Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralizationpotencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.     

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.     

Analysis of a highly immunodominant epitope in the human immunodeficiency virus type 1 transmembrane glycoprotein, gp41, defined by a human monoclonal antibody.

A human monoclonal antibody, 41-7 [immunoglobulin G1(kappa)], directed against the transmembrane glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) has been produced by direct fusion of lymph node cells from an HIV-1-infected individual with a human B-lymphoblastoid cell line. The minimal essential epitope for 41-7 was mapped to a conserved seven-amino acid sequence, N-CSGKLIC-C, located within the N-terminal part of gp41. Antibodies blocking the binding of 41-7 could be detected in the serum of all HIV-1-infected individuals tested, irrespective of the stage of the infection. The epitope is located externally to the plasma membrane, and it is accessible to antibody in the native conformation of the glycoprotein. Despite this, no neutralizing activity of 41-7 could be demonstrated in vitro. These data indicate, directly and indirectly, that this immunodominant epitope on gp41, although exposed on the viral surface, elicits antibodies lacking antiviral activity and, hence, should be avoided in future vaccine candidates.     

Different susceptibility of human immunodeficiency virus type 1 to Env gp41-derived synthetic peptides corresponding to the C-terminal heptad repeat region

Two functional domains, alpha-helical heptad repeat 1 (HR-1) and HR-2, located in the N-terminal and C-terminal regions of human immunodeficiency virus type 1 (HIV-1) Env gp41, respectively, play an important role in the fusion process. Synthetic 34-amino-acid peptide that contains the HR-2 region, named C34, has been shown to inhibit the HIV-1 fusion process. Here, we prepared six representative peptides (C34-B1, -B2, -A, -C1, -C2, and -E from subtypes B, A, C, and E, respectively) according to the sequences from the HIV sequence database of Los Alamos. All the C34 peptides had lower ability to inhibit the primary isolates (subtypes B and CRF01_AE) than subtype B laboratory strain LAI. On the other hand, the L-2 cell clone, isolated from persistently LAI-infected MT-4 cells (MT-4/LAI), showed unique C34 peptide sensitivities. L-2 virus has the same sequences at HR-1 and HR-2 regions as LAI, but showed higher syncytia formation activity than LAI. Interestingly, the sensitivity of L-2 was higher to C34-B2 and -A but slightly lower to C34-C1 at higher concentrations than MT-4/LAI, while C34-B1, -C2, and -E showed similar activity against both viruses. Thus, in addition to the sequences of the C34 peptide as well as of the HR-1 and HR-2 regions in target viruses used for fusion assays, the fusion inhibitory activities of C34 peptides seem to be affected by viral factor(s) other than the gp41 alpha-helical heptad repeats.     

Sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor

T-20 is a synthetic peptide that corresponds to 36 amino acids within the C-terminal heptad repeat region (HR2) of human immunodeficiency virus type 1 (HIV-1) gp41. T-20 has been shown to potently inhibit viral replication of HIV-1 both in vitro and in vivo and is currently being evaluated in a Phase III clinical trial. T-649 is an inhibitory peptide that also corresponds to 36 amino acids within HR2. This sequence overlaps the T-20 sequence but is shifted 10 residues toward the N terminus of gp41. Both inhibitors are thought to exert their antiviral activity by interfering with the conformational changes that occur within gp41 to promote membrane fusion following gp120 interactions with CD4 and coreceptor molecules. We have shown previously that coreceptor specificity defined by the V3 loop of gp120 modulates sensitivity to T-20 and that a critical region within the N-terminal heptad repeat (HR1) of gp41 is the major determinant of sensitivity (C. A. Derdeyn et al., J. Virol. 74:8358-8367, 2000). This report shows that (i) regions within gp41 distinct from those associated with T-20 sensitivity govern the baseline sensitivity to T-649 and (ii) T-649 sensitivity of chimeric viruses that contain sequences derived from CXCR4- and CCR5-specific envelopes is also modulated by coreceptor specificity. Moreover, the pattern of sensitivity of CCR5-specific chimeras with only minor differences in their V3 loop was consistent for both inhibitors, suggesting that the individual affinity for coreceptor may influence accessibility of these inhibitors to their target sequence. Finally, an analysis of the sensitivity of 55 primary, inhibitor-naive HIV-1 isolates found that higher concentrations of T-20 (P < 0.001) and T-649 (P = 0.016) were required to inhibit CCR5-specific viruses compared to viruses that utilize CXCR4. The results presented here implicate gp120-coreceptor interactions in driving the complex conformational changes that occur in gp41 to promote fusion and entry and suggest that sensitivity to different HR1-directed fusion inhibitors is governed by distinct regions of gp41 but is consistently modulated by coreceptor specificity.     

Characterization of a novel human anti-HIV-1 gp41 IgM monoclonal antibody designated clone 37

Human monoclonal antibodies (HuMAbs) demonstrate great potential for passive immunotherapy against HIV-1. The gp41 transmembrane envelope glycoprotein of HIV has an important role in the pathogenicity of AIDS and importantly displays considerably less hypervariability than the gp120 surface envelope HIV glycoprotein, which makes it particularly a better candidate for the development of passive and active immunotherapies. The general aim of this study was to develop HuMAbs to HIV surface glycoproteins and particularly gp41. Peripheral blood mononuclear cells (PBMCs) were isolated from an HIV-seropositive long-term nondisease progressing patient. B-cells from this individual were then immortalized by Epstein-Barr virus (EBV) transformation, and antibody production was stabilized by fusion of transformed cells with a heteromyeloma. Subsets of the human heterohybridomas so generated were analyzed by ELISA. The hybridoma with the highest binding by immunoassay against gp160 was further analyzed. This hybridoma, designated as clone 37 (C37), was determined to be an IgM Kappa antibody and overlapping peptides of HIV envelope proteins (derived from the MN tissue culture line adapted HIV isolate) were used to map the specific binding domain of this HuMAb. Overlapping peptides designated 2026 (SWSNKSLDDIWNN, AA614-626), and 2027 (DDIWNNMTWMQWEREIDNYT, AA621-640) within the HIV-1 gp41 transmembrane glycoprotein were demonstrated to bind to C37 indicating that the specific binding domain for the antibody was DDIWNN. High affinity binding of C37 by ELISA to recombinant gp41 was demonstrated as well. Few IgM HuMAbs against HIV have been generated and characterized. Theoretically, because of the pentameric binding nature of IgM antibodies as well as their very efficient ability to activate complement, such reagents could have potential as anti-HIV agents.     

Mutations of Gln64 in the HIV-1 gp41 N-terminal heptad repeat render viruses resistant to peptide HIV fusion inhibitors targeting the gp41 pocket

To prove that the peptidic HIV-1 fusion inhibitors containing the pocket-binding domain (PBD) mainly target the hydrophobic pocket in the gp41 N-terminal heptad repeat (NHR), we constructed pseudoviruses by replacement of Q64 in the gp41 pocket region with Ala (Q64A) or Leu (Q64L). These viruses were highly resistant to C34 and CP32M containing the PBD, while they were susceptible to T20 (enfuvirtide) lacking the PBD but containing the GIV-motif-binding domain (GBD) and lipid-binding domain (LBD). They were also sensitive to C52L, which contains the PBD, GBD, and LBD. Those mutations may disrupt the hydrophilic interaction between Q64 in the NHR and N113 in the peptides containing the PBD. This report provides insights into the mechanisms of drug resistance, with implications for the design of novel HIV fusion and entry inhibitors.     

Retrocyclin RC-101 overcomes cationic mutations on the heptad repeat 2 region of HIV-1 gp41

Retrocyclin RC-101, a theta-defensin with lectin-like properties, potently inhibits infection by many HIV-1 subtypes by binding to the heptad repeat 2 (HR2) region of glycoprotein 41 (gp41) and preventing six-helix bundle formation. In the present study, we used in silico computational exploration to identify residues of HR2 that interacted with RC-101, and then analyzed the HIV-1 sequence database at Los Alamos National Laboratory (New Mexico, USA) for residue variations in the heptad repeat 1 (HR1) and HR2 segments that could plausibly impart in vivo resistance. Docking RC-101 to gp41 peptides in silico confirmed its strong preference for HR2 over HR1, and implicated residues crucial for its ability to bind HR2. We mutagenized these residues in pseudotyped HIV-1 JR.FL reporter viruses, and subjected them to single-round replication assays in the presence of 1.25-10 microg x mL(-1) RC-101. Apart from one mutant that was partially resistant to RC-101, the other pseudotyped viruses with single-site cationic mutations in HR2 manifested absent or impaired infectivity or retained wild-type susceptibility to RC-101. Overall, these data suggest that most mutations capable of rendering HIV-1 resistant to RC-101 will also exert deleterious effects on the ability of HIV-1 to initiate infections - an interesting and novel property for a potential topical microbicide.     

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.     

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: -15.28 kcal/mol for HK20 Fab (expt. -11.60 kcal/mol) and -17.90 kcal/mol for D5 Fab (expt. -11.70 kcal/mol). Alanine scanning at protein-protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of V(H) region, as well as R30' of V(L) region; whereas for D5 Fab, F54 of V(H) region, as well as W32' and Y94' of V(L) region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing.     

Selection with a peptide fusion inhibitor corresponding to the first heptad repeat of HIV-1 gp41 identifies two genetic pathways conferring cross-resistance to peptide fusion inhibitors corresponding to the first and second heptad repeats (HR1 and HR2) of gp41

We generated four HIV-1 cultures that are resistant to a peptide fusion inhibitor corresponding to the first heptad repeat of gp41 in order to study mechanisms of resistance and gain insights into envelope glycoprotein-mediated membrane fusion. Two genetic pathways emerged that were defined by acquisition of a specific mutation in either the first or second heptad repeat region of gp41 (HR1 or the HR2, respectively). Each pathway was enriched in mutations that clustered in either HR2 and V3 or in HR1 and residues in or near CD4 contact sites. The gp41 mutations in both pathways not only accounted for resistance to the selecting HR1 peptide but also conferred cross-resistance to HR2 peptide fusion inhibitors and enhanced the stability of the six-helix bundle formed by the self-assembly of HR1 and HR2. The gp120 mutations alone enhanced fusion but did not appear to directly contribute to resistance. The implications of these findings for resistance mechanisms and regulation of envelope-mediated fusion are discussed.     

Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding.

Interaction with the CD4 receptor enhances the exposure on the human immunodeficiency type 1 gp120 exterior envelope glycoprotein of conserved, conformation-dependent epitopes recognized by the 17b and 48d neutralizing monoclonal antibodies. The 17b and 48d antibodies compete with anti-CD4 binding antibodies such as 15e or 21h, which recognize discontinuous gp120 sequences near the CD4 binding region. To characterize the 17b and 48d epitopes, a panel of human immunodeficiency virus type 1 gp120 mutants was tested for recognition by these antibodies in the absence or presence of soluble CD4. Single amino acid changes in five discontinuous, conserved, and generally hydrophobic regions of the gp120 glycoprotein resulted in decreased recognition and neutralization by the 17b and 48d antibodies. Some of these regions overlap those previously shown to be important for binding of the 15e and 21h antibodies or for CD4 binding. These results suggest that discontinuous, conserved epitopes proximal to the binding sites for both CD4 and anti-CD4 binding antibodies become better exposed upon CD4 binding and can serve as targets for neutralizing antibodies.     

Conserved salt bridge between the N- and C-terminal heptad repeat regions of the human immunodeficiency virus type 1 gp41 core structure is critical for virus entry and inhibition

The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N- and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp⁶³² located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys⁵⁷⁴ in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys⁵⁷⁴ plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp⁶³² has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp⁶³² substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys⁵⁷⁴ mutations, nonconserved substitutions of Asp⁶³² also severely impaired the α-helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp⁶³² significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N- and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.     

Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable plate-like crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.     

Peptides corresponding to the heptad repeat motifs in the transmembrane protein (gp41) of human immunodeficiency virus type 1 elicit antibodies to receptor-activated conformations of the envelope glycoprotein

Two heptad repeat regions in the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane subunit (gp41) self-assemble into a six-helix bundle structure that is critical for virus entry. Immunizations with peptides corresponding to these regions generated antibodies specific to the receptor-activated conformations of gp41.     

Nonneutralizing HIV-1 gp41 envelope cluster II human monoclonal antibodies show polyreactivity for binding to phospholipids and protein autoantigens

HIV-1 gp41 envelope antibodies, which are frequently induced in HIV-1-infected individuals, are predominantly nonneutralizing. The rare and difficult-to-induce neutralizing antibodies (2F5 and 4E10) that target gp41 membrane-proximal epitopes (MPER) are polyspecific and require lipid binding for HIV-1 neutralization. These results raise the questions of how prevalent polyreactivity is among gp41 antibodies and how the binding properties of gp41-nonneutralizing antibodies differ from those of antibodies that are broadly neutralizing. In this study, we have characterized a panel of human gp41 antibodies with binding specificities within the immunodominant cluster I (gp41 amino acids [aa] 579 to 613) or cluster II (gp41 aa 644 to 667) for reactivity to autoantigens, to the gp140 protein, and with MPER peptide-lipid conjugates. We report that while none of the gp41 cluster I antibodies studied were polyspecific, all three gp41 cluster II antibodies bound either to lipids or autoantigens, thus showing the propensity of cluster II antibodies to manifest polyreactivity. All cluster II gp41 monoclonal antibodies (MAbs), including those that were lipid reactive, failed to bind to gp41 MPER peptide-lipid complexes. Cluster II antibodies bound strongly with nanomolar binding affinity (dissociation constant [K(d)]) to oligomeric gp140 proteins, and thus, they recognize conformational epitopes on gp41 that are distinct from those of neutralizing gp41 antibodies. These results demonstrate that lipid-reactive gp41 cluster II antibodies are nonneutralizing due to their inability to bind to the relevant neutralizing epitopes on gp41.     

Characterization of mutants of human immunodeficiency virus type 1 that have escaped neutralization by a monoclonal antibody to the gp120 V2 loop.

The biologically cloned human immunodeficiency virus type 1 (HIV-1) RF isolate is sensitive to neutralization by the murine monoclonal antibody (MAb) G3-4 to a conformationally sensitive epitope in the V2 loop of HIV-1 gp120. To assess how variation in the V2 amino acid sequence affects neutralization by this MAb, we cultured RF in the presence of G3-4 to select neutralization escape mutants. Three such mutants resistant to G3-4 neutralization were generated from three independent experiments. Solubilized gp120 from each of these escape mutants had a reduced affinity for G3-4 and also for two other V2 MAbs that were able to bind the wild-type RF gp120. PCR sequencing of the entire gp120 of the wild-type RF virus and the escape mutants showed that amino acid substitutions had occurred only at two positions, Y177H and L179P, both in V2. Experimental introduction of the Y177H substitution into the RF V2 loop in the context of the NL4-3 molecular clone re-created the G3-4-resistant phenotype. The L179P mutant was not viable. Thus, our findings confirm that the HIV-1 V2 loop contains the conformationally sensitive neutralization epitope recognized by G3-4 and that a single amino acid substitution within this region can result in escape variants that arise from immune selection pressure.     

Studies on the escape mutants of rabies virus which are resistant to neutralization by a highly conserved conformational epitope-specific monoclonal antibody #1-46-12

We investigated a virus-neutralizing conformational epitope of the rabies virus glycoprotein (G) that is recognized by an anti-G monoclonal antibody (mAb; #1-46-12) and shared by most of the laboratory strains of the virus. To investigate the epitope structure, we isolated escape mutants from the HEP-Flury virus (wild-type; wt) after repeated passages in culture in the presence of the mAb. Immunofluorescence studies indicated that the mutants could be classified into two groups; the Group I lacked the epitope, while Group II preserved the epitope. The latter was dominant under the passage conditions, since Group I disappeared during the continuous passages. G proteins showed different electrophoretic mobilities; G protein of Group I migrated at the same rate as wt G protein, while that of Group II migrated at a slower rate, which was shown to be due to acquisition of an additional oligosaccharide side chain. Nucleotide sequencing of the G gene strongly suggested that amino acid substitutions at Thr-36 by Pro and Ser-39 by Thr of the G protein are responsible for the escape mutations of Groups I and II, respectively. The latter is a unique mutation of the rabies virus that allows the G protein to be glycosylated additionally at Asn-37, a potential glycosylation site that is not glycosylated in the parent virus, in preserving the epitope-positive conformation. These results suggest that to keep the 1-46-12 epitope structure is of greater survival advantage for the virus to escape the neutralization than to destroy it, which could be achieved by acquiring an additional oligosaccharide chain at Asn-37.