HIV-1 Gp120 V3 Loop Research Articles

HIV-1 gp120 V3 Loop for Structure-Based Drug Design

HIV-1 cell entry is mediated by sequential interactions of the envelope protein gp120 with the receptor CD4 and a coreceptor, usually CCR5 or CXCR4, depending on the individual virion. Considerable efforts on exploiting the HIV coreceptors as drug targets have led to the new class of coreceptor antagonists. While these antiretroviral drugs aim at preventing virus/coreceptor interaction by binding to host proteins, neutralizing antibodies directed against the coreceptor-binding sites on gp120 have attracted attention as possible vaccine candidates. However, both approaches are complicated by the multiple protective mechanisms of gp120 which allow for rapid escape from selective pressures exerted by drugs or antibodies. Thus, advances in rational drug and vaccine design rely heavily on improved insights into the relation between genotype and phenotype, the evolution of coreceptor usage, and, ultimately the structural biology of coreceptor usage and inhibition. The third variable (V3) loop of gp120, crucially involved in all these aspects, will be a major focus of this review.

The N-terminal of the V3 loop in HIV type 1 gp120 is responsible for its conformation-dependent interaction with cell surface molecules.

The V3 loop of HIV-1 gp120 plays an important role in the interaction of the viral envelope with cellular coreceptors and/or with other cell surface molecules. To clarify this interaction we used a panel of monoclonal antibodies (MAbs) against V3 loop and synthetic looped V3 peptides V3-BH10, V3-ADA, and V3-89.6, derived from the V3 regions of the BH10 clone of IIIB (X4-tropic), ADA (R5-tropic), and 89.6 (R5X4-tropic), respectively. A linear mutant peptide, V3-BH10/CA, was also synthesized as a control. Biotinylated V3-BH10, -BH10/CA, and-ADA were also made. The binding abilities of the biotinylated and nonbiotinylated peptides to various types of cells were investigated by using flow cytometry. Subsequently, the principal region of the V3 loop involved in cell surface binding was analyzed by using MAbs against the tip (447-52D and 694-98D), N-termini (IIIB-V3-21) or C-termini (IIIB-V3-01) of the V3 loop in flow cytometry and enzyme-linked immunoabsorbent assay. We demonstrate that looped V3 peptides of both X4 and R5X4 HIV (V3-BH10 and V3-89.6) can bind to various types of cells irrespective of their CD4 and/or coreceptor expression in a conformation-dependent manner. In contrast, the V3 loop of R5 HIV (V3-ADA) can scarcely bind to the cells. Using MAbs whose epitopes cover the entire V3 loop we found that MAb IIIB-V3-21 can react with platebound but not cell-bound peptides, and the MAb blocked biotin-V3-BH10 binding suggesting that the N-terminal of the V3 loop interacts directly with cell surface molecule(s).

Expression and functional activity of isotype and subclass switched human monoclonal antibody reactive with the base of the V3 loop of HIV-1 gp120.

Immunoglobulins undergo isotype switching in response to antigenic stimulation. The C(H) domains, in particular the hinge region, impose structural constraints on the interaction of antibody with antigen, especially multivalent antigens such as HIV. We previously showed that switching the IgG1 anti-HIV human monoclonal antibody (HMAb) F105 to an IgG3 resulted in significantly enhanced neutralization of HIV. To further investigate the influence of isotype, including the functional activity of HMAbs switched to IgA, which may be important in mucosal defenses, isotype switched antibodies have been generated for the anti-V3 loop base IgG2 HMAb F425B4e8. Reactivity of the IgG1 antibody was greater than the parental IgG2 antibody for SF2 infected cells but less for primary isolate virions. In contrast, there was less reactivity of the IgG3 with either infected cells or virions. IgA reacted significantly more with infected cells and virions as compared to the IgG subclasses. In contrast to previous studies whereby IgG3 enhanced neutralization, comparable neutralization of primary isolate virus was observed for IgG subclasses (IgG1, IgG2, IgG3) and IgA. This may reflect differences in the exposure of epitopes recognized by the HMAb with antibody flexibility being important to neutralization by antibodies reactive with obscured epitopes (e.g., CD4 binding site). Further analysis of the in vitro activity of isotype or subclass switched antibodies, IgA in particular, alone and in combination with other HMAbs, will provide important information on the role of IgG subclass and IgA antibodies on protective immunity to HIV.

Crystal Structure of a Human Rhinovirus that Displays Part of the HIV-1 V3 Loop and Induces Neutralizing Antibodies against HIV-1

We report the 2.7 Å resolution structure of a chimeric rhinovirus, MN-III-2, that displays part of the HIV-1 gp120 V3 loop and elicits HIV-neutralizing antibodies. The V3 loop insert is dominated by two type I β turns. The structures of two adjacent tripeptides resemble those of analogous segments in three Fab/V3 loop peptide complexes. Although two of the three corresponding antibodies bind and neutralize MN-III-2 well, only one of the three can bind without significant rearrangement. These results suggest that the V3 loop insert: (1) can share some local conformational similarity to V3 loop sequences presented on different structural frameworks; (2) must be able to adopt multiple conformations, even in a relatively constrained environment; and (3) may mimic the conformational variability of the epitope on HIV-1, increasing the likelihood of eliciting appropriate neutralizing immune responses.

Sequence variation and consensus sequence of V3 loop on HIV-1 gp120

Mutation in the V3 loop of HIV-1 gp120 could affect syncytium formation, virus infectivity and neutralization. To acquire more information of the V3 loop mutation, we analyzed amino acid sequences of the V3 loop of 24 504 isolates from most HIV-1 clades (including A, B, C, D, E, F, G and H clades). The consensus sequence of the V3 loop of each subtype with the highest frequency emerging on each position is constituted and the conservation of each amino acid in this region is also calculated. Exploring the restricted mutation of the V3 region could help to understand mechanism of HIV entry and to develop new strategy against HIV-1.

Comparing tandem repeats and multiple antigenic peptides as the antigens to detect antibodies by enzyme immunoassay

Short synthetic peptides are useful alternatives to whole lysate or recombinant proteins as the antigens used for serodiagnosis of bacterial or viral infections. However, certain known antigenic peptides displayed low seroreactivities in direct enzyme immunoassay. This was believed to be due to the low coating efficiency, a constrained orientation, or loss of flexibility required for optimal antibody binding. Using a model peptide system derived from the V3-loop of HIV-1 gp120, we demonstrated that low antigenicity could be overcome by using either tandem repeats (TR) or multiple antigenic peptides (MAPs) which contained the same amino acid sequence as the monomeric peptide. In our model system, a four-branch MAP was a better choice compared to the tandem repeats because of the MAP's slightly higher sensitivity and lower cost of production.

Ion channel activation by SPC3, a peptide derived from the HIV-1 gp120 V3 loop.

SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence. This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains. However, the molecular mode of action of SPC3 remains unclear. Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection. To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors. Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration. This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3. The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor. The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity. Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.

T-tropic sequence of the V3 loop is critical for HIV-1 infection of CXCR4-positive colonic HT-29 epithelial cells.

Some colonic and neuronal cells which are CD4- but galactosyl ceramide-positive are susceptible to infection with HIV-1. We have previously shown that the T-cell tropic V3 loop of HIV-1 gp120 serves as a primary viral determinant for infectivity of CD4- neuronal cells. However, the nature of the V3 loop of HIV-1 needed for infection and the V3 loop's interaction with coreceptors on colonic epithelial cells have not been fully analyzed. By using HIV-1 molecular clones, we show that the T-cell tropic V3 domain is critical for HIV-1 infection of colonic HT-29 epithelial cells. Because T-cell tropic HIV-1 can use CXCR4 as a coreceptor in T cells, we set out to determine the role of CXCR4 during infection of HT-29 cells. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining, we show that these epithelial cells of colonic origin express the chemokine receptor CXCR4. Importantly, antibody against CXCR4 or a neutralizing antibody against HIV-1 gp120 V3 loop blocks T-cell tropic HIV-1 entry into HT-29 cells. These data indicate that the V3 loop of HIV-1 and the chemokine receptor CXCR4 are both critical for HIV-1 infection of colonic HT-29 epithelial cells. An HIV-1 T-tropic virus may be responsible for the infection of human colonic epithelial cells in vivo.

T-Tropic Sequence of the V3 Loop Is Critical for HIV-1 Infection of CXCR4-Positive Colonic HT-29 Epithelial Cells

Some colonic and neuronal cells which are CD4- but galactosyl ceramidepositive are susceptible to infection with HIV-1. We have previously shown that the T-cell tropic V3 loop of HIV-1 gp120 serves as a primary viral determinant for infectivity of CD4- neuronal cells. However, the nature of the V3 loop of HIV-1 needed for infection and the V3 loop's interaction with coreceptors on colonic epithelial cells have not been fully analyzed. By using HIV-1 molecular clones, we show that the T-cell tropic V3 domain is critical for HIV-1 infection of colonic HT-29 epithelial cells. Because T-cell tropic HIV-1 can use CXCR4 as a coreceptor in T cells, we set out to determine the role of CXCR4 during infection of HT-29 cells. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining, we show that these epithelial cells of colonic origin express the chemokine receptor CXCR4. Importantly, antibody against CXCR4 or a neutralizing antibody against HIV-1 gpl20 V3 loop blocks T-cell tropic HIV-1 entry into HT-29 cells. These data indicate that the V3 loop of HIV-1 and the chemokine receptor CXCR4 are both critical for HIV-1 infection of colonic HT-29 epithelial cells. An HIV-1 T-tropic virus may be responsible for the infection of human colonic epithelial cells in vivo.

Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements.

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5beta, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with 15N labels at the eta nitrogen positions of arginine side chains and 13C labels at glycine carbonyl positions and 13C-detected 13C-15N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5beta complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of phi and psi backbone dihedral angles in the RP135/0.53 complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect 13C-15N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.

Phosphorothioate G3T4G3 motifs inhibits the early stage of HIV-1 infection.

It is well known that G-rich phosphorothioate oligonucleotides (G-rich PS) bind to the V3 loop of HIV-1 gp120 and inhibit HIV-1 infection. In this study, we investigated the inhibitory mechanism of a new type of G-rich PS (PS-G3T4G3) on the replication cycle of HIV-1. PS-G3T4G3 inhibits both cell to cell and cell free infections. Binding and entry assays revealed that the inhibitory step of PS-G3T4G3 occurs at the early stage of HIV-1 infection. V3 loop-specific mAb test showed that PS-G3T4G3 binds to the V3 loop and prevents its interaction with chemokine receptors. These results suggest that PS-G3T4G3 may be a novel candidate for an HIV-1 inhibitor.

Solid-state NMR evidence for an antibody-dependent conformation of the V3 loop of HIV-1 gp120.

Solid-state NMR measurements have been carried out on frozen solutions of the complex of a 24-residue peptide derived from the third variable (V3) loop of the HIV-1 envelope glycoprotein gp120 bound to the Fab fragment of an anti-gp120 antibody. The measurements place strong constraints on the conformation of the conserved central GPGR motif of the V3 loop in the antibody-bound state. In combination with earlier crystal structures of V3 peptide-antibody complexes and existing data on the cross-reactivity of the antibodies, the solid-state NMR measurements suggest that the Gly-Pro-Gly-Arg (GPGR) motif adopts an antibody-dependent conformation in the bound state and may be conformationally heterogeneous in unbound, full-length gp120. These measurements are the first application of solid-state NMR methods in a structural study of a peptide-protein complex.

Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing fabs.

The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II beta turn. The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.

The Effect of Low-Profile Serine Substitutions in the V3 Loop of HIV-1 gp120 IIIB/LAI on the Immunogenicity of the Envelope Protein

Many microbial antigens contain powerful hypervariable epitopes that fail to induce broadly protective immunity because they dominate the immune response at the expense of more conserved but weaker epitopes. If the undesired B cell epitopes are eliminated, the immune system could be focused on the conserved epitopes and produce a stronger antibody response to conserved parts of the protein and thus become a more efficacious immunogen for a vaccine. We examined this possibility using the human immunodeficiency virus envelope glycoprotein (gp)120 IIIB/LAI and selectively replaced the amino acids from the V3 region and analyzed the overall immunogenicity of the mutant proteins after nucleic acid immunization in mice. The most variable residues of the human immunodeficiency virus type 1 gp120 V3 loop sequence were replaced with serine, which has a small uncharged hydrophilic side chain and therefore is likely to be less immunogenic than amino acids found in wildtype V3 sequences. The serine substitutions did not affect the ability of soluble CD4 to bind the mutant molecules compared with wildtype gp120 and monoclonal antibodies against both linear and discontinuous epitopes located in the V1/V2, C1, and C4 regions of the molecule. These data suggest that the V3 loop substitutions did not grossly affect the overall conformation of the envelope molecule. Immunization of CBA × BALB/c F1 mice with DNA expression plasmids for the wild-type gp120 sequence induced a predominantly IgGIantibody response with end point titers of 104–5 × 104. The antibodies reacted only with conformationally intact gp120. Serine replacements targeted to both sides of the V3 loop had a major impact on gp120 immunogenicity, with a markedly reduced response in the majority of animals tested. Analysis of the epitope specificity of the responses suggests that N-terminal amino acids in the V3 loop contribute to the major immunodominant epitope and provides no evidence that their removal enhances immunogenicity of the conserved regions.

Dendritic cells from HIV-1-infected patients naturally express HIV-1 gp120 V3 loop-derived peptide ligands.

Little is known of the peptide ligands expressed in vivo on antigen-presenting cells (APC) or of the APC lineages involved. In this study we have addressed this question using HLA-DRbeta1*0101-restricted CD4 T cell clones (TLC) specific for a synthetic peptide based on the HIV-1 gp120 V3 loop consensus sequence for the Clade B isolates predominantly found in European and North American patients. These TLC were found to respond, in a dose-dependent manner, to freshly isolated HIV-infected patient APC in the absence of exogenously added peptides. Further APC purification showed that the naturally expressed peptide ligands were present in both the APC lineages shown to be infected with the virus and were most strongly detectable on purified blood dendritic cells. Peptides based on consensus sequences of viruses isolated from one of the patients over the period when naturally expressed peptide ligands could be detected were all found to stimulate TLC proliferation. These studies, therefore, show that peptide ligands derived from natural infection are detectable on APC lineages, particularly on dendritic cells which play an important role in the immune response to viruses. Even small differences in sequence between the vaccine isolate and the natural infection, if they occur in the key residues of protective T cell epitopes, could therefore have a profound effect on the efficacy of vaccines against viruses with high rates of mutation.

Structure-based design of peptides that recognize the CD4 binding domain of HIV-1 gp120.

Envelope protein-specific antiviral peptides, called mucibodies, that can specifically recognize and bind to the surface unit protein gp120 of HIV-1 were designed. The initial mucibody binding target was the V3 loop of HIV-1 gp120. Here, the gp120-CD4 binding domain was chosen as the site of mucibody binding. The CD4 binding domain of gp120 is known to be a conformational epitope and is involved in the earliest events of viral entry into many cells. The design of the mucibody antivirals was based on previous observations that antibody complementarity determining regions (CDR) are generally similar to the repeating loops or knob structures found in the 20-residue tandem repeat domain of human mucin MUC1. The heavy chain CDR3 from the bacteriophage display antibody b12 was used to construct two mucibodies, b12-CDR1 and b12-26. Peptides corresponding to three tandem repeats were shown to bind directly to the CD4 binding domain of HIV-1 gp120 in a solid-phase enzyme-linked immunosorbent assay. These mucibody peptides also disrupted the gp120-CD4 interaction in a solution-phase inhibition assay. Finally, mucibodies neutralized primary and laboratory macrophage-tropic isolates of HIV-1. There is a potential for medical use of these peptides as topical vaginal microbicides in preventing HIV-1 transmission during sexual contact. These results also suggest that multivalent, non-immunogenic binding proteins of virtually any specificity could be constructed for use in therapeutic applications involving infectious diseases and immune system dysfunction.

Shared Antigenic Epitopes on the V3 Loop of HIV-1 gp120 and Proteins on Activated Human T Cells

Proliferation of HIV-1 in the infected host is characterized by a progressive loss of CD4+T lymphocytes and consequent dysregulation of the immune system. Both direct and indirect mechanisms have been proposed. We show here that proteins with molecular weights 35, 48, and 110 kDa on stimulated primary human T cells are recognized by neutralizing antibodies against the V3 loop of HIV-1 gp120. Recognition is specific since it can be blocked by a recombinant HIV-1 gp120. Furthermore, these V3 monoclonal antibodies, as well as sera from AIDS patients that recognized these V3-like proteins, induced killing of HIV-1-infected as well as uninfected T cells. This killing was also inhibited by HIV-1 gp120 V3 peptides. These results indicate that the V3 loop shares epitopes with proteins on stimulated T cells. This may be an additional autoimmune mechanism contributing to CD4+T cell disappearance in AIDS. V3 antibodies have been proposed as potential prophylactic agents. However, if such vaccines were based on certain epitopes, they might induce cross-reacting immune responses with cellular proteins. Vaccine candidates should be evaluated for such potential effects.

Sequential Interaction of CD4 and HIV-1 gp120 with a Reconstituted Membrane Patch of Ganglioside GM3: Implications for the Role of Glycolipids as Potential HIV-1 Fusion Cofactors

The fusion of HIV-1 with CD4+cells involves, in addition to CD4, specific cell surface molecules acting as fusion cofactors. Recently, we reported that the V3 loop of HIV-1 gp120 binds to GM3, a ganglioside abundantly expressed on CD4+lymphocytes and macrophages. In the present study, we show that CD4 interacts with a reconstituted patch of GM3 by measuring the surface pressure with a Langmuir film balance. A biphasic increase in surface pressure is observed after the sequential addition of CD4 and gp120 under the GM3 monolayer, indicating the formation of the trimolecular complex GM3-CD4-gp120. Neutralization of gp120 with an anti-V3 antibody inhibits the secondary interaction with GM3, suggesting that the CD4-induced conformational change in gp120 allows the V3 loop to interact with GM3. In conclusion, this study supports the concept that glycolipids can function as HIV-1 fusion cofactors.

Thermodynamic Analysis of the Interaction between the 0.5β Fv Fragment and the RP135 Peptide Antigen Derived from the V3 Loop of HIV-1 gp120

The Fv fragment of the 0.5beta monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP135 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5beta Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpy-entropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on DeltaCp and calculated values of DeltaDeltaCp based on the extent of burial of polar and non-polar surface areas of amino acids. The value of DeltaCp for the binding of the 0.5beta Fv fragment to the wild-type RP135 peptide is found to be -5.0 (+/- 0.9) kcal K-1 mol-1 in the presence of 0.1% Tween-20 but only -0.1 (+/- 0.9) kcal K-1 mol-1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents.

NMR study and comparison of the antigenic properties of a peptide recognized by two HIV-1 neutralizing antibodies.

Fab-peptide complexes formed between a 15 residue peptide derived from the HIV-1 gp120 V3 loop and two of its cognate monoclonal antibodies, 5023A and 5025A, were studied using isotope-edited solution nuclear magnetic resonance (NMR) techniques. Since these antibodies neutralize HIV-1 virus with different strain specificities, this study was conducted to better understand the nature of these differences. The amide proton and nitrogen NMR resonances of specific residues were used to monitor the backbone of this peptide in these complexes. Three central residues of this peptide ('RAF') were found to be strongly affected by binding to both antibodies. Several other peptide residues were affected by binding to antibody 5023A but not 5025A. The antibody epitopes mapped by NMR are similar to those obtained previously via PEPSCAN at higher pH. One main difference between the PEPSCAN and NMR determined epitopes for 5023A involved two glycine residues of the peptide. By NMR, one of these glycines was more dramatically affected by antibody binding than predicted by PEPSCAN, while the other was much less so.