Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

A predicted three-dimensional structure for the human immunodeficiency virus binding domains of CD4 antigen

A predicted three-dimensional structure of the two N-terminal extracellular domains of human CD4 antigen, a cell surface glycoprotein, is reported. This region of CD4, particularly the first domain, has been identified as containing the binding region for the envelope gp120 protein of the human immunodeficiency virus. The model was predicted based on the sequence homology of each domain with the variable light chain of immunoglobulins. The framework beta-sheet regions were taken from the crystal coordinates of REI. For one region in the first domain of CD4 there was an ambiguity in the alignment with REI and two alternate models are presented. Loops connecting the framework were modelled from fragments selected from a database of main chain coordinates from all known protein structures. Residues identified as involved in binding gp120 have been located in several other studies within the first domain of CD4. Epitopes from eight monoclonal antibodies have been mapped onto residues in both domains. Competition of these antibodies with each other and with gp120 can be interpreted from the structural model.     

Relationship of the human immunodeficiency virus type 1 gp120 third variable loop to a component of the CD4 binding site in the fourth conserved region.

Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions.     

Discontinuous, conserved neutralization epitopes overlapping the CD4-binding region of human immunodeficiency virus type 1 gp120 envelope glycoprotein.

Monoclonal antibodies have been isolated from human immunodeficiency virus type 1 (HIV-1)-infected patients that recognize discontinuous epitopes on the gp120 envelope glycoprotein, that block gp120 interaction with the CD4 receptor, and that neutralize a variety of HIV-1 isolates. Using a panel of HIV-1 gp120 mutants, we identified amino acids important for precipitation of the gp120 glycoprotein by three different monoclonal antibodies with these properties. These amino acids are located within seven discontinuous, conserved regions of the gp120 glycoprotein, four of which overlap those regions previously shown to be important for CD4 recognition. The pattern of sensitivity to amino acid change in these seven regions differed for each antibody and also differed from that of the CD4 glycoprotein. These results indicate that the CD4 receptor and this group of broadly neutralizing antibodies recognize distinct but overlapping gp120 determinants.     

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.     

Neutralizing antibodies to an immunodominant envelope sequence do not prevent gp120 binding to CD4.

Animals immunized with the human immunodeficiency virus type 1 gp160 glycoprotein or certain recombinant envelope components develop potent virus-neutralizing activity. This activity is principally due to antibodies directed toward a hypervariable region of gp120 between cysteine residues 302 and 337 and is virus isolate specific. These antisera, as well as two neutralizing monoclonal antibodies directed against the same hypervariable sequence, do not appreciably block gp120 from binding CD4. In contrast, serum samples from infected humans possess high titers of antibodies that block gp120-CD4 binding; these titers approximately correlate with the serum neutralization titers. Our results suggest that there are at least two targets on the envelope glycoprotein for virus neutralization. The target responsible for the broader neutralizing activity of human serum may be a conserved region of gp120 involved in CD4 binding. The antibodies directed at the hypervariable region of the envelope inhibit a different step in virus infection which is subsequent to receptor binding. The extent to which these two different epitopes of gp120 may be involved in protection against human immunodeficiency virus infection is discussed.     

Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.

Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently.     

Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1.

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.     

Development of the anti-gp120 antibody response during seroconversion to human immunodeficiency virus type 1.

We have studied the development of the antibody response to the surface glycoprotein gp120 of human immunodeficiency virus type 1 in three individuals who presented with primary human immunodeficiency virus type 1 infection syndrome. Serum anti-gp120 antibodies were first detected 4 to 23 days after presentation, after p24 antigen and infectious-virus titers in the peripheral blood had declined manyfold from their highest values. Whether anti-gp120 antibodies present at undetectable levels are involved in clearance of viremia remains unresolved. Among the earliest detectable anti-gp120 antibodies were those to conformationally sensitive epitopes; these antibodies were able to block the binding of gp120 monomers to soluble CD4 or to a human monoclonal antibody to a discontinuous epitope overlapping the CD4-binding site. Some of these antibodies were type specific to a degree, in that they were more effective at blocking ligand binding to autologous gp120 than to heterologous gp120. However, the appearance of these antibodies did not correlate with that of antibodies able to neutralize the autologous virus in vitro by a peripheral blood mononuclear cell-based assay. Antibodies to the V3 loop were detected at about the same time as, or slightly later than, those to the CD4-binding site. There was a weak correlation between the presence of antibodies to the V3 loop and autologous virus-neutralizing activity in two of three individuals studied. However, serum from the third individual contained V3 antibodies but lacked the ability to neutralize the autologous virus in vitro, even immediately after seroconversion. Thus, no simple, universal correlate of autologous virus-neutralizing activity in a peripheral blood mononuclear cell-based assay is apparent from in vitro assays that rely on detecting antibody interactions with monomeric gp120 or fragments thereof.     

Truncated variants of gp120 bind CD4 with high affinity and suggest a minimum CD4 binding region.

The envelope glycoprotein, gp120, of human immunodeficiency virus type 1 (HIV-1) binds the cellular protein CD4 with high affinity. By deletion we show that 62 N- and 20 C-terminal residues along with the V1, V2 and V3 variable regions of gp 120 are unnecessary for CD4 binding. A 287 residue variant (ENV59), missing those 197 amino acids, binds to CD4 with high affinity. A polyclonal antibody failed to efficiently precipitate ENV59 which is consistent with the loss of immunodominant antigenic structures in the regions deleted. This suggests that ENV59 may have potential as an immunogen, able to elicit antibodies against more conserved regions of gp120. Additionally, complementing co-expressed gp120 fragments as well as a circularly permuted molecule bind CD4, and suggest either that the molecular termini are adjacent in the folded structure, or that an N-terminal region folds into the structure unconstrained by its method of attachment to the rest of the molecule.     

Identification of individual human immunodeficiency virus type 1 gp120 amino acids important for CD4 receptor binding.

The binding of the CD4 receptor by the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein is important for virus entry and cytopathic effect. To investigate the CD4-binding region of the gp120 glycoprotein, we altered gp120 amino acids, excluding cysteines, that are conserved among the primate immunodeficiency viruses utilizing the CD4 receptor. Changes in two hydrophobic regions (Thr-257 in conserved region 2 and Trp-427 in conserved region 4) and two hydrophilic regions (Asp-368 and Glu-370 in conserved region 3 and Asp-457 in conserved region 4) resulted in significant reductions in CD4 binding. For most of the mutations affecting these residues, the observed effects on CD4 binding did not apparently result from global conformational disruption of the gp120 molecule, as assessed by measurements of precursor processing, subunit association, and monoclonal antibody recognition. The two hydrophilic regions exhibit a strong propensity for beta-turn formation, are predicted to act as efficient B-cell epitopes, and are located adjacent to hypervariable, glycosylated regions. This study defines a small number of gp120 residues important for CD4 binding, some of which might constitute attractive targets for immunologic intervention.     

Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor

The primary event in the infection of cells by HIV is the interaction between the viral envelope glycoprotein, gp120, and its cellular receptor, CD4. A recombinant form of gp120 was found to bind to a recombinant CD4 antigen with high affinity. Two gp120-specific murine monoclonal antibodies were able to block the interaction between gp120 and CD4. The gp120 epitope of one of these antibodies was isolated by immunoaffinity chromatography of acid-cleaved gp120 and shown to be contained within amino acids 397-439. Using in vitro mutagenesis, we have found that deletion of 12 amino acids from this region of gp120 leads to a complete loss of binding. In addition, a single amino acid substitution in this region results in significantly decreased binding, suggesting that sequences within this region are directly involved in the binding of gp120 to the CD4 receptor.     

Human immunodeficiency virus type 1 envelope gp120 is cleaved after incubation with recombinant soluble CD4.

Human immunodeficiency virus type 1 (HIV-1) infects human CD4+ cells by a high-affinity interaction between its envelope glycoprotein gp120 and the CD4 molecule on the cell surface. Subsequent virus entry into the cells involves other steps, one of which could be cleavage of the gp120 followed by virus-cell fusion. The envelope gp120 is highly variable among different HIV-1 isolates, but conserved amino acid sequence motifs that contain potential proteolytic cleavage sites can be found. Following incubation with a soluble form of CD4, we demonstrate that gp120 of highly purified HIV-1 preparations is, without addition of exogenous proteinase, cleaved most likely in the V3 loop, yielding two proteins of 50 and 70 kDa. The extent of gp120 proteolysis is HIV-1 strain dependent and correlates with the recombinant soluble CD4 sensitivity to neutralization of the particular strain. The origin of the proteolytic activity in the virus preparations remains unclear. The results support the hypothesis that cleavage of gp120 is required for HIV infection of cells.     

Novel peptides based on HIV-1 gp120 sequence with homology to chemokines inhibit HIV infection in cell culture

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines. In the amino acid sequences of most chemokines there is a Trp residue located at the beginning of the C-terminal α-helix, which is separated by six residues from the fourth Cys residue. The gp120 of all HIV-1 isolates have a similar motif, which includes the C-terminal part of a variable loop 3 (V3) and N-terminal part of a conserved region 3 (C3). Two synthetic peptides, derived from the relevant gp120 sequence inhibited HIV-1 replication in macrophages and T lymphocytes in sequence-dependent manner. The peptides also prevented binding of anti-CXCR4 antibodies to CXCR4, and inhibited the intracellular Ca(2+) influx in response to CXCL12/SDF-1α. Thus these peptides can be used to dissect gp120 interactions with chemokine receptors and could serve as leads for the design of new inhibitors of HIV-1.     

Discovery of small-molecule human immunodeficiency virus type 1 entry inhibitors that target the gp120-binding domain of CD4

The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4(+) cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.     

The V3 region of the envelope glycoprotein of human immunodeficiency virus type 1 binds sulfated polysaccharides and CD4-derived synthetic peptides.

gp120 is the envelope glycoprotein found on the surface of human immunodeficiency virus type 1 (HIV-1), and it binds to human cell surface CD4 receptors to initiate the HIV-1 infection process. It is now well-established that synthetic peptides from the V3 region on gp120 elicit antibodies that block HIV-1 infection and HIV-1-mediated cell fusion. Here we show that synthetic peptides derived from similar V3 regions of several isolates of HIV-1 bind [3H]heparin, and we also demonstrate that [3H]heparin binds to recombinant gp120 IIIB. The binding could be blocked by unlabeled heparin, dextran sulfate, and by a highly anionic benzylated synthetic peptide derived from human CD4 (amino acids 81-92). The nonbenzylated peptides from the same region were considerably less active. Unlabeled heparin, dextran sulfate, and the CD4-derived peptides were able to compete with the binding of soluble gp120 to immobilized antibodies against fragments of the V3 from isolate IIIB, but they had no effect on the binding of gp120 to anti-peptide antibodies targeted against another unrelated region of gp120. Biotin conjugated to the benzylated CD4-peptide bound to gp120 and was blocked from this binding by anti-V3 antibodies. These results indicate that the three materials that have been demonstrated by others to block HIV-1 infection in vitro, sulfated polysaccharides, certain CD4-derived synthetic peptides, and anti-V3 antibodies, may be acting through a common mechanism that includes binding to the V3 region of gp120 on HIV-1.     

The Highly Conserved Layer-3 Component of the HIV-1 gp120 Inner Domain Is Critical for CD4-Required Conformational Transitions

The trimeric envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) mediates virus entry into host cells. CD4 engagement with the gp120 exterior envelope glycoprotein subunit represents the first step during HIV-1 entry. CD4-induced conformational changes in the gp120 inner domain involve three potentially flexible topological layers (layers 1, 2, and 3). Structural rearrangements between layer 1 and layer 2 have been shown to facilitate the transition of the envelope glycoprotein trimer from the unliganded to the CD4-bound state and to stabilize gp120-CD4 interaction. However, our understanding of CD4-induced conformational changes in the gp120 inner domain remains incomplete. Here, we report that a highly conserved element of the gp120 inner domain, layer 3, plays a pivot-like role in these allosteric changes. In the unliganded state, layer 3 modulates the association of gp120 with the Env trimer, probably by influencing the relationship of the gp120 inner and outer domains. Importantly, layer 3 governs the efficiency of the initial gp120 interaction with CD4, a function that can also be fulfilled by filling the Phe43 cavity. This work defines the functional importance of layer 3 and completes a picture detailing the role of the gp120 inner domain in CD4-induced conformational transitions in the HIV-1 Env trimer.     

Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.

Forty-six monoclonal antibodies (MAbs) able to bind to the native, monomeric gp120 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) LAI (HXBc2) strain were used to generate a competition matrix. The data suggest the existence of two faces of the gp120 glycoprotein. The binding sites for the viral receptor, CD4, and neutralizing MAbs appear to cluster on one face, which is presumably exposed on the assembled, oligomeric envelope glycoprotein complex. A second gp120 face, which is presumably inaccessible on the envelope glycoprotein complex, contains a number of epitopes for nonneutralizing antibodies. This analysis should be useful for understanding both the interaction of antibodies with the HIV-1 gp120 glycoprotein and neutralization of HIV-1.     

Mutations in human immunodeficiency virus type 1 gp41 affect sensitivity to neutralization by gp120 antibodies.

Three closely related molecular human immunodeficiency virus type 1 (HIV-1) clones, with differential neutralization phenotypes, were generated by cloning of an NcoI-BamHI envelope (env) gene fragment (HXB2R nucleotide positions 5221 to 8021) into the full-length HXB2 molecular clone of HIV-1 IIIB. These env gene fragments, containing the complete gp120 coding region and a major part of gp41, were obtained from three different biological clones derived from a chimpanzee-passaged HIV-1 IIIB isolate. Two of the viruses thus obtained (4.4 and 5.1) were strongly resistant to neutralization by infection-induced chimpanzee and human polyclonal antibodies and by HIV-1 IIIB V3-specific monoclonal antibodies and weakly resistant to soluble CD4 and a CD4-binding-site-specific monoclonal antibody. The third virus (6.8) was sensitive to neutralization by the same reagents. The V3 coding sequence and the gp120 amino acid residues important for the discontinuous neutralization epitope overlapping the CD4-binding site were completely conserved among the clones. However, the neutralization-resistant clones 4.4 and 5.1 differed from neutralization-sensitive clone 6.8 by two mutations in gp41. Exchange experiments confirmed that the 3' end of clone 6.8 (nucleotides 6806 to 8021; amino acids 346 to 752) conferred a neutralization-sensitive phenotype to both of the neutralization-resistant clones 4.4 and 5.1. From our study, we conclude that mutations in the extracellular portion of gp41 may affect neutralization sensitivity to gp120 antibodies.     

Neutralization of human immunodeficiency virus type 1 by sCD4-17b,a single-chain chimeric protein,based on sequential interaction of gp120 with CD4 and coreceptor

We designed a novel single-chain chimeric protein, designated sCD4-17b, for neutralization of human immunodeficiency virus type 1 (HIV-1). The recombinant protein contains domains 1 and 2 of soluble CD4 (sCD4), connected via a flexible polypeptide linker to a single-chain variable region construct of 17b, a human monoclonal antibody that targets a conserved CD4-induced epitope on gp120 overlapping the coreceptor binding region. We hypothesized that the sCD4 moiety would bind gp120 and expose the 17b epitope; the 17b moiety would then bind, thereby blocking coreceptor interaction and neutralizing infection. The sCD4-17b protein, expressed by a recombinant vaccinia virus, potently neutralized a prototypic R5 clade B primary isolate, with a 50% inhibitory concentration of 3.2 nM (0.16 microg/ml) and >95% neutralization at 32 nM (1.6 microg/ml). The individual components (sCD4 and 17b, singly or in combination) had minimal effects at these concentrations, demonstrating that the activity of sCD4-17b reflected the ability of a single chimeric molecule to bind gp120 simultaneously via two independent moieties. sCD4-17b was highly potent compared to the previously characterized broadly cross-reactive neutralizing monoclonal antibodies IgGb12, 2G12, and 2F5. Multiple primary isolates were neutralized, including two previously described as antibody resistant. Neutralization occurred for both R5 and X4 strains and was not restricted to clade B. However, several primary isolates were insensitive over the concentration range tested, despite the known presence of binding sites for both CD4 and 17b. sCD4-17b has potential utility for passive immunization against HIV-1 in several contexts, including maternal transmission, postexposure prophylaxis, and sexual transmission (topical microbicide).