Bispecific chimeric antigen receptors targeting the CD4 binding site and high-mannose Glycans of gp120 optimized for anti-human immunodeficiency virus potency and breadth with minimal immunogenicity.

BACKGROUND AIMS: Chimeric antigen receptors (CARs) offer great potential toward a functional cure of human immunodeficiency virus (HIV) infection. To achieve the necessary long-term virus suppression, we believe that CARs must be designed for optimal potency and anti-HIV specificity, and also for minimal probability of virus escape and CAR immunogenicity. CARs containing antibody-based motifs are problematic in the latter regard due to epitope mutation and anti-idiotypic immune responses against the variable regions. METHODS: We designed bispecific CARs, each containing a segment of human CD4 linked to the carbohydrate recognition domain of a human C-type lectin. These CARs target two independent regions on HIV-1 gp120 that presumably must be conserved on clinically significant virus variants (i.e., the primary receptor binding site and the dense oligomannose patch). Functionality and specificity of these bispecific CARs were analyzed in assays of CAR-T cell activation and spreading HIV-1 suppression. RESULTS: T cells expressing a CD4-dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DCSIGN) CAR displayed robust stimulation upon encounter with Env-expressing targets, but negligible activity against intercellular adhesion molecule (ICAM)-2 and ICAM-3, the natural dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin ligands. Moreover, the presence of the lectin moiety prevented the CD4 from acting as an entry receptor on CCR5-expressing cells, including CD8+ T cells. However, in HIV suppression assays, the CD4-DCSIGN CAR and the related CD4-liver/lymph node-specific intercellular adhesion molecule-3-grabbing non-integrin CAR displayed only minimally increased potency compared with the CD4 CAR against some HIV-1 isolates and reduced potency against others. By contrast, the CD4-langerin and CD4-mannose binding lectin (MBL) CARs uniformly displayed enhanced potency compared with the CD4 CAR against all the genetically diverse HIV-1 isolates examined. Further experimental data, coupled with known biological features, suggest particular advantages of the CD4-MBL CAR. DISCUSSION: These studies highlight features of bispecific CD4-lectin CARs that achieve potency enhancement by targeting two distinct highly conserved Env determinants while lacking immunogenicity-prone antibody-based motifs.

Novel CD4-Based Bispecific Chimeric Antigen Receptor Designed for Enhanced Anti-HIV Potency and Absence of HIV Entry Receptor Activity.

UNLABELLED: Adoptive transfer of CD8 T cells genetically engineered to express "chimeric antigen receptors" (CARs) represents a potential approach toward an HIV infection "functional cure" whereby durable virologic suppression is sustained after discontinuation of antiretroviral therapy. We describe a novel bispecific CAR in which a CD4 segment is linked to a single-chain variable fragment of the 17b human monoclonal antibody recognizing a highly conserved CD4-induced epitope on gp120 involved in coreceptor binding. We compared a standard CD4 CAR with CD4-17b CARs where the polypeptide linker between the CD4 and 17b moieties is sufficiently long (CD4-35-17b CAR) versus too short (CD4-10-17b) to permit simultaneous binding of the two moieties to a single gp120 subunit. When transduced into a peripheral blood mononuclear cell (PBMC) or T cells thereof, all three CD4-based CARs displayed specific functional activities against HIV-1 Env-expressing target cells, including stimulation of gamma interferon (IFN-γ) release, specific target cell killing, and suppression of HIV-1 pseudovirus production. In assays of spreading infection of PBMCs with genetically diverse HIV-1 primary isolates, the CD4-10-17b CAR displayed enhanced potency compared to the CD4 CAR whereas the CD4-35-17b CAR displayed diminished potency. Importantly, both CD4-17b CARs were devoid of a major undesired activity observed with the CD4 CAR, namely, rendering the transduced CD8(+) T cells susceptible to HIV-1 infection. Likely mechanisms for the superior potency of the CD4-10-17b CAR over the CD4-35-17b CAR include the greater potential of the former to engage in the serial antigen binding required for efficient T cell activation and the ability of two CD4-10-17b molecules to simultaneously bind a single gp120 subunit. IMPORTANCE: HIV research has been energized by prospects for a cure for HIV infection or, at least, for a "functional cure" whereby antiretroviral therapy can be discontinued without virus rebound. This report describes a novel CD4-based "chimeric antigen receptor" (CAR) which, when genetically engineered into T cells, gives them the capability to selectively respond to and kill HIV-infected cells. This CAR displays enhanced features compared to previously described CD4-based CARs, namely, increased potency and avoidance of the undesired rendering of the genetically modified CD8 T cells susceptible to HIV infection. When adoptively transferred back to the individual, the genetically modified T cells will hopefully provide durable killing of infected cells and sustained virus suppression without continued antiretroviral therapy, i.e., a functional cure.

Hyperglycosylated stable core immunogens designed to present the CD4 binding site are preferentially recognized by broadly neutralizing antibodies.

UNLABELLED: The HIV-1 surface envelope glycoprotein (Env) trimer mediates entry into CD4(+) CCR5(+) host cells. Env possesses conserved antigenic determinants, such as the gp120 primary receptor CD4 binding site (CD4bs), a known neutralization target. Env also contains variable regions and protein surfaces occluded within the trimer that elicit nonneutralizing antibodies. Here we engineered additional N-linked glycans onto a cysteine-stabilized gp120 core (0G) deleted of its major variable regions to preferentially expose the conformationally fixed CD4bs. Three, 6, 7, and 10 new NXT/S glycan (G) motifs were engineered into 0G to encode 3G, 6G, 7G, and 10G cores. Following purification, most glycoproteins, except for 10G, were recognized by broadly neutralizing CD4bs-directed antibodies. Gel and glycan mass spectrometry confirmed that additional N-glycans were posttranslationally added to the redesigned cores. Binding kinetics revealed high-affinity recognition by seven broadly neutralizing CD4bs-directed antibodies and low to no binding by non-broadly neutralizing CD4bs-directed antibodies. Rabbits inoculated with the hyperglycosylated cores elicited IgM and IgG responses to each given protein that were similar in their neutralization characteristics to those elicited by parental 0G. Site-specific glycan masking effects were detected in the elicited sera, and the antisera competed with b12 for CD4bs-directed binding specificity. However, the core-elicited sera showed limited neutralization activity. Trimer priming or boosting of the core immunogens elicited tier 1-level neutralization that mapped to both the CD4bs and V3 and appeared to be trimer dependent. Fine mapping at the CD4bs indicated that conformational stabilization of the cores and addition of N-glycans altered the molecular surface of Env sites of vulnerability to neutralizing antibody, suggesting an explanation for why the elicited neutralization was not improved by this rational design strategy. IMPORTANCE: Major obstacles to developing an effective HIV-1 vaccine include the variability of the envelope surface glycoproteins and its high-density glycan shield, generated by incorporation of host (human) glycosylation. HIV-1 does harbor highly conserved sites on the exposed envelope protein surface of gp120, one of which is the virus receptor (CD4) binding site. Several broadly neutralizing antibodies elicited from HIV patients do target this gp120 CD4 binding site (CD4bs); however, gp120 immunogens do not elicit broadly neutralizing antibodies. In this study, we targeted the CD4bs by conformational stabilization and additional glycan masking. We used the atomic-level structure to reengineer gp120 cores to preferentially present the cysteine-stabilized CD4bs and to mask (by glycan) nonneutralizing determinants. Importantly, glycan masking did successfully focus antibody responses to the CD4bs; however, the elicited CD4bs-directed antibodies did not neutralize HIV or bind to unmodified gp120, presumably due to the structure-guided modifications of the modified gp120 core.

Structural definition of a conserved neutralization epitope on HIV-1 gp120.

The remarkable diversity, glycosylation and conformational flexibility of the human immunodeficiency virus type 1 (HIV-1) envelope (Env), including substantial rearrangement of the gp120 glycoprotein upon binding the CD4 receptor, allow it to evade antibody-mediated neutralization. Despite this complexity, the HIV-1 Env must retain conserved determinants that mediate CD4 binding. To evaluate how these determinants might provide opportunities for antibody recognition, we created variants of gp120 stabilized in the CD4-bound state, assessed binding of CD4 and of receptor-binding-site antibodies, and determined the structure at 2.3 A resolution of the broadly neutralizing antibody b12 in complex with gp120. b12 binds to a conformationally invariant surface that overlaps a distinct subset of the CD4-binding site. This surface is involved in the metastable attachment of CD4, before the gp120 rearrangement required for stable engagement. A site of vulnerability, related to a functional requirement for efficient association with CD4, can therefore be targeted by antibody to neutralize HIV-1.

Factors associated with cesarean delivery in Bangladesh: A multilevel modeling.

BACKGROUND: In recent decades, the number of C-section deliveries has increased over the world, including Bangladesh. The study aimed to identify individual- and community-level factors associated with C-section childbirth in Bangladesh and investigate the annual average increase rate of C-section childbirth. C-section. METHODS: Data were derived from four waves of the Bangladesh Demographic and Health Survey (BDHS) conducted between 2007 and 2017-18. Chi-square test of association was run to check the bivariate association between dependent and independent factors. For the individual- and community-level factors deliveries among Bangladeshi married women, a multilevel logistic regression model was carried out. RESULT: Over the last ten years, the average annual increase of C-section rates during delivery was 13.09% in Bangladesh, while this rate was 33.25%according to 2017-18 BDHS.C-section 33.25% of women have access to C-section birth. Women who had four or more than four ANC visits, women and their husbands with secondary and above education, middle and rich-wealth households, and community-level factors such as high media access, secondary and above educational experience women used C-sectionC-section delivery compared to their respective counterpart. CONCLUSION: The findings of the study revealed the association of some individual and community-level factors that need to be taken into account to minimize the rising rate of C-section deliveries in Bangladesh, which has increased drastically over the survey years.

Structure-based stabilization of HIV-1 gp120 enhances humoral immune responses to the induced co-receptor binding site.

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions) into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.

sCD4-17b bifunctional protein: extremely broad and potent neutralization of HIV-1 Env pseudotyped viruses from genetically diverse primary isolates.

BACKGROUND: We previously described a potent recombinant HIV-1 neutralizing protein, sCD4-17b, composed of soluble CD4 attached via a flexible polypeptide linker to an SCFv of the 17b human monoclonal antibody directed against the highly conserved CD4-induced bridging sheet of gp120 involved in coreceptor binding. The sCD4 moiety of the bifunctional protein binds to gp120 on free virions, thereby enabling the 17b SCFv moiety to bind and block the gp120/coreceptor interaction required for entry. The previous studies using the MAGI-CCR5 assay system indicated that sCD4-17b (in concentrated cell culture medium, or partially purified) potently neutralized several genetically diverse HIIV-1 primary isolates; however, at the concentrations tested it was ineffective against several other strains despite the conservation of binding sites for both CD4 and 17b. To address this puzzle, we designed variants of sCD4-17b with different linker lengths, and tested the neutralizing activities of the immunoaffinity purified proteins over a broader concentration range against a large number of genetically diverse HIV-1 primary isolates, using the TZM-bl Env pseudotype assay system. We also examined the sCD4-17b sensitivities of isogenic viruses generated from different producer cell types. RESULTS: We observed that immunoaffinity purified sCD4-17b effectively neutralized HIV-1 pseudotypes, including those from HIV-1 isolates previously found to be relatively insensitive in the MAGI-CCR5 assay. The potencies were equivalent for the original construct and a variant with a longer linker, as observed with both pseudotype particles and infectious virions; by contrast, a construct with a linker too short to enable simultaneous binding of the sCD4 and 17b SCFv moieties was much less effective. sCD4-17b displayed potent neutralizing activity against 100% of nearly 4 dozen HIV-1 primary isolates from diverse genetic subtypes (clades A, B, C, D, F, and circulating recombinant forms AE and AG). The neutralization breadth and potency were superior to what have been reported for the broadly neutralizing monoclonal antibodies IgG b12, 2G12, 2F5, and 4E10. The activity of sCD4-17b was found to be similar against isogenic virus particles from infectious molecular clones derived either directly from the transfected producer cell line or after a single passage through PBMCs; this contrasted with the monoclonal antibodies, which were less potent against the PMBC-passaged viruses. CONCLUSIONS: The results highlight the extremely potent and broad neutralizing activity of sCD4-17b against genetically diverse HIV-1 primary isolates. The bifunctional protein has potential applications for antiviral approaches to combat HIV infection.

Human immunodeficiency virus type 1 env trimer immunization of macaques and impact of priming with viral vector or stabilized core protein.

Currently there is limited information about the quality of immune responses elicited by candidate human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogens in primates. Here we describe a comprehensive analysis of neutralizing antibody and T-cell responses obtained in cynomolgus macaques by three selected immunization regimens. We used the previously described YU2-based gp140 protein trimers administered in an adjuvant, preceded by two distinct priming strategies: either alphavirus replicon particles expressing matched gp140 trimers or gp120 core proteins stabilized in the CD4-bound conformation. The rationale for priming with replicon particles was to evaluate the impact of the expression platform on trimer immunogenicity. The stable core proteins were chosen in an attempt to expand selectively lymphocytes recognizing common determinants between the core and trimers to broaden the immune response. The results presented here demonstrate that the platform by which Env trimers were delivered in the priming (either protein or replicon vector) had little impact on the overall immune response. In contrast, priming with stable core proteins followed by a trimer boost strikingly focused the T-cell response on the core sequences of HIV-1 Env. The specificity of the T-cell response was distinctly different from that of the responses obtained in animals immunized with trimers alone and was shown to be mediated by CD4(+) T cells. However, this regimen showed limited or no improvement in the neutralizing antibody responses, suggesting that further immunogen design efforts are required to successfully focus the B-cell response on conserved neutralizing determinants of HIV-1 Env.

B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

Interdomain Stabilization Impairs CD4 Binding and Improves Immunogenicity of the HIV-1 Envelope Trimer.

The HIV-1 envelope (Env) spike is a trimer of gp120/gp41 heterodimers that mediates viral entry. Binding to CD4 on the host cell membrane is the first essential step for infection but disrupts the native antigenic state of Env, posing a key obstacle to vaccine development. We locked the HIV-1 Env trimer in a pre-fusion configuration, resulting in impaired CD4 binding and enhanced binding to broadly neutralizing antibodies. This design was achieved via structure-guided introduction of neo-disulfide bonds bridging the gp120 inner and outer domains and was successfully applied to soluble trimers and native gp160 from different HIV-1 clades. Crystallization illustrated the structural basis for CD4-binding impairment. Immunization of rabbits with locked trimers from two different clades elicited neutralizing antibodies against tier-2 viruses with a repaired glycan shield regardless of treatment with a functional CD4 mimic. Thus, interdomain stabilization provides a widely applicable template for the design of Env-based HIV-1 vaccines.

Structure-activity relationships in the binding of chemically derivatized CD4 to gp120 from human immunodeficiency virus.

The first step in HIV infection is the binding of the envelope glycoprotein gp120 to the host cell receptor CD4. An interfacial "Phe43 cavity" in gp120, adjacent to residue Phe43 of gp120-bound CD4, has been suggested as a potential target for therapeutic intervention. We designed a CD4 mutant (D1D2F43C) for site-specific coupling of compounds for screening against the cavity. Altogether, 81 cysteine-reactive compounds were designed, synthesized, and tested. Eight derivatives exceeded the affinity of native D1D2 for gp120. Structure-activity relationships (SAR) for derivatized CD4 binding to gp120 revealed significant plasticity of the Phe43 cavity and a narrow entrance. The primary contacts for compound recognition inside the cavity were found to be van der Waals interactions, whereas hydrophilic interactions were detected in the entrance. This first SAR on ligand binding to an interior cavity of gp120 may provide a starting point for structure-based assembly of small molecules targeting gp120-CD4 interaction.

Towards an HIV cure based on targeted killing of infected cells: different approaches against acute versus chronic infection.

PURPOSE OF REVIEW: Current regimens of combination antiretroviral therapy (cART) offer effective control of HIV infection, with maintenance of immune health and near-normal life expectancy. What will it take to progress beyond the status quo, whereby infectious virus can be eradicated (a 'sterilizing cure') or fully controlled without the need for ongoing cART (a 'functional cure')? RECENT FINDINGS: On the basis of therapeutic advances in the cancer field, we propose that targeted cytotoxic therapy to kill HIV-infected cells represents a logical complement to cART for achieving an HIV cure. This concept is based on the fact that cART effectively blocks replication of the virus, but does not eliminate cells that are already infected; targeted cytotoxic therapy would contribute precisely this missing component. We suggest that different modalities are suited for curing primary acute versus established chronic infection. For acute infection, relatively short-acting potent agents such as recombinant immunotoxins might prove sufficient for HIV eradication, whereas for chronic infection, a long-lasting (lifelong?) modality is required to maintain full virus control, as might be achieved with genetically modified autologous T cells. SUMMARY: We present perspectives for complementing cART with targeted cytotoxic therapy, whereby HIV infection is either eradicated or fully controlled, thereby eliminating the need for lifelong cART.

Blocking HIV-1 gp120 at the Phe43 cavity: if the extension fits .

The Phe43 cavity is a mysterious feature in crystallographic structures of HIV-1 gp120-CD4 complexes. In this issue of Structure, Acharya and colleagues provide structural explanations for the potent neutralization by CD4 mimetic miniproteins with chemical extensions that fit into this cavity.

Protein-based HIV-1 microbicides.

Although the development of a protective vaccine remains the most effective strategy for the global control of HIV/AIDS, another practical form of medical intervention would be a microbicide capable of preventing HIV-1 transmission at the mucosal level. A broad spectrum of antiviral molecules have demonstrated in vitro efficacy in proofof- principle studies, and a selected few have already been tested in pre-clinical and clinical microbicide trials. Nevertheless, major hurdles remain to be overcome and there is still much uncertainty about the choice of inhibitors, formulations and administration vehicles for obtaining a safe and effective microbicide. A special category of HIV-1 microbicides are those based on proteins or peptides that interfere with the earliest steps in the viral infectious cycle. Besides a high degree of target specificity and a limited, if any, systemic absorption, proteinbased microbicides offer the unique advantage of being suitable to in vivo expression by engineered bacteria or viral vectors, which might ensure prolonged protection without the need for planned, intercourse-coordinated application. In this respect, vaginal or rectal microbiota such as Lactobacillus spp. represent ideal expression systems as they would not only produce the inhibitor of choice at the mucosal surface, but also easily blend within the resident microflora and offer additional valuable homeostatic effects. In this article, we review the current state of the art on protein-based microbicides.

Characterization of human immunodeficiency virus type 1 monomeric and trimeric gp120 glycoproteins stabilized in the CD4-bound state: antigenicity, biophysics, and immunogenicity.

The human immunodeficiency virus type 1 exterior gp120 envelope glycoprotein is highly flexible, and this flexibility may contribute to the inability of monomeric gp120 immunogens to elicit broadly neutralizing antibodies. We previously showed that an S375W modification of a critical interfacial cavity central to the primary receptor binding site, the Phe43 cavity, stabilizes gp120 into the CD4-bound state. However, the immunological effects of this cavity-altering replacement were never tested. Subsequently, we screened other mutations that, along with the S375W alteration, might further stabilize the CD4-bound state. Here, we define a selected second cavity-altering replacement, T257S, and analyze the double mutations in several gp120 envelope glycoprotein contexts. The gp120 glycoproteins with the T257S-plus-S375W double mutation (T257S+S375W) have a superior antigenic profile compared to the originally identified single S375W replacement in terms of enhanced recognition by the broadly neutralizing CD4 binding-site antibody b12. Isothermal titration calorimetry measuring the entropy of the gp120 interaction with CD4 indicated that the double mutant was also stabilized into the CD4-bound state, with increasing relative fixation between core, full-length monomeric, and full-length trimeric versions of gp120. A significant increase in gp120 affinity for CD4 was also observed for the cavity-filling mutants relative to wild-type gp120. The most conformationally constrained T257S+S375W trimeric gp120 proteins were selected for immunogenicity analysis in rabbits and displayed a trend of improvement relative to their wild-type counterparts in terms of eliciting neutralizing antibodies. Together, the results suggest that conformational stabilization may improve the ability of gp120 to elicit neutralizing antibodies.

Vaccinia-based reporter gene cell-fusion assays to quantitate functional interactions of HIV envelope glycoprotein with receptors.

This unit describes quantitation of functional interactions between HIV envelope glucoprotein and target cell receptors, using assay of cell fusion-dependent reporter gene activation. The method is particularly useful since it isolates the fusion reaction from the rest of the HIV replication cycle, and obviates the need for infectious HIV particles. Reporter Gene Cell Fusion Assays to Quantitate Functional Interactions of HIV Envelope Glycoprotein with Receptors.

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).