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Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.
The development of vaccine immunogens that can elicit high-titer, potent, and broadly cross-reactive human immunodeficiency virus type 1-neutralizing antibodies (HIV-1 NAbs) remains a major challenge. Such antibodies are rare in HIV-infected individuals, and despite extensive research efforts, only a limited number of envelope (Env)-specific broadly cross-reactive NAbs (51), including antibodies against functionally important receptor and coreceptor binding sites in the gp120 subunit (8, 33, 39, 50), and antibodies against the ectodomain of gp41 subunit (34, 46, 52), have been identified. In general, it appears that antibodies against gp120 are more potent than, but not as broadly neutralizing as, antibodies against the fusion subunit gp41, which is more conserved than gp120 (4, 13).
The three gp41-specific cross-reactive NAbs, 2F5, 4E10, and Z13 bind peptides derived from the gp41 membrane-proximal external region (MPER). Immunogens based on these peptides, however, have failed to elicit NAbs against primary isolates. 2F5, Z13, and 4E10 appear to be polyspecific autoantibodies reactive with the phospholipid cardiolipin (CL) (1, 2, 7, 20, 35, 40, 41), indicating that the MPER could mimic human self-antigens, another possible mechanism for HIV immune evasion in addition to the multiple mechanisms described previously (47). Attempts to identify antibodies by immunizing mice or panning nonimmune antibody libraries against Env fusion intermediate structures, including the six-helix bundle (6HB), five-helix bundle (5HB), and N trimer, have been made, but they have resulted in nonneutralizing antibodies or antibodies with neutralizing activity significantly lower than that of 2F5 or 4E10 (19, 22, 28, 31).
Recently, we have identified two cross-reactive, HIV-1-neutralizing gp41-specific human monoclonal antibodies (hMAbs), m48 (48) and m46 (10), by competitive antigen panning (CAP) of an HIV-1-immune library derived from the bone marrows of three long-term nonprogressors whose sera had high titers of cross-reactive NAbs. m46 exhibited potency in peripheral blood mononuclear cell (PBMC)-based assays that was significantly higher than that in cell line-based assays, and its activity was greatly increased in cells with a decreased level of coreceptor (CCR5) (10).
Identification of novel broadly cross-reactive NAbs and characterization of their conserved epitopes may have implications for development of vaccines and therapeutics and for an understanding of the mechanisms of HIV entry and evasion of immune responses. Here, we describe the identification and characterization of a novel gp41-specific cross-reactive hMAb, m44, which was selected from an HIV-1-immune library (see above) by using uncleaved Env ectodomains, gp140s, which contain both gp120s and truncated gp41s lacking transmembrane domains and cytoplasmic tails, as antigens for panning and screening. In PBMC-based assays, this antibody in both formats, Fab and immunoglobulin G (IgG), neutralized HIV-1 primary isolates from different clades with potency significantly higher than that of 4E10 or Fab Z13. IgG1 m44 also neutralized a clade C simian/human immunodeficiency virus (SHIV) isolate, SHIV-1157ipd3N4, more potently than 2F5 and b12. Importantly, m44 did not bind to human self-antigens. Its epitope is conformational and conserved, which may help in the design of vaccine immunogens capable of eliciting this antibody or similar antibodies in vivo.
293T cells were purchased from ATCC. Free-style 293 cells were purchased from Invitrogen. TZM-bl cells and HIV-1 isolates were obtained from the NIH AIDS Research and Reference Reagent Program. Recombinant gp140s from primary isolates were produced as described previously (49); gp140/gp12089.6, gp140/gp120CM243, and gp140/gp120R2 were produced by recombinant vaccinia virus (89.6 virus was a gift from R. Doms, University of Pennsylvania, Philadelphia, PA) with a combination of lentil lectin affinity chromatography and size exclusion chromatography. Wild-type gp41-Fc and mutants generated by alanine-scanning mutagenesis were produced as follows. gp41 derived from 89.6 was cloned in pEAK10 plasmid upstream of human Fc. Mutants created by alanine-scanning mutagenesis were generated using QuikChange II site-directed mutagenesis kits (Stratagene). All mutations were confirmed by DNA sequencing. The recombinant plasmid DNA of wild-type gp41-Fc and mutants was introduced into Escherichia coli by using the Qiagen plasmid Giga kit and used for transient transfection of freestyle 293 cells using 293fectin as the transfection reagent (Invitrogen). The supernatants were harvested three days posttransfection, and gp41-Fc fusion proteins were purified from the supernatants by using protein A affinity purification. The hMAbs Z13, m44, m46, m48, and m14, mouse MAbs D61 (cluster I), D17, D40, and D50 (cluster II), T3 (cluster IV), T30 (cluster VI), and D47 (anti-V3 loop), and 6HB-specific mouse antibody NC-1 were produced in our laboratories. The hMAbs 2F5 and 4E10 were provided by Hermann Katinger. The plasmid encoding Z13 was provided by M. Zwick and D. Burton (The Scripps Research Institute). gp41-derived peptides, anti-p24 MAb (183-12H-5C), and HIV Ig were obtained from the NIH AIDS Research and Reference Reagent Program. PBMCs were isolated from the blood of healthy donors by standard density gradient centrifugation using Histopaque-1077 (Sigma). The following antibodies were purchased: horseradish peroxidase (HRP)-conjugated monoclonal mouse anti-M13 antibody (Pharmacia, Uppsala, Sweden), HRP-conjugated polyclonal anti-human IgG F(ab′)2 antibodies (Jackson ImmunoResearch, West Grove, PA), and HRP-conjugated streptavidin (Zymed Laboratories Inc., San Francisco, CA).
The phage library was constructed using pComb3H phagemid vector and 30 ml of bone marrow obtained from three long-term nonprogressors (A, H, and K) (15) whose sera exhibited the broadest (against six primary isolates) (32) and most potent (against JR-FL at 1:40 dilution) HIV-1 neutralization among 37 HIV-infected individuals (provided by T. Evans, currently at Novartis, Boston, MA) (49). CAP facilitates identification of high-affinity hMAbs targeting gp41 in the context of whole Env protein and was performed as described previously (48). Briefly, the phage library (5 × 1012 CFU/ml) was preadsorbed on streptavidin-M280-Dynabeads in phosphate-buffered saline (PBS) for 1 h at room temperature and incubated with 50 nM biotinylated HIV-1 Env glycoprotein gp140CM243 and 250 nM nonbiotinylated gp120CM243 (fivefold more on a molar level than biotinylated gp140CM243) for 2 h at room temperature with gentle agitation. The panning against tethered Envs gp14089.6 and gp140R2 was done in parallel in the same manner in which panning against gp140CM243 was performed. Phage binding to biotinylated Env was separated from the phage library by using streptavidin-M280-Dynabeads and a magnetic separator (Dynal). After being washed 20 times with 1 ml of PBS containing 0.1% Tween 20 and another 20 times with 1 ml of PBS, bound phage was eluted from the beads by using 100 mM triethanolamine followed by neutralization with 1 M Tris-HCl, pH 7.5. For the second and third rounds of panning, 10 nM (second round) or 2 nM (third round) of biotinylated gp140CM243, gp14089.6, and gp140R2 and fivefold excesses of nonbiotinylated gp120CM243, gp12089.6, and gp120R2 were used as antigens. After the third round of panning, 96 individual clones from each panned library were screened for binding to gp140CM243, gp14089.6, gp140R2, gp120CM243, gp12089.6, and gp120R2 by phage enzyme-linked immunosorbent assay (ELISA).
Competition ELISAs of free gp12089.6 with immobilized gp14089.6 for binding to anti-gp41 antibodies were performed by using 1 μg/ml gp14089.6 to coat 96-well Maxisorp plates, PBS-Tween 20 to wash the plates, and 3% bovine serum albumin (BSA) in PBS to block the wells, followed by the addition of 10-fold serially diluted free gp12089.6 and soluble anti-gp41 Fabs at constant concentrations corresponding to 70% maximum binding. Bound Fabs were detected using HRP conjugated to anti-human IgG, F(ab′)2, and ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] substrate. ELISAs of soluble Fabs to recombinant HIV-1 gp140s from different isolates were performed by using gp140s to directly coat 96-well plates, followed by the addition of threefold serially diluted soluble anti-gp41 Fabs. In cases using denatured gp140s, purified gp140s were diluted in 1% sodium dodecyl sulfate-50 mM dithiothreitol to 10 μg/ml, boiled for 5 min, and then diluted 1:10 in PBS and coated on 96-well plates. Binding of Fab m44 and IgG1 2F5 to mutants generated by alanine-scanning mutagenesis of the gp41-Fc fusion protein was measured by diluting the wild type and each mutant fusion protein to 1 μg/ml and using 50 μl of each diluted fusion protein to coat 96-well plates, followed by the addition of threefold serially diluted anti-gp41 antibodies. The bound antibodies were detected using HRP conjugated to anti-human IgG, F(ab′)2, and ABTS substrate. The binding of anti-gp41 antibodies to gp41 peptides was tested as described previously (52). A lipid binding assay was done by using 10 μg/well of phosphatidylserine (PS) or CL to coat microwell plates, followed by the addition of twofold serially diluted anti-gp41 antibodies. Bound antibodies were detected by using alkaline phosphatase-conjugated goat anti-human IgG (Fc-specific) antibody (Sigma) and nitrophenyl phosphate (Sigma) as the substrate. Optical densities at 405 nm were measured after color development at room temperature for 45 min. Western blotting with gp41 intermediate structures was done as previously described (27-29).
Three neutralization assays based on spreading infection of PBMCs were carried out in three independent research laboratories. The PBMC-based p24 assay was carried out in S. Jiang's laboratory by using the protocol previously described (see Table Table2)2) (23). PBMCs isolated from the blood of healthy donors were plated in 75-cm2 plastic flasks and incubated at 37°C for 2 h. The nonadherent cells were collected and resuspended (5 × 106) in 10 ml of RPMI 1640 medium containing 10% fetal bovine serum, 5 μg/ml of phytohemagglutinin (PHA), and 100 U/ml of interleukin-2 (IL-2), followed by incubation at 37°C for 3 days. The PHA-stimulated cells were infected with the corresponding primary HIV-1 isolates at a multiplicity of infection of 0.01 in the absence or presence of an antibody at graded concentrations. Culture media were changed every 3 days. The supernatants were collected seven days postinfection and tested for p24 antigen by ELISA. The percent inhibition of p24 production and the concentrations for 50% inhibition (IC50) and 90% inhibition (IC90) were calculated by using the software Calcusyn. The PBMC-based reverse transcriptase (RT) assay was carried out by R. Buckheit's group at ImQuest Biosciences (initially at Therimmune) (see Table Table3).3). In this assay, 100 μl of antibodies diluted in complete RPMI with IL-2 was incubated with 50 μl of virus containing 100 times the 50% tissue culture infective dose for 30 min at 37°C and added to 50 μl of PHA-activated PBMC (1 × 106) in complete RPMI 1640 with IL-2. The levels of calculated neutralization activity refer to the antibody concentrations present during this incubation step. Triplicate samples were taken on day 7 for the RT assay, and percent neutralization was calculated by dividing the RT activity of the tested samples by the RT activity of mock culture without antibody. Zidovudine was used as a positive control with 100% inhibition at a concentration equal to 0.1 μM, at which the measured cell toxicity was undetectable (IC50 = 0.002 μM; 50% tissue culture infective dose > 1 μM). The PBMC-based p27 assay was performed in the R. Ruprecht laboratory with the clade C SHIV chimera SHIV-1157ipd3N4 (see Fig. Fig.1)1) as previously described (45). Briefly, antibodies were serially diluted in triplicate in 96-well plates, starting at a concentration of 80 μg/ml (2×) in 50 μl; an equal volume of diluted virus (7 ng of p27) was added and incubated for 1 h at 37°C with 5% CO2. PHA-activated human PBMCs (5 μg/ml for 3 days) grown in RPMI supplemented with 15% fetal calf serum and IL-2 were added (5 × 105 cells/well in 50 μl; the virus-only control received medium only). The plates were incubated for 2 h at 37°C in 5% CO2, followed by the addition of 100 μl medium supplemented with 20 U/ml IL-2. From day 3 onward, 100 μl supernatant was harvested daily and replaced with fresh medium. Virus growth kinetics as determined by measuring p27 levels with p27 ELISA kits (Perkin Elmer, Boston, MA) were used as a guideline for the selection of the time point to assess virus neutralization (8 to 15 ng/ml of p27 in virus-plus-cell wells). The percent neutralization was calculated using the following equation: [1 − (E − N)/(P − N)] × 100, where E is the measurement obtained from the experimental group (antibody plus virus plus cells), P is that obtained from the positive control (virus plus cells), and N is that obtained from the negative control (virus only).
The TZM-bl cell line-based assay whose results are shown in Table Table44 was carried out in triplicate in the laboratory of D. C. Montefiori, following the protocol described in reference 44, and the TZM-bl cell line-based assay whose results are shown in Table Table55 was carried out in duplicate in the laboratory of J. R. Mascola, following the protocol described in reference 24. Two viruses used in this assay, RW20.2 and ZA12.29, were described previously (25). Both assays were performed by using an HIV-1 Env-pseudotyping system and TZM-bl target cells containing a tat-inducible luciferase reporter and expressing CD4, CCR5, and CXCR4. To measure the effect of target cells on antibody neutralization activity, we also used the same pseudoviruses to infect PBMCs (see Table Table5).5). Note that in this case, in contrast to the three assays based on PBMCs described above, the initial infection does not lead to subsequent rounds of infection. Thus, this assay was used for the purpose of comparison with the cell line-based assay, with most parameters identical except the target cells. In this assay, the pseudoviruses were prepared by cotransfecting 293T cells with an Env expression plasmid containing a full gp160 Env gene and an env-deficient HIV-1 backbone vector (pNL4-3.luc.E-R-) containing a luciferase reporter gene (26). Viral stocks were harvested and used to infect PHA- and IL-2-stimulated PBMCs that had been depleted of CD8+ T cells. The rest of the protocol is the same as that described previously for the TZM-bl cell line-based assay (24).
To identify antibodies specific for gp41 in the context of Env, we used the CAP methodology (48) and recombinant gp140s from three different isolates, CM243 from clade E and 89.6 and R2 from clade B (denoted gp140CM243, gp14089.6, and gp140R2, respectively), as antigens in three independent panning procedures. One of the antibody clones, designated m44, was enriched in libraries obtained after panning against gp140CM243, gp14089.6, and gp140R2, indicating that its epitope is conserved among these three isolates. The antibody was expressed as a fragment (Fab) for characterization of its binding. Fab m44 did not bind gp120, as measured by a competition ELISA using free gp12089.6 (Fig. (Fig.1),1), but bound with high affinity to recombinant gp140s from primary isolates of different clades except the clade A isolate, UG037.8 (Table (Table1)1) . These results suggest that m44 is a highly cross-reactive gp41-specific antibody.
To investigate the neutralizing activity of the newly selected antibody, we converted Fab m44 to IgG1 m44 and tested both Fab m44 and IgG1 m44 with primary HIV-1 isolates in neutralization assays based on infection of PBMCs and measurement of p24 (PBMC-based p24 assay), RT (PBMC-based RT assay), or p27 (PBMC-based p27 assay) produced by infected cells. Fab m44 was tested with a panel of primary isolates from different clades in a PBMC-based p24 assay in comparison with Fab Z13 (Table (Table2).2). Fab m44 neutralized isolates from clades B, C, D, E, and F. Z13 neutralized only the clade B isolate 92US657. Two clade A isolates, one clade G isolate, and one group O isolate were not neutralized by Fab m44 (Table (Table2).2). IgG1 m44 was also tested at one concentration with another panel of primary isolates in the PBMC-based RT assay side by side with IgG1 4E10 and Fab Z13 (Table (Table3).3). IgG1 m44 neutralized all four clade C isolates tested, as well as isolates from clades B, E, F, and G, as well as one isolate from group O. The median percent neutralization by IgG1 m44 was higher than that by IgG1 4E10 or Fab Z13.
We further tested IgG1 m44 and Fab m44 with a clade C SHIV strain, SHIV-1157ipd3N4, in a PBMC-based p27 assay (45). Both Fab m44 and IgG1 m44 neutralized this virus, but IgG1 m44 (IC50 < 0.1 μg/ml) was more potent than Fab m44 (IC50 = 1.2 μg/ml) (Fig. (Fig.2).2). IgG1 m44 was also more potent than IgG1 2F5 and IgG1 b12 (IC50s of 0.68 and 0.85 μg/ml, respectively). The potency of IgG1 4E10 (IC50 = 0.17 μg/ml) was comparable to that of IgG1 m44 in neutralizing this clade C SHIV strain. Fab m44 and IgG1s 2F5, 4E10, b12, and 2G12 were further tested against the same SHIV strain in a cell line (TZM-bl)-based assay. Interestingly, none of the antibodies neutralized this virus at the highest concentration tested (40 μg/ml) (the percentages of antibody neutralization at the highest antibody concentration were 20, 21, 23, 13, and 22% for Fab m44, IgG1 2F5, IgG1 4E10, IgG1 b12, and IgG1 2G12, respectively). IgG1 m44 was not tested against this SHIV strain in the cell line-based assay. However, it was tested in a TZM-bl cell line-based assay against a panel of primary isolates from clades A, B, and C. IgG1 m44 exhibited weak neutralizing activity at concentrations up to 50 μg/ml in this assay against the panel of primary isolates (Table (Table4).4). It neutralized more than 50% of the activity of only one of the clade B isolates (SF162.LS) tested. To further compare the potency of IgG1 m44 in PBMC-based assays to its potency in cell line-based assays, it was tested against a panel of pseudoviruses by using protocols that were identical except for their different target cells (Table (Table5).5). IgG1 m44 was significantly weaker in the TZM-bl cell line-based assay than in the PBMC-based assay up to the highest concentration (50 μg/ml) used. IgG1 m44 neutralized five out of six isolates tested in the PBMC-based assay, while it did not neutralize any of the four isolates tested in the TZM-bl cell line-based assay. Of note is its high level of activity against the clade C isolate DU123 in the PBMC-based assay (IC50 = 0.3 μg/ml). Taken together, these results show that m44 can neutralize primary isolates from different clades in assays based on spreading infection in PBMCs with potency on average comparable to or higher than that of Z13, 4E10, and 2F5 for the panels of isolates tested here but is significantly less potent in TZM-bl cell line-based assays.
The epitopes of the three known broadly neutralizing gp41-specific antibodies, 2F5, 4E10, and Z13, include the MPER of gp41. They bind to linear peptide sequences located in the MPER. To find out whether m44 binds to the same region or other linear epitopes on gp41, we measured in the ELISA its binding to denatured recombinant gp14089.6 and 34 peptides derived from gp41. Fab m44 bound to gp140 (Fig. (Fig.3A)3A) but did not bind to dithiothreitol-treated gp140 (Fig. (Fig.3B)3B) or to gp41-derived peptides to any significant degree (data not shown), suggesting that m44 recognizes a conformational epitope and that disulfide bonds are important for the structural integrity of its epitope. The reducing condition did not affect 2F5 binding but slightly affected Z13 binding to gp140 (Fig. 3A and B). IgG1 m44 was further tested in a Biacore assay with an MPER peptide containing the 2F5 and 4E10 epitopes (QQEKNEQELLELDKWASLWNSLWNWFNITNWLWYIK) (Fig. (Fig.3C).3C). Unlike 2F5 and 4E10, m44 did not bind to this long peptide even at high concentrations (100 μg/ml). In an attempt to further localize the m44 epitope, we measured the antibody binding to fusion intermediate structures, including N36/C34-formed 6HB, single-chain protein of 5HB, and the N-trimer-containing polypeptides NCCG-gp41 (27), N35CCG-N13 (29), and N34CCG (29) by using Western blotting (28) (Fig. (Fig.3D).3D). Fab m44 bound to the 5HB (lane 5) and the 6HB (lane 4). Fab m44 did not bind to the N trimer for lack of reactivity with N35CCG-N13 or N34CCG (lane 2 and 3), suggesting that the C-heptad repeat (C-HR) of gp41 is involved in m44 binding. Fab m44 bound weakly to the NCCG-gp41 (lane 1) that comprises 6HB and N-helix. To test whether m44 can also bind membrane-associated Env, we used flow cytometry and cells expressing uncleaved gp160 from HIVHTLV-IIIB. IgG1 m44 bound to the Env at about the same level as IgG1 b12 (Fig. (Fig.3E),3E), suggesting that the m44 epitope is exposed in membrane-associated Env.
The binding of Fab m44 to the 5HB was confirmed by ELISA (Fig. (Fig.4).4). Unlike the control mouse MAb NC-1 raised against the 6HB and another mouse MAb T3 that bound strongly to both 5HB and 6HB, the binding of m44 to the 5HB was stronger than that to the 6HB, suggesting that the m44 epitope in the C-HR is exposed in the 5HB but partially buried in the 6HB. Z13 and 2F5 were used as controls in this binding assay. Z13 and 2F5 did not bind to 5HB, but for some reason, Z13 bound to 6HB at the same affinity as that for m44 (Fig. (Fig.4).4). In contrast to the nonneutralizing mouse MAb NC-1, m44 bound strongly to recombinant gp140s (Fig. (Fig.3A;3A; Table Table1).1). We noticed that the m44 binding to gp140 (the concentration at which antibody has half-maximum binding [EC50] was 2.3 nM for gp14089.6) was stronger than that to 5HB (EC50 = 11 nM), indicating that other structures of gp140 could also be involved in the m44 binding. To identify such a region(s) that could contribute to the m44 binding, we performed competition ELISAs for m44 with known anti-gp41 antibodies recognizing different regions of gp41 and a control antibody (m14) against gp120 (Table (Table6).6). The results showed that m44 did not compete with any of the antibodies tested except a cluster IV mouse MAb, T3 (16). T3 has characteristics similar to those of NC-1. Both T3 and NC-1 bind strongly to 5HB and 6HB (Fig. (Fig.44).
In an attempt to further localize the m44 epitope, we constructed a fusion protein of gp41 (derived from 89.6) with human Fc (gp41-Fc) and performed alanine-scanning mutagenesis in the C-HR and the adjacent region upstream of C-HR. The antigenic structure of gp41-Fc protein was intact, as demonstrated by its ability to bind to a panel of known anti-gp41 antibodies, including 2F5, 4E10, and Z13 recognizing linear epitopes in the MPER, and the conformation-dependent human HIV-1 NAbs m44, m46, and m48 (data not shown). The site-directed alanine-scanning mutagenesis targeted mostly the loop region and the N-terminal portion of the C-HR in gp41-Fc. Thirty-three mutants were generated by alanine-scanning mutagenesis and expressed as soluble proteins (Table (Table7).7). The EC50s of Fab m44 to these mutants were measured by ELISA, and the relative affinities to wild-type gp41-Fc fusion protein were calculated (Table (Table7).7). 2F5 was used as a control. Mutations causing >10-fold changes in m44 binding (considered significant changes) are indicated in Table Table7.7. In accordance with the ELISA data described above, it appears from these alanine-scanning mutagenesis data that the C-HR and a stretch of residues at the C-terminal portion of the loop could be involved in the m44 binding.
Taken together, these results suggest that m44 binds to a conformational epitope formed partly by the C-HR and portion of the loop upstream from it. There are probably other regions involved in the m44 binding. Cocrystallization of this antibody with gp140 may provide more detailed information about the epitope of this new anti-gp41 antibody.
2F5 and 4E10 have been shown to be reactive with phospholipids, including CL (1, 20, 40, 41). 4E10 binds to CL and PS, as well as to the Rose antigen (Ro; or Sjogren's syndrome antigen A [SSA]) and the histidyl-tRNA synthase (Jo1) (20). 2F5 binds weakly to CL and to three other self-antigens, Ro (SSA), centromere B, and histones, as measured by an AtheNA antinuclear autoantibody assay (ANA) (20). We compared side by side in the same experiment binding of m44, 2F5, and 4E10 in lipid binding assays. m44 did not bind to CL (Fig. (Fig.5),5), whereas in agreement with previous results, 2F5 and 4E10 bound to CL with 340 nM and 23 nM apparent affinities, respectively. m44 also did not react with any of the other self-antigens tested in the Luminex AtheNA ANA (Table (Table8),8), indicating that m44 is a not a polyspecific antibody.
The major finding of this study is the identification of a gp41-specific cross-reactive HIV-1-neutralizing hMAb, which binds to a novel conformational epitope on gp41 and does not exhibit reactivity to self-antigens. A number of anti-gp41 hMAbs have been identified and characterized, but only several of them are neutralizing (51). Of those, 2F5, 4E10, and Z13 bind peptides and denatured gp140s (Fig. 3A and B) (52), and another anti-gp41 HIV-1 NAb, CL3, recognizes a linear epitope that includes the disulfide bond in the immunodominant loop of gp41 (GCSGKLICTT) (12). It appears that immunogens based on these linear epitopes do not lead to elicitation of antibodies neutralizing primary isolates. For example, the use of ELDKWA inserted into a carrier protein did not induce HIV-1 NAbs, possibly due to the lack of an appropriate environment (absence of the lipid of the viral membrane) to support the structure of this peptide in the context of gp41 (11, 36). In contrast to these previously characterized neutralizing anti-gp41 hMAbs, the newly identified antibody m44 does not bind peptides and Envs without conformational integrity, nor does it bind to lipids or the other autoantigens tested. Its epitope is conformational and conserved. The m44 VH is derived from the M29811 IGHV4-61*01 germ line, and the VL is derived from the X12686 IGKV3-20*01 germ line. m44 has a relatively short heavy-chain CDR3 (HCDR3) (m44 HCDR3, ARGTRGGSTLDS) compared to 2F5 and 4E10, which have long HCDR3s (2F5 HCDR3, AHRRGPTTLFGVPIARGPVNAMDV; 4E10 HCDR3, AREGTTGWGWLGKPIGAFAH). Arginines in HCDR3s were reported to be associated with antibody polyreactivity (18). 2F5, 4E10, and m44 have three arginines, one arginine, and two arginines, respectively, in the HCDR3s. While several groups have found the lipid reactivities of 4E10 (1, 2, 7, 20, 35, 43) and 2F5 (1, 20, 40, 41), one group has not found 2F5 reactivity with lipids, although they found 2F5 reactivity with glyceraldehyde phosphate dehydrogenase (43). This difference is likely due to the weak apparent affinity of 2F5 for lipids and to assay differences. In both ELISAs and surface plasmon resonance assays, m44 reactivities in direct comparison to 2F5 and 4E10 reactivities were negative. The structural basis of lack of polyreactivity of m44 remains to be determined, and as well, it remains to be determined if m44-like antibodies will be difficult to elicit. Meffre et al. have suggested that most hMAbs with long CDR3s are depleted in the bone marrow (30). Certainly, the lack of binding to self-antigens raises the hope that elicitation of m44 or m44-like antibodies may not be regulated by tolerance mechanisms in humans.
m44 does not compete significantly with 2F5 or 4E10 or with many of the mouse antibodies previously developed to map epitopes on gp41 (Table (Table4)4) (5, 6) but competes strongly with the cluster IV conformation-dependent mouse MAb T3 (16), probably due to steric interference. In our study, we found that T3 bound to 5HB and 6HB (Fig. (Fig.4)4) in a way that is very similar to the binding of NC-1. However, the m44 epitope is distinct from the epitopes of NC-1 and T3, based on their different capacities for binding to 5HB, N36/C34-formed 6HB (Fig. (Fig.4),4), and gp140 (Fig. (Fig.3A).3A). To localize the m44 epitope on gp41, we constructed a gp41-Fc fusion protein and performed alanine-scanning mutagenesis. We characterized the wild-type gp41-Fc for antigenicity and immunogenicity prior to generation of a panel of mutants by alanine-scanning mutagenesis. Our preliminary data based on immunization of rabbits with purified gp41-Fc showed that the gp41-Fc is highly immunogenic. The titers of immune serum reached 102,400 for the gp41-Fc fusion protein and 5,120 for the recombinant gp14089.6. In addition, purified rabbit IgG from one of the four immunized rabbits showed broad neutralization activity against several HIV isolates from different clades in a pseudovirus assay (data not shown), suggesting that gp41-Fc retains its antigenicity and immunogenicity. We emphasize that the alanine-scanning mutagenesis data presented in this work suggest but do not definitely prove that the m44 epitope is located in the C-HR and the upstream adjacent region. However, the agreement of the mutagenesis data with the data obtained by measuring binding to 5HB and 6HB (Fig. (Fig.4)4) increases our confidence that the m44 epitope involves portions of the C-HR and adjacent regions. Only the crystal structure of m44 in complex with gp41 will definitely and precisely define its epitope.
Alanine-scanning mutagenesis focused on six residues in the loop region (M61, G62, L70, W78, V80, and W82) also caused significant decrease in the 2F5 binding. It was previously reported that alanine-scanning mutagenesis focused on residues M61, L70, W78, and W82 resulted in abolishment or significant reduction of viral entry (21). Thus, mutations at these positions could change the overall structure of gp41, and it is difficult to evaluate their contribution to the antibody epitope. Mutations at G62 and V80 may also affect the gp41 structure, but this fact does not exclude the possibility that these residues are involved in m44 and 2F5 binding. In the C-HR region, five alanine mutations (M94, W96, M97, R101, and I103) also affected 2F5 binding. These residues could be involved in stabilizing the gp41 structure, and/or their mutations may affect the exposure of the 2F5 epitope in the MPER.
It has been previously reported that some antibodies exhibit large variations in neutralizing activities, depending on the assay used. For example, 4E10 exhibits much broader neutralizing activity when tested in an assay based on pseudovirus entry into a cell line than when tested in a PBMC-based assay (4). We have also found that in some cases, 4E10 exhibits different activities when measured by the assays used in this study in comparison to previously reported results (e.g., it did not neutralize the 92UG029 isolate in our PBMC-based assay but did neutralize the same isolate in another PBMC-based assay, although relatively weakly , and two other isolates [BR025 and G3] were better neutralized by 4E10 in another study ). These differences could be related to the donor of PBMCs used or other details of the assays used, including the use of p24 as a marker of replication instead of RT activity, as in our assay. The RT activity is correlated with the number of virions in culture supernatant (14), while p24 can also be released by infected cells as a free non-virion-associated protein. In addition, other details in the protocol, including whether the infection is spreading (e.g., if it includes at least two infection cycles) and whether the antibody is present for the subsequent cycles of infection could significantly affect the neutralizing activity of the antibody (for a recent analysis of possible causes of discrepancies, see reference 38). Our data from three different assays based on spreading HIV-1 infection in PBMCs consistently showed that the neutralizing activity of m44 in such assays is on average comparable to or better than that of 4E10 or 2F5. However, the potency and breadth of neutralization exhibited by m44 in cell line/pseudovirus-based assays are significantly lower than those it exhibited in PBMC-based assays. Experiments with animal models would be required to evaluate the in vivo neutralizing efficacy of this antibody.
We noticed that m44 was weak in neutralizing HIV-1 primary isolates from clade A. In an attempt to find possible cause of this difference between clade A and other clades (B, C, and D), we performed an analysis of all HIV-1 Env sequences available in the HIV Sequence Database (http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html) by calculating the frequency of most common residues in each position (data not shown). We specifically looked at the regions corresponding to the alanine-scanning mutagenesis we performed and noticed two unique differences between the clade A consensus sequence and two clade A primary isolates, 92UG037 and 92UG029, that were not neutralized by m44. One position with different residues is 629, for which the clade A residue is leucine, while the clade B, C, and D residues are methionine. The other one is position 633, for which the residue for clade A is lysine, while the residues for clades B, C, and D are arginine. We have no evidence to directly show that these changes are related to the lack of neutralization activity of m44 against clade A isolates. Further experiments with more clade A isolates are required.
The identification of this new antibody with unique properties could be attributed to the antibody library we used for panning, the antigens, and the CAP methodology we have developed and used here. The antibody library was previously constructed (Tom Evans, personal communication) by using bone marrow obtained from three long-term nonprogressors (A, H, and K) (15) whose sera exhibited the broadest (against six primary isolates) (32) and most potent (against JR-FL at a 1:40 dilution) HIV-1 neutralization among 37 HIV-infected individuals (49), suggesting that m44 may exist in vivo. CAP methodology facilitates the identification of gp41-reactive antibodies in the context of native Env (48). Further studies are needed to elucidate the mechanism of neutralization by the newly identified anti-gp41 antibody m44. One can speculate that it does bind to native Env and sterically prevents virus from binding to receptor-expressing cells, as previously proposed, as a mechanism of virus neutralization by antibodies (9, 37). Another possible mechanism is that this antibody binds structures important for fusion either before or after engagement of CD4 and coreceptors or both (3, 17, 42, 52). This unique antibody could help in the elucidation of the mechanisms of evasion of immune response by HIV and in the development of new candidate vaccine immunogens.
We thank Michael Zwick and Dennis Burton for providing DNA encoding Z13; Peter Kwong, Lai-Xi Wang, and Jilad Ofek for gp41 peptides; David C. Chan for 5HB; and Marius Clore and Carole Bewley for 6HB, NCCG-gp41, N35CCG-N13, and N34CCG. We thank John M. Louis for providing the data shown in Fig. Fig.3D.3D. We also thank Igor Sidorov for data analysis and Robert Blumenthal, Peter Kwong, Gary Nabel, and Hana Golding for helpful discussions.
This research was supported by funding from the NIH Intramural AIDS Targeted Antiviral Program (IATAP) and the Gates Foundation to D.S.D., by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, by funding from the Intramural Research Program of the Vaccine Research Center, NIAID, NIH, by NIH grants AI48280 to C.C.B., AI46221 to S.J., AI47434 to G.Q. and R37 AI34266 to R.M.R., by federal funds from the NIH, National Cancer Institute, under contract no. NO1-CO-12400, and in part by funding from the IATAP to Marius Clore and Carole Bewley.
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Published ahead of print on 14 May 2008.