Neutralizing cross-reactive and non-neutralizing monoclonal antibodies to HIV-1 gp120.

Amino acid sequences inducing neutralizing antibodies to HIV-1 were sought. Murine monoclonal antibodies (MAbs) were characterized by their reactivity with the envelope precursor gp160 or the Escherichia coli recombinant DNA products pB1 and pE3 representing the carboxy- and amino-terminal halves of mature envelope gp120. Fine mapping of the MAb determinants was performed using defined 15-mer synthetic peptides spanning the entire envelope gp120 region of HIV-1. One group of MAbs recognizes epitopes (amino acids 304-323) occurring in a small region with variable and conserved amino acid sequences of gp120. These MAbs mediate neutralization of the HIV-1 strain HTLV-IIIB (HIV-1IIIB) which was used for immunization. Nine out of 11 primary HIV-1 isolates were neutralized well or moderately well. In addition, prominent serological reactivity was noted with peptide sequences of strains of various European or American origins, but not with two HIV-1 strains of African origin. The cross-reactivity contrasts with previously described type-specific reactions to other sequences of this region. The reactivity to the short conserved site GPGR with its flanking amino acids may explain the broad sequence cross-reactivity seen with our neutralizing MAbs. Two other MAbs recognize conserved epitopes (amino acids 79-103) situated in the amino-terminal region of gp120. These MAbs did not neutralize HIV-1IIIB.     

Characterization of recombinant gp120 and gp160 from HIV-1: binding to monoclonal antibodies and soluble CD4

We compared four preparations of recombinant HIV-1 envelope glycoprotein: mammalian (Chinese hamster ovary cells) gp120 (Celltech); baculovirus gp120 from American Biotechnologies Inc. (ABT) and from MicroGeneSys (MGS); and baculovirus gp160 (Institute of Virology, Oxford, UK). Each envelope glycoprotein binds to a neutralizing monoclonal antibody (MAb) directed against the V3 loop, confirming the integrity of this type-specific neutralization epitope. MGS gp120 binds abnormally well to a MAb which recognizes an epitope preferentially exposed on denatured gp120. Consistent with this finding, MGS gp120 binds to soluble CD4 (sCD4) with an affinity 50-100-fold lower than that of Celltech gp120. The affinity of Celltech gp120 from sCD4 is 2.3 nM, indistinguishable from that of gp120 extracted from HIV-1 virions. Baculovirus gp120 (ABT) and gp160 also have a high affinity for sCD4. A significant proportion of anti-gp120 antibodies in HIV-positive human sera recognize epitopes that are dependent on the mammalian glycosylation pattern, and a human HIV-positive serum inhibits the binding of mammalian gp120 to sCD4 five- to 10-fold more potently than it inhibits baculovirus gp120 binding to sCD4.     

Energetics of the HIV gp120-CD4 binding reaction

HIV infection is initiated by the selective interaction between the cellular receptor CD4 and gp120, the external envelope glycoprotein of the virus. We used analytical ultracentrifugation, titration calorimetry, and surface plasmon resonance biosensor analysis to characterize the assembly state, thermodynamics, and kinetics of the CD4-gp120 interaction. The binding thermodynamics were of unexpected magnitude; changes in enthalpy, entropy, and heat capacity greatly exceeded those described for typical protein-protein interactions. These unusual thermodynamic properties were observed with both intact gp120 and a deglycosylated and truncated form of gp120 protein that lacked hypervariable loops V1, V2, and V3 and segments of its N and C termini. Together with previous crystallographic studies, the large changes in heat capacity and entropy reveal that extensive structural rearrangements occur within the core of gp120 upon CD4 binding. CD spectral studies and slow kinetics of binding support this conclusion. These results indicate considerable conformational flexibility within gp120, which may relate to viral mechanisms for triggering infection and disguising conserved receptor-binding sites from the immune system.     

CCR5/CD4/CXCR4 oligomerization prevents HIV-1 gp120IIIB binding to the cell surface

CCR5 and CXCR4, the respective cell surface coreceptors of R5 and X4 HIV-1 strains, both form heterodimers with CD4, the principal HIV-1 receptor. Using several resonance energy transfer techniques, we determined that CD4, CXCR4, and CCR5 formed heterotrimers, and that CCR5 coexpression altered the conformation of both CXCR4/CXCR4 homodimers and CD4/CXCR4 heterodimers. As a result, binding of the HIV-1 envelope protein gp120_IIIB to the CD4/CXCR4/CCR5 heterooligomer was negligible, and the gp120-induced cytoskeletal rearrangements necessary for HIV-1 entry were prevented. CCR5 reduced HIV-1 envelope-induced CD4/CXCR4-mediated cell-cell fusion. In nucleofected Jurkat CD4 cells and primary human CD4⁺ T cells, CCR5 expression led to a reduction in X4 HIV-1 infectivity. These findings can help to understand why X4 HIV-1 strains infection affect T-cell types differently during AIDS development and indicate that receptor oligomerization might be a target for previously unidentified therapeutic approaches for AIDS intervention.For HIV-1 to enter a target cell, the viral envelope glycoprotein gp120 must interact with a set of cell surface molecules that include the primary receptor, CD4 (1), and a chemokine receptor (CCR5 or CXCR4) that acts as a coreceptor (2, 3). These molecules form CD4/chemokine receptor complexes, as deduced from coprecipitation data for CXCR4 or CCR5 with CD4 (4–8).Most HIV-1 variants isolated from newly infected individuals use CCR5 and CD4 to enter host cells; these M-tropic R5 strains are predominant in acute and asymptomatic phases of HIV infection. CD4⁺ T helper type 1 (Th1) cells, which express high CCR5 levels (9, 10), are implicated in maintaining asymptomatic status (11, 12). The “viral shift” from R5 to T-tropic X4 HIV-1 strains correlates with AIDS progression (13, 14). X4 strains infect mainly CD4⁺ Th2 cells, which express little CCR5 and whose CXCR4 levels resemble those of Th1 cells (15, 16), which suggests that cell susceptibility to HIV-1 infection depends on the CD4/coreceptor ratio and on receptor levels during cell activation and/or differentiation (17). CXCR4 and CCR5 are present as homodimers and heterodimers at the plasma membrane (18–20). In addition, gp120-mediated CD4/CXCR4 and CD4/CCR5 association and clustering is reported (21–23). Nonetheless, little is known of how CCR5 expression influences the CD4/CXCR4 interaction, or of the molecular basis that underlies the differences in X4 strains infection relative to CCR5 levels at the cell surface.Here, we identify CD4/CXCR4/CCR5 oligomers at the cell membrane, even in the absence of ligands. CCR5 expression in these complexes modifies the heterodimeric CD4/CXCR4 conformation and blocks gp120_IIIB binding, without altering binding of the CXCR4 ligand CXCL12 and its subsequent signaling. gp120_IIIB-triggered LIMK1 activation, cofilin dephosphorylation, and the actin cytoskeleton rearrangement necessary for cell-cell fusion were impeded in CD4/CXCR4/CCR5-expressing cells. The data obtained using recombinant gp120_IIIB glycoprotein were confirmed by experiments showing that X4 HIV-1 infection of Jurkat and primary T cells is regulated by CCR5 expression.     

Human domain antibodies to conserved sterically restricted regions on gp120 as exceptionally potent cross-reactive HIV-1 neutralizers

The antibody access to some conserved structures on the HIV-1 envelope glycoprotein (Env) is sterically restricted. We have hypothesized that the smallest independently folded antibody fragments (domains) could exhibit exceptionally potent and broadly cross-reactive neutralizing activity by targeting hidden conserved epitopes that are not accessible by larger antibodies. To test this hypothesis, we constructed a large (size 2.5 x 10(10)), highly diversified library of human antibody variable domains (domain antibodies) and used it for selection of binders to conserved Env structures by panning sequentially against Envs from different isolates. The highest affinity binder, m36, neutralized all tested HIV-1 isolates from clades A- D with an activity on average higher than that of C34, a peptide similar to the fusion inhibitor T20, which is in clinical use, and that of m9, which exhibits a neutralizing activity superior to known potent cross-reactive antibodies. Large-size fusion proteins of m36 exhibited diminished neutralizing activity but preincubation of virions with soluble CD4 restored it, suggesting that m36 epitope is sterically restricted and induced by CD4 (CD4i). M36 bound to gp120-CD4 complexes better than to gp120 alone and competed with CD4i antibodies. M36 is the only reported representative of a promising class of potent, broadly cross-reactive HIV-1 inhibitors based on human domain antibodies. It has potential for prevention and therapy and as an agent for exploration of the closely guarded conserved Env structures with implications for design of small molecule inhibitors and elucidation of mechanisms of virus entry and evasion of immune responses.     

Thermodynamic and kinetic analysis of sCD4 binding to HIV-1 virions and of gp120 dissociation.

Kinetic and thermodynamic aspects of the binding of sCD4 to intact virions of human immunodeficiency virus type 1 (HIV-1 RF), and of the subsequent induction of gp120 dissociation were studied. sCD4 binding to virions at 4 and 37 degrees C is half-maximal at approximately 40 and 10 nM, respectively. The transition between low-affinity and high-affinity binding of sCD4 to virions occurs over a narrow temperature range between 20 and 25 degrees C. Shedding of gp120 from virions after sCD4 binding is also temperature dependent, being initiated above approximately 20 degrees C. The minimum temperatures for the sCD4 affinity transition and gp120 shedding are, therefore, similar and we suggest how the two processes might be related mechanistically.     

Antibody-dependent cell-mediated cytotoxicity directed by a human monoclonal antibody reactive with gp120 of HIV-1.

We used a human monoclonal antibody (MAb; 15e) to identify an antibody-dependent cell-mediated cytotoxicity (ADCC) epitope on HIV-1 gp120. 15e has been shown to recognize a conformation-dependent epitope on gp120 which is important in both CD4 binding and neutralizing of HIV-1 infection. 15e binds to gp120 of HIV-1IIIB but not HIV-1RF. Using a standard ADCC assay, 15e was found to mediate ADCC against cells infected with HIV-1IIIB but not HIV-1RF. 15e did not mediate ADCC against cells with recombinant gp120 bound to surface CD4, indicating that 15e does not mediate innocent bystander ADCC against uninfected CD4 cells. To better define the 15e epitope, we performed ADCC against target cells infected with a vaccinia vector which expresses processed HIV-1IIIB gp160 from which the third variable region was deleted (amino acids, 312-328). MAb 15e efficiently mediated ADCC against cells expressing this altered form of gp120, indicating that this region is not contributing to the conformational epitope defined by 15e. 15e defines an important epitope in the human immune response to HIV-1 infection. Antibodies with 15e-like activity may be useful in immunoprophylaxis or immunotherapy of HIV-1 infection.     

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope

HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a "hot spot" for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.     

Simple methods for monitoring HIV-1 and HIV-2 gp120 binding to soluble CD4 by enzyme-linked immunosorbent assay: HIV-2 has a 25-fold lower affinity than HIV-1 for soluble CD4

Sensitive enzyme-linked immunosorbent assay-based methods are described for monitoring the binding of envelope glycoproteins from HIV-1 and HIV-2 to soluble CD4 (sCD4). Each of the assays has different properties suitable for different applications, but all can be used to characterize recombinant antigens and to screen for inhibitors of the gp120-CD4 interaction. Recombinant mammalian gp120 (Celltech) binds to sCD4 with high affinity (3 nM); this interaction is inhibited by sera from HIV-infected individuals and by specific monoclonal and polyclonal antibodies raised to a component of the CD4 binding site on gp120. The affinity for sCD4 of HIV-2 viral gp120 is shown to be approximately 25-fold lower than that of HIV-1 gp120 (viral or recombinant).     

Alpha-defensins block the early steps of HIV-1 infection: interference with the binding of gp120 to CD4

Alpha-defensins are antibiotic peptides that act as natural inhibitors of HIV-1 infection. However, the mechanisms of such inhibition are still unclear. Here we demonstrate that alpha-defensins block the earliest steps in the viral infectious cycle, as documented using an HIV-1 envelope-mediated cell-fusion assay. A broad-spectrum inhibitory activity was observed on primary and laboratory-adapted HIV-1 isolates irrespective of their coreceptor specificity and genetic subtype. A primary mechanism of such inhibition was identified as the ability of alpha-defensins to bind specifically both to the primary HIV-1 cellular receptor, CD4, and to the viral envelope glycoprotein, gp120. Moreover, treatment of CD4+ T cells with alpha-defensins caused a dramatic downmodulation of CD4 expression. By monoclonal antibody competition, the regions of interaction with alpha-defensins were mapped to the D1 domain of CD4 and to a surface contiguous to the CD4- and coreceptor-binding sites of gp120. Consistent with these findings, alpha-defensins inhibited the binding of gp120 to CD4. These data demonstrate that alpha-defensins specifically block the initial phase of the HIV infectious cycle and modulate the expression of CD4, a critical receptor in the physiology of T-cell activation.     

A molecular mechanism of inhibition of HIV-1 binding to CD4+ cells by monoclonal antibodies to gp110

We have investigated the possible involvement in the interaction between HIV gp110 and its CD4 receptor of epitopes different from the currently known binding site(s) of the molecule. Four monoclonal antibodies (MAbs) to gp110 were used (Genetic Systems Corporation, Seattle, Washington, USA): one (110-1) recognized a peptide corresponding to the C-terminal part of gp110 (494-517); the other three (110-3, 110-4, 110-5) recognized the same peptide located at position 308-328. HIV or purified gp110 obtained from a vaccinia recombinant (Transgene S.A., Strasbourg, France) were pre-incubated with the MAb prior to addition to CD4+ cells. Specific binding of viral particles or of the soluble molecule was then determined by flow cytometer analysis, compared with that of control preparations where the MAb was added after HIV or gp 110 had been allowed to bind CD4+ cells. Significant inhibition of HIV binding was noted with the three MAbs to peptide (308-328), but not with 110-1. At the molecular level, these same MAbs decreased the affinity of interaction between CD4 and soluble gp110, although they could still label the latter molecule after it had bound to CD4+ cells. Therefore, steric hindrance may account for neutralization of HIV binding by antibodies that are actually directed to epitopes topographically distinct from the site of binding of gp110 to CD4.     

The immunoglobulin superantigen-binding site of HIV-1 gp120 activates human basophils

OBJECTIVE: To investigate the mechanism whereby HIV-1 envelope glycoprotein gp120 from four different isolates obtained in three different countries induces proinflammatory mediator release from normal human basophils. METHODS: Histamine, cysteinyl leukotriene C4 (LTC4) and interleukin 4 (IL-4) release into the supernatant was measured in gp120-stimulated peripheral blood basophils from HIV-1 and HIV-2 negative subjects. RESULTS: The HIV glycoprotein was a potent stimulus for release of these mediators in basophils purified from donors negative for HIV-1 and HIV-2. There was also a correlation (r = 0.58; P < 0.01) between the maximum IL-4 release from basophils induced by gp120 and by anti-IgE. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released histamine in response to gp120 and anti-IgE. Anti-IgE specifically desensitized basophils to a subsequent challenge with anti-IgE and gp120. Human monoclonal IgM carrying the VH3 domain, but not that carrying the VH6 domain, inhibited gp120-induced secretion of histamine from basophils in a concentration-dependent manner. Synthetic peptides identical to regions distant from the N- and C-termini of gp120MN inhibited its activating capacity. CONCLUSIONS: gp120 acts as a viral superantigen interacting with the VH3 domain of IgE to induce the release of preformed and de novo synthesized mediators from human cells carrying the Fc fragment Fc epsilonRI receptor.     

N-terminal residues 105-117 of HIV-1 gp120 are not involved in CD4 binding.

Syu et al. recently reported that deletion of residues Ile-108 to Leu-116 from the amino terminus of gp120 abolished CD4 binding. The authors have investigated the role of this region using a monospecific antipeptide antibody. As assessed by a microtiter plate-based radioimmunoassay, the antibody, raised in sheep against a synthetic peptide encompassing this deleted region, does not inhibit the gp120-CD4 association. The reported loss of CD4 binding ability, resulting from the deletion in this region of gp120, is likely to be due to indirect structural changes in gp120 rather than representing an integral part of the CD4 binding domain.     

The Src kinase Lyn is required for CCR5 signaling in response to MIP-1beta and R5 HIV-1 gp120 in human macrophages

CCR5 is a receptor for several beta chemokines and the entry coreceptor used by macrophage-tropic (R5) strains of HIV-1. In addition to supporting viral entry, CCR5 ligation by the HIV-1 envelope glycoprotein 120 (gp120) can activate intracellular signals in macrophages and trigger inflammatory mediator release. Using a combination of in vitro kinase assay, Western blotting for phospho-specific proteins, pharmacologic inhibition, CCR5 knockout (CCR5Delta32) cells, and kinase-specific blocking peptide, we show for the first time that signaling through CCR5 in primary human macrophages is linked to the Src kinase Lyn. Stimulation of human monocyte-derived macrophages with either HIV-1 gp120 or MIP-1beta results in the CCR5-mediated activation of Lyn and the concomitant Lyn-dependent activation of the mitogen-activated protein (MAP) kinase ERK-1/2. Furthermore, activation of the CCR5/Lyn/ERK-1/2 pathway is responsible for gp120-triggered production of TNF-alpha by macrophages, which is believed to contribute to HIV-1 pathogenesis. Thus, Lyn kinase may play an important role both in normal CCR5 function in macrophages and in AIDS pathogenesis in syndromes such as AIDS dementia where HIV-1 gp120 contributes to inappropriate macrophage activation, mediator production, and secondary injury.     

Sensitive ELISA for the gp120 and gp160 surface glycoproteins of HIV-1

We have used a panel of polyclonal and monoclonal antibodies against gp120 and gp160, the envelope glycoproteins of human immunodeficiency virus type 1, to create rapid, simple, and sensitive twin-site sandwich ELISA specific for gp120 and gp160 or for gp160 alone. These assays can detect 500 COS cells in a population transiently transfected with a construct encoding gp120 and gp160, or 50 pg of recombinant gp160. We estimate that the mean amount of gp120 + gp160 in the transfected population is equivalent to 2.5 x 10(6) molecules per cell, 40-50% of which can be recovered from the culture medium as gp120 after 24 hours. The ELISA can be adapted to assess whether gp120 is detectable in the sera of HIV-1-infected persons: we show that gp120/gp160 is completely stable in normal human serum for at least 24 hours at 37 degrees C.     

Mapping of two new human B-cell epitopes on HIV-1 gp120.

OBJECTIVE: To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of these antibodies. DESIGN AND METHODS: Serum from HIV-1-antibody-positive individuals was used to screen a random fragment expression library representing gp120 from the HIVIIIB clone BH10. The library was based on the pUEX1 expression vector. Serum was tested for in vitro neutralizing activity using H9 cells and the HIVIIIB isolate. RESULTS: Four different epitopes defined by human antibodies were mapped on gp120. Two of these have not previously been reported and are located within amino acids (aa) 90-100 in the C1 region and aa 355-365 in the semi-conserved region between V3 and V4. The other two are located within aa 140-145 and aa 286-309. These epitopes are situated in regions that have been shown to demarcate human epitopes. Three serum samples with neutralization titres > or = 1024 were identified. None of the purified antibody fractions defining the mapped epitopes on gp120 had any neutralizing capacity against HIVIIIB. CONCLUSIONS: This study is the first demonstration of the applicability of random fragment expression libraries for the direct screening of human serum in order to map epitopes on gp120. Two new epitopes and two previously identified epitopes were mapped in this way. However, none of the linear epitopes was defined by antibody fractions neutralizing HIVIIIB, and it was not possible to map epitopes defined by neutralizing antibodies in the serum samples capable of neutralizing HIVIIIB infection of H9 cells. Thus, it appears that the neutralizing activity of serum in this study was not due to anti-gp120 antibodies defining linear epitopes.     

Synergistic neutralization of HIV-1 by human monoclonal antibodies against the V3 loop and the CD4-binding site of gp120.

Two distinct regions or epitope clusters of human immunodeficiency virus type 1 (HIV-1) gp120 have been shown to elicit neutralizing antibodies: the V3 loop and the CD4-binding site. We have isolated neutralizing human monoclonal antibodies (HuMAbs) against conserved epitopes in both of these regions. In this study, we demonstrate that an equimolar mixture of two of these HuMAbs, one directed against the V3 loop and the other against the CD4-binding site, neutralizes HIV-1 at much lower concentrations than does either of the individual HuMAbs. Mathematical analysis of this effect suggests cooperative neutralization of HIV-1 by the two HuMAbs and demonstrates a high level of synergy, with combination indices (CIs) of 0.07 and 0.16 for 90% neutralization of the MN and SF-2 strains, respectively. The dose reduction indices (DRIs) for each of the two HuMAbs at 99% neutralization range approximately from 10 to 150. A possible mechanism for this synergism is suggested by binding studies with recombinant gp160 of the MN strain; these show enhanced binding of the anti-CD4 binding site HuMAb in the presence of the anti-V3 loop HuMAb. These results demonstrate the advantage of including both V3 loop and CD4-binding site epitopes in a vaccine against HIV-1 and indicate that combinations of HuMAbs against these two sites may be particularly effective in passive immunotherapy against the virus.     

HIV-1 Protein gp120 Induces Mouse Lung Fibroblast-to-Myofibroblast Transdifferentiation via CXCR4 Activation

BackgroundIndividuals with HIV have ～2-fold increased risk of developing pulmonary fibrosis. The mechanism(s) by which this occurs has yet to be determined. HIV-1 protein gp120 activates CXCR4 in the lymphocyte, promoting a variety of intracellular signaling pathways including those common to TGFβ1 associated with lung fibroblast-to-myofibroblast transdifferentiation. We hypothesized that gp120 promotes pulmonary fibrotic changes via activation of CXCR4 in the lung fibroblast.MethodsMouse primary lung fibroblasts (PLFs) were cultured ± gp120, then analyzed for α-SMA expression and stress fiber formation. In parallel, PLFs were cultured ± gp120 ± AMD3100 (a CXCR4 antagonist), and α-SMA, pan and phospho-Akt, and total and phospho-MAPK (or ERK1/2) protein expression was quantified. Finally, lungs and PLFs from wild-type and HIV-1 transgenic mice were analyzed for hydroxyproline and α-SMA content.Resultsgp120 treatment increased α-SMA expression and myofibroblast differentiation in PLFs. gp120 treatment activated phosphorylation of ERK1/2, but not PI3K-Akt. Pretreatment with AMD3100 inhibited gp120-induced ERK1/2 phosphorylation and gp120-induced α-SMA expression. In parallel, there was a significant increase in hydroxyproline content in lungs from older HIV-1 transgenic mice and a >3-fold increase in α-SMA expression in PLFs isolated from HIV-1 transgenic mice.Conclusionsgp120 induces α-SMA expression and fibroblast-to-myofibroblast transdifferentiation by activating the CXCR4-ERK1/2 signaling pathway in mouse PLFs. Lungs of older HIV-1 transgenic mice contain higher hydroxyproline content and their PLFs have a striking increase in α-SMA expression. These results suggest a mechanism by which individuals with HIV are at increased risk of developing pulmonary fibrotic changes as they age.     

HIV-1 gp120 and chemokines activate ion channels in primary macrophages through CCR5 and CXCR4 stimulation

HIV type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry into target cells. Here we show that the HIV-1 envelope gp120 (Env) activates multiple ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. Env from both CCR5-dependent JRFL (R5) and CXCR4-dependent IIIB (X4) HIV-1 opened calcium-activated potassium (K(Ca)), chloride, and calcium-permeant nonselective cation channels in macrophages. These signals were mediated by CCR5 and CXCR4 because macrophages lacking CCR5 failed to respond to JRFL and an inhibitor of CXCR4 blocked ion current activation by IIIB. MIP-1beta and SDF-1alpha, chemokine ligands for CCR5 and CXCR4, respectively, also activated K(Ca) and Cl(-) currents in macrophages, but nonselective cation channel activation was unique to gp120. Intracellular Ca(2+) levels were also elevated by gp120. The patterns of activation mediated by CCR5 and CXCR4 were qualitatively similar but quantitatively distinct, as R5 Env activated the K(Ca) current more frequently, elicited Cl(-) currents that were approximately 2-fold greater in amplitude, and elevated intracellular Ca(+2) to higher peak and steady-state levels. Env from R5 and X4 primary isolates evoked similar current responses as the corresponding prototype strains. Thus, the interaction of HIV-1 gp120 with CCR5 or CXCR4 evokes complex and distinct signaling responses in primary macrophages, and gp120-evoked signals differ from those activated by the coreceptors' chemokine ligands. Intracellular signaling responses of macrophages to HIV-1 may modulate postentry steps of infection and cell functions apart from infection.     

Immunogenicity of HIV type 1 gp120 CD4 binding site phage mimotopes

The conserved domain of the CD4 binding site (CD4bs) on the human immunodeficiency virus type 1 (HIV- 1) envelope represents a potential target for vaccine development. Here we describe selection of peptide mimotopes by panning a phage peptide library on the HIV-1 CD4bs-specific, broadly neutralizing anti-HIV-1 monoclonal antibody, IgG(1) b12. We identified an initial consensus sequence for IgG1 b12 binding (M/VThetaSD, where Theta represents an aromatic amino acid). A molecular evolution approach, using second- and third-generation libraries, led us to identify a refined consensus sequence (GLLVWSDEL). The resulting IgG1 b12 phage mimotopes compete with gp160 for the IgG1 b12 antigen-binding site, but the phage coat protein (pIII) may play an important structural role, since both free peptides and KLH-conjugated peptides have no detectable binding activity. Mice immunized with IgG1 b12 phage mimotopes elicited a weak but persistent humoral response directed against the HIV-1 envelope. An antibody fragment was isolated from the antibody repertoires of these animals. It is noteworthy that while it has a relatively low affinity for HIV-1 gp160, the antibody targets an epitope that overlaps with that of IgG1 b12. Our data therefore suggest that engineered IgG1 b12 mimotopes share immunogenic features with the CD4bs. However, these peptidic structures will require further improvement in order to generate broad specificity neutralizing antibodies like IgG1 b12.