Membrane HIV-1 envelope glycoproteins stabilized more strongly in a pretriggered conformation than natural virus Envs.

The pretriggered conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ((gp120/gp41)3) is targeted by virus entry inhibitors and broadly neutralizing antibodies (bNAbs). The lability of pretriggered Env has hindered its characterization. Here, we produce membrane Env variants progressively stabilized in pretriggered conformations, in some cases to a degree beyond that found in natural HIV-1 strains. Pretriggered Env stability correlated with stronger trimer subunit association, increased virus sensitivity to bNAb neutralization, and decreased capacity to mediate cell-cell fusion and virus entry. For some highly stabilized Env mutants, after virus-host cell engagement, the normally inaccessible gp120 V3 region on an Env intermediate became targetable by otherwise poorly neutralizing antibodies. Thus, evolutionary pressure on HIV-1 Env to maintain trimer integrity, responsiveness to the CD4 receptor, and resistance to antibodies modulates pretriggered Env stability. The strongly stabilized pretriggered membrane Envs reported here will facilitate further characterization of this functionally important conformation.

Bacterial Spermosphere Inoculants Alter N. benthamiana-Plant Physiology and Host Bacterial Microbiome.

In this study, we investigated the interplay between the spermosphere inoculum, host plant physiology, and endophytic compartment (EC) microbial community. Using 16S ribosomal RNA gene sequencing of root, stem, and leaf endophytic compartment communities, we established a baseline microbiome for Nicotiana sp. Phenotypic differences were observed due to the addition of some bacterial inoculants, correlated with endogenous auxin loads using transgenic plants expressing the auxin reporter pB-GFP::P87. When applied as spermosphere inoculants, select bacteria were found to create reproducible variation within the root EC microbiome and, more systematically, the host plant physiology. Our findings support the assertion that the spermosphere of plants is a zone that can influence the EC microbiome when applied in a greenhouse setting.

Three families of CD4-induced antibodies are associated with the capacity of plasma from people living with HIV to mediate ADCC in presence of CD4-mimetics.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation which occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in presence of CD4mc.

The combination of three CD4-induced antibodies targeting highly conserved Env regions with a small CD4-mimetic achieves potent ADCC activity.

The majority of naturally-elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs), because they are unable to recognize the Env timer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by Antibody-Dependent Cellular Cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly-neutralizing antibodies and even showed activity against HIV-1 infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies.

Discovery and Characterization of Fluopipamine, a Putative Cellulose Synthase 1 Antagonist within Arabidopsis.

Herbicide-resistant weeds are increasingly a problem in crop fields when exposed to similar chemistry over time. To avoid future yield losses, identifying herbicidal chemistry needs to be accelerated. We screened 50,000 small molecules using a liquid-handling robot and light microscopy focusing on pre-emergent herbicides in the family of cellulose biosynthesis inhibitors. Through phenotypic, chemical, genetic, and in silico methods we uncovered 6-{[4-(2-fluorophenyl)-1-piperazinyl]methyl}-N-(2-methoxy-5-methylphenyl)-1,3,5-triazine-2,4-diamine (fluopipamine). Symptomologies support fluopipamine as a putative antagonist of cellulose synthase enzyme 1 (CESA1) from Arabidopsis (Arabidopsis thaliana). Ectopic lignification, inhibition of etiolation, phenotypes including loss of anisotropic cellular expansion, swollen roots, and live cell imaging link fluopipamine to cellulose biosynthesis inhibition. Radiolabeled glucose incorporation of cellulose decreased in short-duration experiments when seedlings were incubated in fluopipamine. To elucidate the mechanism, ethylmethanesulfonate mutagenized M2 seedlings were screened for fluopipamine resistance. Two loci of genetic resistance were linked to CESA1. In silico docking of fluopipamine, quinoxyphen, and flupoxam against various CESA1 mutations suggests that an alternative binding site at the interface between CESA proteins is necessary to preserve cellulose polymerization in compound presence. These data uncovered potential fundamental mechanisms of cellulose biosynthesis in plants along with feasible leads for herbicidal uses.

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins.

IMPORTANCE: CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

Alterations in gp120 glycans or the gp41 fusion peptide-proximal region modulate the stability of the human immunodeficiency virus (HIV-1) envelope glycoprotein pretriggered conformation.

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates entry into host cells by binding receptors, CD4 and CCR5/CXCR4, and fusing the viral and cell membranes. In infected cells, cleavage of the gp160 Env precursor yields the mature Env trimer, with gp120 exterior and gp41 transmembrane Env subunits. Env cleavage stabilizes the State-1 conformation, which is the major target for broadly neutralizing antibodies, and decreases the spontaneous sampling of more open Env conformations that expose epitopes for poorly neutralizing antibodies. During HIV-1 entry into cells, CD4 binding drives the metastable Env from a pretriggered (State-1) conformation into more "open," lower-energy states. Here, we report that changes in two dissimilar elements of the HIV-1 Env trimer, namely particular gp120 glycans and the gp41 fusion peptide-proximal region (FPPR), can independently modulate the stability of State 1. Individual deletion of several gp120 glycans destabilized State 1, whereas removal of a V1 glycan resulted in phenotypes indicative of a more stable pretriggered Env conformation. Likewise, some alterations of the gp41 FPPR decreased the level of spontaneous shedding of gp120 from the Env trimer and stabilized the pretriggered State-1 Env conformation. State-1-stabilizing changes were additive and could suppress the phenotypes associated with State-1-destabilizing alterations in Env. Our results support a model in which multiple protein and carbohydrate elements of the HIV-1 Env trimer additively contribute to the stability of the pretriggered (State-1) conformation. The Env modifications identified in this study will assist efforts to characterize the structure and immunogenicity of the metastable State-1 conformation. IMPORTANCE The elicitation of antibodies that neutralize multiple strains of HIV-1 is an elusive goal that has frustrated the development of an effective vaccine. The pretriggered shape of the HIV-1 envelope glycoprotein (Env) spike on the virus surface is the major target for such broadly neutralizing antibodies. The "closed" pretriggered Env shape resists the binding of most antibodies but is unstable and often assumes "open" shapes that elicit ineffective antibodies. We identified particular changes in both the protein and the sugar components of the Env trimer that stabilize the pretriggered shape. Combinations of these changes were even more effective at stabilizing the pretriggered Env than the individual changes. Stabilizing changes in Env could counteract the effect of Env changes that destabilize the pretriggered shape. Locking Env in its pretriggered shape will assist efforts to understand the Env spike on the virus and to incorporate this shape into vaccines.

Soluble CD4 and low molecular weight CD4-mimetic compounds sensitize cells to be killed by anti-HIV cytotoxic immunoconjugates.

IMPORTANCE: HIV infection can be effectively treated to prevent the development of AIDS, but it cannot be cured. We have attached poisons to anti-HIV antibodies to kill the infected cells that persist even after years of effective antiviral therapy. Here we show that the killing of infected cells can be markedly enhanced by the addition of soluble forms of the HIV receptor CD4 or by mimics of CD4.

Total Synthesis of the Reported Structure of Neaumycin B.

The stereoselective total synthesis of structure 1 assigned to the macrolide natural product neaumycin B is reported in a 2.3% overall yield on 90 mg scale. The synthesis features a gram-scale nickel-catalyzed reductive cross-coupling/spiroketalization tactic to construct the spiroketal core of neaumycin B. The stereostructures of the C3-C6, C8-C14, and C20-C41 segments of synthetic neaumycin B were unambiguously verified by X-ray crystallography.

Piperidine CD4-Mimetic Compounds Expose Vulnerable Env Epitopes Sensitizing HIV-1-Infected Cells to ADCC.

The ability of the HIV-1 accessory proteins Nef and Vpu to decrease CD4 levels contributes to the protection of infected cells from antibody-dependent cellular cytotoxicity (ADCC) by preventing the exposure of Env vulnerable epitopes. Small-molecule CD4 mimetics (CD4mc) based on the indane and piperidine scaffolds such as (+)-BNM-III-170 and (S)-MCG-IV-210 sensitize HIV-1-infected cells to ADCC by exposing CD4-induced (CD4i) epitopes recognized by non-neutralizing antibodies that are abundantly present in plasma from people living with HIV. Here, we characterize a new family of CD4mc, (S)-MCG-IV-210 derivatives, based on the piperidine scaffold which engages the gp120 within the Phe43 cavity by targeting the highly conserved Asp368 Env residue. We utilized structure-based approaches and developed a series of piperidine analogs with improved activity to inhibit the infection of difficult-to-neutralize tier-2 viruses and sensitize infected cells to ADCC mediated by HIV+ plasma. Moreover, the new analogs formed an H-bond with the α-carboxylic acid group of Asp368, opening a new avenue to enlarge the breadth of this family of anti-Env small molecules. Overall, the new structural and biological attributes of these molecules make them good candidates for strategies aimed at the elimination of HIV-1-infected cells.

Conformations of Human Immunodeficiency Virus Envelope Glycoproteins in Detergents and Styrene-Maleic Acid Lipid Particles.

The mature human immunodeficiency virus (HIV) envelope glycoprotein (Env) trimer, which consists of noncovalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanoparticles (styrene-maleic acid lipid particles [SMALPs]). The integrity of the Env trimer in SMALPs was maintained at both 4°C and room temperature. In contrast, Envs solubilized in Cymal-5, a nonionic detergent, were unstable at room temperature, although their stability was improved at 4°C and/or after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4°C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region, consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA-solubilized Envs was remarkably similar, both in the absence and in the presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4°C alters the pretriggered membrane Env conformation in consistent ways, suggesting that Env assumes default conformations when its association with the membrane is disrupted. IMPORTANCE The human immunodeficiency virus (HIV) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.

Pharmacophore Variants of the Macrocyclic Peptide Triazole Inactivator of HIV-1 Env.

Previously we established a family of macrocyclic peptide triazoles (cPTs) that inactivate the Env protein complex of HIV-1, and identified the pharmacophore that engages Env's receptor binding pocket. Here, we examined the hypothesis that the side chains of both components of the triazole Pro - Trp segment of cPT pharmacophore work in tandem to make intimate contacts with two proximal subsites of the overall CD4 binding site of gp120 to stabilize binding and function. Variations of the triazole Pro R group, which previously had been significantly optimized, led to identification of a variant MG-II-20 that contains a pyrazole substitution. MG-II-20 has improved functional properties over previously examined variants, with Kd for gp120 in the nM range. In contrast, new variants of the Trp indole side chain, with either methyl- or bromo- components appended, had disruptive effects on gp120 binding, reflecting the sensitivity of function to changes in this component of the encounter complex. Plausible in silico models of cPT:gp120 complex structures were obtained that are consistent with the overall hypothesisof occupancy by the triazole Pro and Trp side chains, respectively, into the ß20/21 and Phe43 sub-cavities. The overall results strengthen the definition of the cPT-Env inactivator binding site and provide a new lead composition (MG-II-20) as well as structure-function findings to guide future HIV-1 Env inactivator design.

Piperidine CD4-mimetic compounds expose vulnerable Env epitopes sensitizing HIV-1-infected cells to ADCC.

The ability of HIV-1 accessory proteins Nef and Vpu to decrease CD4 levels contributes to the protection of infected cells from antibody-dependent cellular cytotoxicity (ADCC) by preventing the exposure of Env vulnerable epitopes. Small-molecule CD4 mimetics (CD4mc) based on the indane and piperidine scaffolds such as (+)-BNM-III-170 and ( S )-MCG-IV-210 sensitize HIV-1 infected cells to ADCC by exposing CD4-induced (CD4i) epitopes recognized by non-neutralizing antibodies abundantly present in plasma from people living with HIV. Here, we characterize a new family of CD4mc, ( S )-MCG-IV-210 derivatives, based on the piperidine scaffold which engage the gp120 within the Phe43 cavity by targeting the highly-conserved Asp 368 Env residue. We utilized structure-based approaches and developed a series of piperidine analogs with improved activity to inhibit infection of difficult-to-neutralize tier-2 viruses and sensitize infected cells to ADCC mediated by HIV+ plasma. Moreover, the new analogs formed an H-bond with the α-carboxylic acid group of Asp 368 , opening a new avenue to enlarge the breadth of this family of anti-Env small molecules. Overall, the new structural and biological attributes of these molecules make them good candidates for strategies aimed at the elimination HIV-1-infected cells.

Indoline CD4-mimetic compounds mediate potent and broad HIV-1 inhibition and sensitization to antibody-dependent cellular cytotoxicity.

Binding to the host cell receptors, CD4 and CCR5/CXCR4, triggers large-scale conformational changes in the HIV-1 envelope glycoprotein (Env) trimer [(gp120/gp41)3] that promote virus entry into the cell. CD4-mimetic compounds (CD4mcs) comprise small organic molecules that bind in the highly conserved CD4-binding site of gp120 and prematurely induce inactivating Env conformational changes, including shedding of gp120 from the Env trimer. By inducing more "open," antibody-susceptible Env conformations, CD4mcs also sensitize HIV-1 virions to neutralization by antibodies and infected cells to antibody-dependent cellular cytotoxicity (ADCC). Here, we report the design, synthesis, and evaluation of novel CD4mcs based on an indoline scaffold. Compared with our current lead indane scaffold CD4mc, BNM-III-170, several indoline CD4mcs exhibit increased potency and breadth against HIV-1 variants from different geographic clades. Viruses that were selected for resistance to the lead indane CD4mc, BNM-III-170, are susceptible to inhibition by the indoline CD4mcs. The indoline CD4mcs also potently sensitize HIV-1-infected cells to ADCC mediated by plasma from HIV-1-infected individuals. Crystal structures indicate that the indoline CD4mcs gain potency compared to the indane CD4mcs through more favorable π-π overlap from the indoline pose and by making favorable contacts with the vestibule of the CD4-binding pocket on gp120. The rational design of indoline CD4mcs thus holds promise for further improvements in antiviral activity, potentially contributing to efforts to treat and prevent HIV-1 infection.

Temsavir blocks the immunomodulatory activities of HIV-1 soluble gp120.

While HIV-1-mediated CD4 downregulation protects infected cells from antibody-dependent cellular cytotoxicity (ADCC), shed gp120 binds to CD4 on uninfected bystander CD4+ T cells, sensitizing them to ADCC mediated by HIV+ plasma. Soluble gp120-CD4 interaction on multiple immune cells also triggers a cytokine burst. The small molecule temsavir acts as an HIV-1 attachment inhibitor by preventing envelope glycoprotein (Env)-CD4 interaction and alters the overall antigenicity of Env by affecting its processing and glycosylation. Here we show that temsavir also blocks the immunomodulatory activities of shed gp120. Temsavir prevents shed gp120 from interacting with uninfected bystander CD4+ cells, protecting them from ADCC responses and preventing a cytokine burst. Mechanistically, this depends on temsavir's capacity to prevent soluble gp120-CD4 interaction, to reduce gp120 shedding, and to alter gp120 antigenicity. This suggests that the clinical benefits provided by temsavir could extend beyond blocking viral entry.

Small CD4 mimetics sensitize HIV-1-infected macrophages to antibody-dependent cellular cytotoxicity.

HIV-1 envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to antibody-dependent cellular cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu, and Env. Limited data exist, however, of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu, and Env are all required to efficiently downregulate CD4 on infected CD4+ T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared with CD4+ T cells. However, treatment of infected macrophages with small CD4 mimetics exposes vulnerable CD4-induced Env epitopes and sensitizes them to ADCC.

Structural and Functional Characterization of Indane-Core CD4-Mimetic Compounds Substituted with Heterocyclic Amines.

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer on the virion surface interacts with the host receptors, CD4 and CCR5/CXCR4, to mediate virus entry into the target cell. CD4-mimetic compounds (CD4mcs) bind the gp120 Env, block CD4 binding, and inactivate Env. Previous studies suggested that a C(5)-methylamino methyl moiety on a lead CD4mc, BNM-III-170, contributed to its antiviral potency. By replacing the C(5) chain with differentially substituted pyrrolidine, piperidine, and piperazine ring systems, guided by structural and computational analyses, we found that the 5-position of BNM-III-170 is remarkably tolerant of a variety of ring sizes and substitutions, both in regard to antiviral activity and sensitization to humoral responses. Crystallographic analyses of representative analogues from the pyrrolidine series revealed the potential for 5-substituents to hydrogen bond with gp120 Env residue Thr 283. Further optimization of these interactions holds promise for the development of CD4mcs with greater potency.

Microtubule-Stabilizing 1,2,4-Triazolo[1,5-a]pyrimidines as Candidate Therapeutics for Neurodegenerative Disease: Matched Molecular Pair Analyses and Computational Studies Reveal New Structure-Activity Insights.

Microtubule (MT)-stabilizing 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) hold promise as candidate therapeutics for Alzheimer's disease (AD) and other neurodegenerative conditions. However, depending on the choice of substituents around the TPD core, these compounds can elicit markedly different cellular phenotypes that likely arise from the interaction of TPD congeners with either one or two spatially distinct binding sites within tubulin heterodimers (i.e., the seventh site and the vinca site). In the present study, we report the design, synthesis, and evaluation of a series of new TPD congeners, as well as matched molecular pair analyses and computational studies, that further elucidate the structure-activity relationships of MT-active TPDs. These studies led to the identification of novel MT-normalizing TPD candidates that exhibit favorable ADME-PK, including brain penetration and oral bioavailability, as well as brain pharmacodynamic activity.

Characterization of a Novel CD4 Mimetic Compound YIR-821 against HIV-1 Clinical Isolates.

Small CD4-mimetic compound (CD4mc), which inhibits the interaction between gp120 with CD4, acts as an entry inhibitor and induces structural changes in the HIV-1 envelope glycoprotein trimer (Env) through its insertion within the Phe43 cavity of gp120. We recently developed YIR-821, a novel CD4mc, that has potent antiviral activity and lower toxicity than the prototype NBD-556. To assess the possibility of clinical application of YIR-821, we tested its antiviral activity using a panel of HIV-1 pseudoviruses from different subtypes. YIR-821 displayed entry inhibitor activity against 53.5% (21/40) of the pseudoviruses tested and enhanced neutralization mediated by coreceptor binding site (CoRBS) antibodies in 50% (16/32) of these. Furthermore, when we assessed the antiviral effects using a panel of pseudoviruses and autologous plasma IgG, enhancement of antibody-mediated neutralization activity was observed for 48% (15/31) of subtype B strains and 51% (28/55) of non-B strains. The direct antiviral activity of YIR-821 as an entry inhibitor was observed in 53% of both subtype B (27/51) and non-B subtype (40/75) pseudoviruses. Enhancement of antibody-dependent cellular cytotoxicity was also observed with YIR-821 for all six selected clinical isolates, as well as for the transmitted/founder (T/F) CH58 virus-infected cells. The sequence diversity in the CD4 binding site as well as other regions, such as the gp120 inner domain layers or gp41, may be involved in the multiple mechanisms related to the sensitive/resistant phenotype of the virus to YIR-821. Our findings may facilitate the clinical application of YIR-821. IMPORTANCE Small CD4-mimetic compound (CD4mc) interacts with the Phe43 cavity and triggers conformational changes, enhancing antibody-mediated neutralization and antibody-dependent cellular cytotoxicity (ADCC). Here, we evaluated the effect of YIR-821, a novel CD4mc, against clinical isolates, including both subtype B and non-B subtype viruses. Our results confirm the desirable properties of YIR-821, which include entry inhibition, enhancement of IgG-neutralization, binding, and ADCC, in addition to low toxicity and long half-life in a rhesus macaque model, that might facilitate the clinical application of this novel CD4mc. Our observation of primary viruses that are resistant to YIR-821 suggests that further development of CD4mcs with different structural properties is required.