Identification and synthesis of the epitope for a human monoclonal antibody which can neutralize human T-cell leukemia/lymphotropic virus type I

A monoclonal antibody (mAb), designated 0.5 alpha, derived from a patient with adult T-cell leukemia was found previously to neutralize the human T-cell leukemia/lymphotropic type I (HTLV-I) virus in in vitro assays and bind to the major envelope glycoprotein (gp46) of HTLV-I (Matsushita, S., Guroff, M.R., Trepel, J., Crossman, J., Mitsuya, H., and Broder, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2671-2676). We have designed experiments to determine the epitope for this mAb. Using simultaneous multiple peptide synthesis, we synthesized 481 overlapping octapeptides which corresponded to the sequence of gp46. We mapped the epitope for mAb 0.5 alpha to lie between residues 186 and 195 of gp46. This result was confirmed by independently synthesizing a peptide containing this epitope which bound specifically to mAb 0.5 alpha with an approximate Ka = 4 x 10(7) M-1. In addition, the peptide inhibited mAb 0.5 alpha binding to gp46 derived from T-cells infected with HTLV-I. This epitope containing peptide may facilitate understanding HTLV-1 infection of T-cells.     

Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

Background

The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported.

Methods and Results

This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched ²¹³SVQYHPL²¹⁹ of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that ²¹³SVQYHPL²¹⁹ was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group.

Conclusions and Significance

We identified ²¹³SVQYHPL²¹⁹ as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.     

The HIV-neutralizing monoclonal antibody 4E10 recognizes N-terminal sequences on the native antigen

Characterization of the epitope recognized by the broadly neutralizing anti-HIV Ab 4E10 has, heretofore, focused on a linear sequence from the gp41 pretransmembrane region (PTMR). Attempts to generate neutralizing Abs based on this linear epitope sequence have been unsuccessful. We have characterized the antigenic determinants on recombinant glycosylated full-length Ags, and nonglycosylated and truncated Ags recognized by 4E10 using epitope extraction and excision assays in conjunction with MALDI mass spectrometry. The mAb recognized the peptides (34)LWVTVYYGVPVWK(46) and (512)AVGIGAVFLGFLGAAGSTMGAASMTLTVQAR(542) located at the N-terminal region of gp120 and gp41, respectively. Immunoassays verified AV(L/M)FLGFLGAA as the gp41 epitope core. Recognition of the peptide from the gp41 PTMR was detected only in constructs in which the N termini of the mature envelope proteins were missing. In this region, the epitope core is located in the sequence (672)WFDITNWLWY(681). We hypothesize that the hydrophobic surface of the paratope functions as a "trap" for the viral sequences, which are responsible for insertion into the host cell membrane. As the N-terminal region of gp120, the fusogenic peptide of gp41, and the PTMR of gp41 show high sequence homology among various HIV strains, this model is consistent with the broadly neutralizing capabilities of 4E10.     

Characterization of a conserved T cell epitope in HIV-1 gp41 recognized by vaccine-induced human cytolytic T cells.

A human CTL epitope located in a region of the HIV-1 envelope protein gp41 that is highly conserved among various HIV-1 strains was identified. This epitope was recognized by CD4+ CTL clones that were induced in seronegative humans by immunization with recombinant gp160. Fusion proteins carrying portions of the HIV-1 env gene and synthetic peptides were used to localize this epitope to amino acids 584-595 of the HIV-1 BRU env sequence. Only two positions within this epitope showed variation among North American HIV-1 isolates, and the substitutions were conservative in nature. The Lys to Arg substitution at position 593 abolished recognition, probably by interfering with the peptide-MHC interactions. This epitope was recognized in association with at least one subtype of the widely distributed human class II MHC specificity DPw4, namely DPw4.2. The relatively high frequency of this allele (27.2% among Caucasians) makes it likely that a larger fraction of the population would generate a response directed at this epitope than would be the case for epitopes recognized in the context of gene products of most other class II and class I loci. Interestingly, the closely related DP beta-chain allele types 4.1 and 2.1, which differ from 4.2 by 3 and 1 amino acids, respectively, were unable to present this gp41 peptide to DPw4.2-restricted clones. Comparison of the structure of this epitope with that of other peptides recognized in the context of DPw4.2 led to the identification of a consensus sequence for DPw4.2 binding peptides. Because the gp41 CTL epitope 584-595 identified here is highly conserved and is recognized in the context of a common DP allele, it may represent an important target region for vaccine development. Our results indicate that vaccines containing this epitope may induce in a significant fraction of those immunized CTL active against at least half of all HIV-1 strains.     

Induction of a Mucosal Cytotoxic T-Lymphocyte Response by Intrarectal Immunization with a Replication-Deficient Recombinant Vaccinia Virus Expressing Human Immunodeficiency Virus 89.6 Envelope Protein

To improve the safety of recombinant vaccinia virus vaccines, modified vaccinia virus Ankara (MVA) has been employed, because it has a replication defect in most mammalian cells. Here we apply MVA to human immunodeficiency virus type 1 (HIV-1) vaccine development by incorporating the envelope protein gp160 of HIV-1 primary isolate strain 89.6 (MVA 89.6) and use it to induce mucosal cytotoxic-T-lymphocyte (CTL) immunity. In initial studies to define a dominant CTL epitope for HIV-1 89.6 gp160, we mapped the epitope to a sequence, IGPGRAFYAR (from the V3 loop), homologous to that recognized by HIV MN loop-specific CTL and showed that HIV-1 MN-specific CTLs cross-reactively recognize the corresponding epitope from strain 89.6 presented by H-2D^d. Having defined the CTL specificity, we immunized BALB/c mice intrarectally with recombinant MVA 89.6. A single mucosal immunization with MVA 89.6 was able to elicit long-lasting antigen-specific mucosal (Peyer’s patch and lamina propria) and systemic (spleen) CTL responses as effective as or more effective than those of a replication-competent vaccinia virus expressing 89.6 gp160. Immunization with MVA 89.6 led to (i) the loading of antigen-presenting cells in vivo, as measured by the ex vivo active presentation of the P18-89.6 peptide to an antigen-specific CTL line, and (ii) the significant production of the proinflammatory cytokines (interleukin-6 and tumor necrosis factor alpha) in the mucosal sites. These results indicate that nonreplicating recombinant MVA may be at least as effective for mucosal immunization as replicating recombinant vaccinia virus.     

Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies.

The humoral immune response to human cytomegalovirus (CMV) membrane glycoprotein gp58/116 (gB) has been studied by establishing cell lines producing specific human monoclonal antibodies. These cell lines were generated from peripheral blood lymphocytes obtained from a healthy carrier. Hybridomas producing gp58/116-specific antibodies were detected by reactivity to procaryotically expressed proteins containing the major neutralizing epitopes of this glycoprotein complex. One antibody, ITC88, which recognized an epitope located between amino acid residues 67 and 86 of gp116, potently neutralized the virus at 1 to 2 micrograms of immunoglobulin G per ml. Only four of the six human antibodies detecting the major neutralizing domain of gp58 neutralized the virus, and none of them required complement for activity. All antibodies that bound mature, processed gp58 recognized a conformational epitope involving sequences between residues 549 and 635. However, small differences existed between the antibodies in the actual minimal requirement for C- and N-terminal parts of this epitope. By peptide mapping with several of the antibodies, the epitope was shown to consist mainly of residues between amino acids 570 to 579 and 606 to 619. Despite the conformational nature of the epitope, the antibodies recognized both reduced and denatured native antigen. Presence of carbohydrates was not required for antigen binding of these gp58-specific human antibodies, but in at least one case, it greatly enhanced antigen recognition, indicating an importance of carbohydrate structures in some epitopes within the major neutralizing specificity of gp58.     

Identification of a MHC class II-restricted human gp100 epitope using DR4-IE transgenic mice

CD4+ T cells play a central role in the induction and persistence of CD8+ T cells in several models of autoimmune and infectious disease. To improve the efficacy of a synthetic peptide vaccine based on the self-Ag, gp100, we sought to provide Ag-specific T cell help. To identify a gp100 epitope restricted by the MHC class II allele with the highest prevalence in patients with malignant melanoma (HLA-DRB1*0401), we immunized mice transgenic for a chimeric human-mouse class II molecule (DR4-IE) with recombinant human gp100 protein. We then searched for the induction of CD4+ T cell reactivity using candidate epitopes predicted to bind to DRB1*0401 by a computer-assisted algorithm. Of the 21 peptides forecasted to bind most avidly, murine CD4+ T cells recognized the epitope (human gp10044-59, WNRQLYPEWTEAQRLD) that was predicted to bind best. Interestingly, the mouse helper T cells also recognized human melanoma cells expressing DRB1*0401. To evaluate whether human CD4+ T cells could be generated from the peripheral blood of patients with melanoma, we used the synthetic peptide h-gp10044-59 to sensitize lymphocytes ex vivo. Resultant human CD4+ T cells specifically recognized melanoma, as measured by tumor cytolysis and the specific release of cytokines and chemokines. HLA class II transgenic mice may be useful in the identification of helper epitopes derived from Ags of potentially great clinical utility.     

Quaternary protein mimetics of gp41 elicit neutralizing antibodies against HIV fusion-active intermediate state

During viral entry, the fusogenic state of human immunodeficiency virus Type 1 (HIV-1) envelope protein gp41 is a quaternary structure consisting of three gp41 glycoproteins, each with two conserved helical domains (N-HR and C-HR). Thus far, the examination of monomeric gp41 peptides as an immunologically focused approach to vaccine design has not been successful. Here we report an approach using quaternary protein mimetics (called 3alpha mimetics) that are based on the gp41 N-HR and C-HR domains to closely mimic the fusogenic state and overcome the deficiencies of the monomeric peptide approach for synthetic vaccine design. The 3alpha mimetics are conveniently prepared by chemoselective ligation of unprotected monomeric peptides to an interstrand linker, and display enhanced conformational stability compared to the corresponding monomers. The 3alpha mimetics with or without a covalently attached T-helper epitope were immunogenic and elicited antisera that bound both recombinant gp160, which contains gp41, and HIV-1 virions and immunoprecipitated recombinant gp41. Anti-3alpha mimetic antisera neutralized viral infectivity against R5- and X4-tropic strains of HIV-1 at 31.5 degrees C. The results suggest that a quaternary protein approach to mimic conserved and functional domains of viral envelope proteins is desirable for HIV vaccine development as such antigens are more likely to produce immunologically-focused and broadly neutralizing antibody responses.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs). Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries, displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and 4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4 mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized a continoous topographic epitope.     

Mapping of HLA epitopes recognized by H-2-restricted cytotoxic T lymphocytes specific for HLA using recombinant genes and synthetic peptides

Immunization of DBA/2 (H-2d) mice with syngeneic P815 tumor cell transfectants that express HLA class I genes elicits CTL that recognize HLA in the context of H-2Kd molecules. Anti-HLA-CW3 CTL cross-react to a variable extent on the related alleles A3 and A24. Using a panel of target cells expressing native or recombinant HLA genes, we could map the epitope recognized by a CTL clone specific for CW3 to the second external (alpha 2) domain of CW3. Moreover, the epitope recognized by this clone could be mimicked by incubating P815 (HLA negative) target cells with a synthetic peptide corresponding to the C-terminal 12 amino acids of the CW3 alpha 2 domain (residues 171 to 182). Other independent anti-CW3 CTL clones with different fine specificities recognized the same CW3 peptide. In contrast, CTL clones specific for HLA-A24 or HLA-A3 that did not lyse P815-CW3 transfectants did not recognize this peptide. The CW3 peptide could be recognized on other tumor cell targets that were also of H-2d origin, but not on those of H-2b or H-2k origin. The requirement for the expression of H-2Kd by the target cells was directly demonstrated using L cell Kd transfectants. Our results suggest that the CTL response of DBA/2 mice immunized with P815-CW3 transfectants is predominantly Kd restricted and focused on epitopes contained within the 12 C-terminal amino acids of the alpha 2 domain.     

Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.     

Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox

Flavocytochrome b558 is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91phox and p22phox subunits. Human flavocytochrome b558 is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22phox. 7D5 has proven useful in rapid screening of individuals for X-linked chronic granulomatous disease by flow-cytometry. Therefore, we re-evaluated the location of the 7D5 epitope using gene-engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp91phox. Flow-cytometric analyses showed that 7D5 consistently bound to cells expressing human gp91phox. In addition, 7D5 immunoprecipitated the approximately 58 kDa unglycosylated gp91phox protein from solubilized membrane fractions of tunicamycin-treated PLB-985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91phox but not p22phox were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91phox and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome b558-deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b558.     

Common immunologic determinant between human immunodeficiency virus type 1 gp41 and astrocytes.

Monoclonal antibodies against a synthetic 12-amino-acid peptide that comprises the immunodominant domain of human immunodeficiency virus type 1 gp41 (amino acids 598 through 609) reacted with astrocytes found in human and rodent central nervous system tissue. The monoclonal antibodies bound to a 43-kDa protein found in central nervous system tissue preparations. These results indicate that human immunodeficiency virus type 1 gp41 contains a common epitope with astrocytes and that an immune response to human immunodeficiency virus type 1 gp41 could generate antibodies that are cross-reactive to astrocytes. Furthermore, anti-astrocyte antibodies, which were directed at a common epitope with the gp41 sequence, were found to be present in cerebrospinal fluid from some AIDS patients with central nervous system complications. Astrocytes regulate the environment for appropriate neuronal function, and astrocyte hyperactivity (astrocytosis) is known to be the common and early pathologic event in brains from patients with central nervous system AIDS. We suggest that antibody-induced effect(s) on astrocytes could lead to the physiologic neuronal dysfunctions observed in AIDS patients.     

HIV neutralizing IgA in exposed seronegative subjects recognise an epitope within the gp41 coiled-coil pocket

Human immunodeficiency virus (HIV)-specific IgA can be detected in cervical secretions, saliva, and sera of HIV-infected and HIV-uninfected individuals with a known exposure to the virus. IgA from HIV-uninfected exposed seronegative individuals (ESN) neutralize in vitro primary strains of HIV-1. We analyzed the epitopes of HIV recognized by serum HIV-specific IgA of ESN individuals to identify the antigenic correlates of HIV neutralization in exposed-uninfected subjects, and to verify whether different epitopes would be recognized by HIV-specific IgA of ESN and of HIV-infected patients. Results confirmed that HIV-neutralizing IgA are detected in sera of ESN and showed that neutralization of primary HIV strains is mediated by the recognition of different epitopes in HIV-infected patients and ESN. Thus, whereas IgA of HIV+ individuals recognize epitopes expressed both within gp120 and gp41, IgA of ESN exclusively bind to gp41-expressed epitopes. Epitope mapping revealed that the epitope recognized by serum IgA of ESN on gp41 is restricted to aa 581-584 (LQAR) and corresponds to coiled coil pocket in the alpha helic region. In contrast, the epitope seen by IgA of HIV-infected patients on gp41 is identified by two regions; the first is contained within the cystein loop (aa 589-618), the second correspond to C terminal region in the extra membrane region of gp 41 (aa 642-673). Thus, we have identified and characterized the epitopes that mediate neutralization of HIV in individuals in whom infection does not occur despite multiple exposures to the virus. These results have important implications for the development of a new therapy against HIV infection.     

Structure‐antigenicity of the V3 region of SIVmac envelope glycoprotein

The objective of this study was to analyze the immunogenicity and antigenicity of the V3 domain (Cys313–Cys346) of the external envelope glycoprotein gp125 of SIVmac251. The corresponding peptide was synthesized and characterized as linear and cyclic peptides. Our results showed that this region, as for HIV‐1, contained an immunodominant epitope. The antigenicity was similar for the linear and cyclic peptides when tested against a panel of 15 sera from SIV infected macaques. Similarly, both peptide structures presented similar immunogenicity as shown by the characterization of the anti‐peptide antibodies produced in rabbits against the cyclic and linear forms. But, unexpectedly, the antibodies produced against linear peptides recognized with a relatively higher intensity the native envelope gp140 than those produced against the cyclic structure. Furthermore, we showed that these antibodies recognized better the deglycosylated form of the glycoprotein. But, in contrast to the neutralizing activity obtained with anti‐V3 peptides from HIV‐1, no antiviral activity was obtained with antibodies generated against linear or cyclic SIVmac V3 peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Immunoglobulins against gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus,are directed against charged O-linked oligosaccharide chains bearing a Lewis-like structure and interact with epitopes of the human zona pellucida

In oocytes of the mollusc bivalve Unio elongatulus, gp273 is the ligand molecule for sperm-egg interaction and binding is mediated by its O-glycans. A serum raised against this protein enabled its localization in the crater region, the area of the vitelline coat where sperm recognition occurs, and showed that after cyanogen bromide fragmentation, the anti-gp273 epitope(s) was retained by a peptide where the O-glycans are localized. In this article, we utilized purified anti-gp273 immunoglobulins to characterize the corresponding epitope by: (i) immunoblotting analysis of the protein after removal of O- and N-glycans; (ii) solid phase binding analysis of anti-gp273 IgG to gp273 N- and O-glycans; and (iii) binding analysis of the same antibody to commercially available oligosaccharides. The results showed that the epitope consists of O-glycans and contains a Lewis-like structure with fucose as determinant. Anti-gp273 IgG were then used to investigate human zona pellucida by immunoelectronmicroscopy and immunoblotting. Epitopes recognized by the antibody were demonstrated on the outer surface of the zona pellucida and shown to belong to a zona pellucida protein having electrophoretic mobility similar to human ZP3. Since human sperm specifically bind to gp273, and anti-gp273 interferes with this binding a functional role for these epitopes is suggested.     

NMR structure of an anti-gp120 antibody complex with a V3 peptide reveals a surface important for co-receptor binding

BACKGROUND: The protein 0.5beta is a potent strain-specific human immunodeficiency virus type 1 (HIV-1) neutralizing antibody raised against the entire envelope glycoprotein (gp120) of the HIV-1(IIIB) strain. The epitope recognized by 0.5beta is located within the third hypervariable region (V3) of gp120. Recently, several HIV-1 V3 residues involved in co-receptor utilization and selection were identified. RESULTS: Virtually complete sidechain assignment of the variable fragment (Fv) of 0.5beta in complex with the V3(IIIB) peptide P1053 (RKSIRIQRGPGRAFVTIG, in single-letter amino acid code) was accomplished and the combining site structure of 0.5beta Fv complexed with P1053 was solved using multidimensional nuclear magnetic resonance (NMR). Five of the six complementarity determining regions (CDRs) of the antibody adopt standard canonical conformations, whereas CDR3 of the heavy chain assumes an unexpected fold. The epitope recognized by 0.5beta encompasses 14 of the 18 P1053 residues. The bound peptide assumes a beta-hairpin conformation with a QRGPGR loop located at the very center of the binding pocket. The Fv and peptide surface areas buried upon binding are 601 A and 743 A(2), respectively, in the 0.5beta Fv-P1053 mean structure. The surface of P1053 interacting with the antibody is more extensive and the V3 peptide orientation in the binding site is significantly different compared with those derived from the crystal structures of a V3 peptide of the HIV-1 MN strain (V3(MN)) complexed to three different anti-peptide antibodies. CONCLUSIONS: The surface of P1053 that is in contact with the anti-protein antibody 0.5beta is likely to correspond to a solvent-exposed region in the native gp120 molecule. Some residues of this region of gp120 are involved in co-receptor binding, and in discrimination between different chemokine receptors utilized by the protein. Several highly variable residues in the V3 loop limit the specificity of the 0.5beta antibody, helping the virus to escape from the immune system. The highly conserved GPG sequence might have a role in maintaining the beta-hairpin conformation of the V3 loop despite insertions, deletions and mutations in the flanking regions.     

The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5

The human monoclonal antibody 2F5 neutralizes primary human immunodeficiency virus type 1 (HIV-1) with rare breadth and potency. A crystal structure of a complex of 2F5 and a peptide corresponding to its core epitope on gp41, ELDKWAS, revealed that the peptide interacts with residues at the base of the unusually long (22-residue) third complementarity-determining region of the heavy chain (CDR H3) but not the apex. Here, we perform alanine-scanning mutagenesis across CDR H3 and make additional substitutions of selected residues to map the paratope of Fab 2F5. Substitution of residues from the base of the H3 loop or from CDRs H1, H2, and L3, which are proximal to the peptide, significantly diminished the affinity of Fab 2F5 for gp41 and a short peptide containing the 2F5 core motif. However, nonconservative substitutions to a phenylalanine residue at the apex of the H3 loop also markedly decreased 2F5 binding to both gp41 and the peptide, suggesting that recognition of the core epitope is crucially dependent on features at the apex of the H3 loop. Furthermore, substitution at the apex of the H3 loop had an even more pronounced effect on the neutralizing activity of 2F5 against three sensitive HIV-1. These observations present a challenge to vaccine strategies based on peptide mimics of the linear epitope.     

Identification of a neutralization epitope on the envelope gp46 antigen of human T cell leukemia virus type I and induction of neutralizing antibody by peptide immunization.

We have generated a number of mAb against various epitopes on the external envelope glycoprotein, gp46, of human T cell leukemia virus type I (HTLV-I) from a WKA rat immunized with a recombinant vaccinia virus containing the HTLV-I env gene. Among these mAb, one group of mAb, represented by a mAb designated LAT-27, could neutralize the infectivity of HTLV-I, as determined by a HTLV-I-mediated cell fusion inhibition assay. LAT-27 also interfered with transformation of normal T lymphocytes by HTLV-I in vitro. An antibody-binding assay using overlapping synthetic oligopeptides showed that LAT-27 bound specifically to 10-mer peptides that contained the gp46 amino acid sequence 191-196 (Leu-Pro-His-Ser-Asn-Leu). Antibodies from HTLV-I+ humans interfered with the binding of LAT-27 to gp46 Ag. Sera from rabbits immunized with a LAT-27-reactive peptide, 190-199, conjugated with OVA, but not sera from OVA-immunized rabbits, reacted with gp46 Ag and neutralized infectivity of HTLV-I. These results show that the HTLV-I neutralization epitope recognized by LAT-27 locates to the gp46 amino acids 191-196, and that immunization with a peptide containing the LAT-27 epitope can elicit an HTLV-I neutralizing antibody response.