Epitope mapping, V-region DNA sequence, and neutralizing Fab fragments of two monoclonal antibodies against the HIV-1 V3 loop (original) (raw)
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Journal of Virology, 2002
The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3 JR-CSF sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.
Protein Expression and Purification, 2002
enables studies of the same epitope bound to different Most human immunodeficiency virus type 1 (HIV-1) HIV-1 neutralizing antibodies. ᭧ 2002 Elsevier Science (USA) neutralizing antibodies in infected individuals and in Key Words: peptide expression; isotope labeling; antiimmunized animals are directed against the third varibody; NMR. able loop (V3) of the envelope glycoprotein (gp120) of the virus. This loop plays a crucial role in phenotypic Most neutralizing antibodies against human immudetermination, cytopathicity (syncytium induction), nodeficiency virus (HIV) in infected individuals or in and coreceptor usage of HIV-1. The human monoclonal immunized animals are directed against a determinant antibody 447-52D was found to neutralize a broad speclocated within the third hypervariable region (V3 loop) trum of HIV-1 strains. In order to solve the solution structure of the V3 MN peptide bound to the 447-52D Fab of the virus glycoprotein gp120 (1-3). This determinant fragment by NMR, large quantities of labeled peptide was termed the principal neutralizing determinant and a protocol for the purification of the Fab fragment (PND) (4) and was mapped to a 24-amino-acid sequence were needed. An expression plasmid coding for the between 2 conserved cysteine residues (Cys303 and 23-residue V3 peptide of the HIV-1 MN strain (V3 MN Cys338) which form a disulfide bridge. The V3 loop peptide, YNKRKRIHIGPGRAFYTTKNIIG) linked to a contains major determinants responsible for the phenoderivative of the RNA-binding domain of hnRNCP1 was type of the virus and cell tropism. This loop is one of constructed. The fusion protein attached to the V3 pepthe most variable sites on the virus, differing by as tide prevents its degradation. Using this system, U-15 N, much as 50% among isolates (4, 5); however, residues at U-13 C, 15 N, and U-13 C, 15 N, 50% 2 H labeled fusion protein the tip of the loop (GPGR) are highly conserved among molecules were expressed in Escherichia coli grown various subtypes. This region is a major target for HIVon rich Celtone medium with yields of about 240 mg/ 1 neutralizing antibodies, which inhibit the binding of liter. The V3 MN peptide was released by CNBr cleavage gp120 to the chemokine receptor (6). and purified by RP-HPLC, giving final yields of 6-13 One of the most broadly neutralizing and potent antimg/liter. This expression system is generally applicable V3 antibodies identified to date is the human monofor biosynthesis of V3-related peptides and was also clonal antibody 447-52D (7). It binds to a broad specused to prepare the V3 JR-FL. The 447-52D Fab fragment trum of highly divergent V3 peptides from six clades was obtained by a short enzymatic papain cleavage of (A, B, D, F, G, and H) (8) and is capable of neutralizing the whole antibody. Preliminary NMR spectra demonprimary isolates (macrophage-tropic), which are more strate that full structural analysis of the V3 MN comresistant than T-cell tropic strains to neutralization by plexed to the 447-52D Fab is feasible. This system monoclonal antibodies (9-11). These characteristics make 447-52D a very intriguing antibody. Antibody
Archives of Virology, 1992
Monoclonal antibodies raised against viral lysate of HIV-1 (strain LAV-1) and against recombinant gp 160 of HIV-1 (strain HTLV IIIB) which neutralized HIV-1 in a type specific manner were mapped with the aid ofpeptides (Pepscan analysis). Each of these monoclonal antibodies bound to peptides located on the principal neutralizing domain (PND) of HIV-1. We found that the antigenic sites of the MAbs described in this paper are represented by linear peptides of at least 10 amino acids long. The affinity of the MAbs is high for these peptides and in the same order of magnitude as for native gp 160. The fine mapping of the epitopes may reflect structural features of the PND, for instance which amino acid side chains are exposed and which are buried in the protein. Furthermore the fine mapping of the epitopes explained the HIV typespecifc neutralizing activity of the MAbs. Antibodies that bound to the tip of the loop (amino acids QRGPGRAF) have a higher neutralizing activity than antibodies that bound to amino acids towards the N-terminal side of the loop (amino acids KSIRI). Furthermore, MAbs that bound to virtually the same amino acids on the tip of the loop (amino acids IQRGPGRAF and RGPGRAFV) had different neutralizing activities due to different affinities for native gp 160. These data reveal that neutralizing activity not only is determined by the affinity of an antibody to the neutralizing site but also by its fine binding specificities to the V 3 loop of gp 120.
1991
The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gpl20 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The aim of this study was to obtain such site-specific antibodies. By using synthetic peptides derived from the V3 domain, a group-specific neutralizing PAb, two high-affinity HIV-1 IIIB neutralizing MAb, and two nonneutralizing MAb were raised. A 15-amino-acid peptide overlapping the tip of the V3 domain of H1V-1 MN was used to produce a rabbit PAb (W0/07). This PAb inhibited syncytium formation induced by HIV-1 IIIB and four field isolates. A similar IllB-derived peptide was used to generate two mur...
Journal of General Virology, 1991
Monoclonal antibodies (MAbs) raised against a 15-mer peptide representing the centre of the principal neutralization domain of human immunodeficiency virus type 1 (strain BH 10) showed wide variations in neutralizing activity against the homologous strain. The nature of this difference in neutralizing activity was studied by measuring antibody concentration, their affinity for peptide and specificity, by reaction with peptides which differed in the extent of sequence overlap, length and the presence of single amino acid replacements. All MAbs bound to approximately the same region in the principal neutralization domain, within the sequence RIQRGPGRAFV. The peptides with which each antibody was able to react differed by only a few amino acids. The neutralizing activity of each MAb preparation was related to its affinity and concentration; the affinity is related in part to the fine structure of the epitope recognized. MAbs with high affinity for the peptide tended to react only with peptides in which amino acid replacements did not affect the /?-turn potential of the peptide, whereas the reactivity of MAbs with low affinity was relatively insensitive to amino acid replacements affecting the/?-turn potential.
Journal of General Virology, 1996
Monoclonal antibody (MAb) ICR41 .li (rat IgG2a) is specific for a conformation-dependent epitope of human immunodeficiency virus type 1 (HIV-1) V3 , and MAb F58 (mouse 19G1) recognizes the peptide IXXGPGR, at the tip of the V3 loop. Both MAbs neutralized HIV-1 strain IIIB in C8166 and HeLa-T4(CD4) cells. Neutralization by either MAb did not inhibit attachment of virus to target cells as determined by FACS analysis, ELISA or immunofluorescence, and such attachment was absolutely dependent on the availability of CD4 molecules. F58 inhibited virus-induced cell-cell fusion, and reduced internalization of virions in direct proportion to neutralization. In contrast, ICR41.1i had no effect on HIV-l-mediated cell fusion or on internalization of virus. It was concluded that MAb F58 neutralized infectivity by inhibiting fusion of the virus with the cell and internalization of the viral core, and that ICR41.1i neutralized by inhibiting a post-fusion-internalization event. The possible mechanism by which a neutralizing antibody binds to the V3 loop and affects the function(s) of structures inside the virion is discussed. Lastly, postattachment neutralization (PAN) was investigated. F58 mediated PAN at 21 °C and 35 °C. However, ICR41.1i gave PAN at 21 °C but not at 35°C, suggesting that a temperature-dependent event affecting the V3 loop had abrogated neutralization. Overall, it appears that antibodies to different epitopes within the V3 loop neutralize by affecting very different functions of the virus.
A V1V2 neutralizing epitope is conserved in divergent non-M groups of HIV-1
Journal of acquired immune deficiency syndromes (1999), 2015
Highly potent broadly neutralizing monoclonal antibodies (bNabs) have been obtained from individuals infected by HIV-1 group M variants. We analyzed the cross-group neutralization potency of these bNabs towards non-M primary isolates (PI). The sensitivity to neutralization was analyzed in a neutralization assay using TZM-bl cells. Twenty three bNabs were used, including reagents targeting the CD4 binding site (CD4bs), the N160 glycan-V1V2 site, the N332 glycan-V3 site, the membrane proximal external region of gp41, and complex epitopes spanning both Env subunits. Two bispecific antibodies that combine the inhibitory activity of an anti-CD4 with that of PG9 or PG16 (BibNabs) were included in the study (PG9-iMab and PG16-iMab). Cross-group neutralization was observed only with the bNabs targeting the N160 glycan-V1V2 site. Four group O PIs, one group N PI and the group P PI were neutralized by PG9 and/or PG16 or PGT145 at low concentrations (0.04-9.39 µg/mL). None of the non-M PIs was...