this loop with no contributions from residues in other domains of the molecule. This is the first complete characterization of a conserved invasion-inhibitory epitope on PfAMA1. Our results will aid in the design of subunit vaccines designed to generate a broadly effective, focused anti-PfAMA1 protective immune response and may help elucidate the function of PfAMA1.     

A monoclonal antibody recognizes a phosphorylated epitope shared by proteins of the cell nucleus and the erythrocyte membrane skeleton

Monoclonal antibody 3C5 recognizes a family of proteins in the nuclei of cultured cells [(1985) Eur. J. Cell Biol. 38, 344]. This antibody has now been shown to recognize equivalent proteins in liver nuclei and in the Triton-insoluble fraction of tissue extracts. In human erythrocytes the antibody recognized a single protein, present in the membrane skeleton fraction and with the molecular mass and extraction properties of beta-spectrin. The epitope recognized by 3C5 was destroyed by alkaline phosphatase. We conclude that this antibody recognizes a phosphorylation site shared by nuclear proteins and a protein of the erythrocyte membrane skeleton, probably beta-spectrin.     

Conformational fluctuations of proteins revealed by variable pressure NMR

With the high-resolution variable-pressure NMR spectroscopy, one can study conformational fluctuations of proteins in a much wider conformational space than hitherto explored by NMR and other spectroscopic techniques. This is because a protein in solution generally exists as a dynamic mixture of conformers mutually differing in partial molar volume, and pressure can select the population of a conformer according to its relative volume. In this review, we describe how variable-pressure NMR can be used to probe conformational fluctuations of proteins in a wide conformational space from the folded to the fully unfolded structures, with actual examples. Furthermore, the newly emerging technique "NMR snapshots" expresses amply fluctuating protein structures as changes in atomic coordinates. Finally, the concept of conformational fluctuation is extended to include intermolecular association leading to amyloidosis.     

Novel polysaccharide antigen of Orientia tsutsugamushi revealed by a monoclonal antibody

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates in the cytosol of host cells. Although several protein antigens have been characterized and cloned, little information exists regarding the polysaccharide antigen of this bacterium. In this study, we identified and characterized a novel antigen defined by a monoclonal antibody (MAb), NT19, against O. tsutsugamushi . Immunofluorescence microscopic studies showed that the NT19 antigen is released from the bacteria in the cytosol of host cells forming aggregates with bacteria. Immunoblot analysis showed that MAb NT19 recognized a strong band with a molecular mass of 20 kDa that was resistant to proteinase K digestion and sensitive to periodate oxidation, suggesting that the NT19 antigen is a polysaccharide. The function of this polysaccharide is not known, but considering its distribution within a bacterial microcolony, it is suspected to be involved in forming a biofilm-like structure within host cells.     

Ultrastructural characterization of embryonic chick cartilage proteoglycan core protein and the mapping of a monoclonal antibody epitope.

The ultrastructure of embryonic chick cartilage proteoglycan core protein was investigated by electron microscopy of specimens prepared by low angle shadowing. The molecular images demonstrated a morphological substructural arrangement of three globular and two linear regions within each core protein. The internal globular region (G2) was separated from two terminally located globular regions (G1 and G3) by two elongated strands with lengths of 21 +/- 3 nm (E1) and 105 +/- 22 nm (E2). The two N-terminal globular regions, separated by the 21-nm segment, were consistently visualized in well spread molecules and showed little variation in the length of the linear segment connecting them. The E2 segment, however, was quite variable in length, and the C-terminal globular region (G3) was detected in only 53% of the molecules. The G1, G2, and G3 regions in chick core protein were 10.1 +/- 1.7 nm, 9.7 +/- 1.3 nm, and 8.3 +/- 1.3 nm in diameter, respectively. These results are similar to those described previously for proteoglycan core proteins isolated from rat chondrosarcoma, bovine nasal cartilage, and pig laryngeal cartilage (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D., Hardingham, T., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). However, a significant difference was detected between the length of the elongated strand (E2) of core proteins isolated from chick cartilage, E2 length = 105 +/- 22 nm, compared to bovine nasal cartilage, E2 length = 260 +/- 39 nm. The epitope of the proteoglycan core protein-specific monoclonal antibody, S103L, was visualized by electron microscopy, and the distance from the core protein N terminus to the S103L binding site was measured. The S103L binding site was localized to the E2 region, 111 +/- 20 nm from the G1 (N terminus) domain and 34 nm from the G3 (C terminus) domain. cDNA clones selected from an expression vector library of chicken cartilage mRNA also show this epitope to be located near the C-terminal region (R. C. Krueger, T. A. Fields, J. Mensch, and B. Schwartz (1990) J. Biol. Chem. 265, 12088-12097).