Safety, pharmacokinetics, and biological activity of CD4-mimetic BNM-III-170 in SHIV-infected rhesus macaques

Journal of Virology · 2025-04-07 · 3 citations

ABSTRACT Anti-HIV-1 antibodies capable of mediating ADCC are elicited by the majority of people with HIV-1 and preferentially target the “open,” CD4-bound conformation of HIV-1 envelope glycoproteins (Env). However, due to the “closed” conformation sampled by unliganded HIV-1-Envs, these antibodies are ineffective at eliminating infected cells. BNM-III-170 is a small-molecule CD4-mimetic compound that binds the Phe43 cavity of the gp120 subunit of Env, forcing Env to “open up,” thus exposing epitopes targeted by CD4-induced (CD4i), ADCC-mediating antibodies. Here, we assessed the safety, pharmacokinetics, and biological activity of BNM-III-170 in uninfected and SHIV-AD8-EO-infected rhesus macaques (RMs). In uninfected RMs, single subcutaneous administrations of 3–36 mg/kg BNM-III-170 were well-tolerated, with serum half-lives ranging from 3 to 6 h. In SHIV-infected RMs, four different regimens were evaluated: 2 × 36 mg/kg daily, 1 × 24 mg/kg, 3 × 36 mg/kg every 7 days, and 3 × 36 mg/kg every 3 days. While toxicity was observed with daily doses, all other regimens demonstrated reasonable safety profiles. No changes in plasma viral loads were observed in SHIV-infected RMs following any of the evaluated BNM-III-170 dosing regimens. However, plasma collected following BNM-III-170 administration was shown to have increased binding to infected cells and to sensitize SHIV AD8-EO virions to neutralization by otherwise non-neutralizing antibodies. In addition, the plasma of treated animals mediated ADCC in the presence of BNM-III-170. These results establish a well-tolerated BNM-III-170 dosing regimen in SHIV-infected RMs and serve as proof of concept for its biological activity in promoting the targeting of infected cells by CD4i ADCC-mediating antibodies. Thus, they inform future studies evaluating CD4mc treatment in ART-treated animals. IMPORTANCE A therapeutic regimen able to eradicate or functionally cure HIV-1 remains elusive and may require a “shock-and-kill” approach to reactivate and then purge the latent HIV-1 reservoir. The small-molecule CD4-mimetic compound BNM-III-170 has previously been shown to (i) sensitize HIV-1-infected cells to ADCC mediated by plasma from people with HIV-1 (PWH) in vitro and (ii) significantly delay the time to viral rebound following ART interruption when combined with anti-CoRBS + anti-cluster A Abs or plasma from PWH in humanized mice. To evaluate the use of BNM-III-170 as part of a kill approach, we characterized the safety, pharmacokinetics, and biological activity of BNM-III-170 in uninfected and SHIV-infected RMs. Our study identifies a tolerable BNM-III-170 dosing regimen in SHIV-infected RMs and provides insights into its antiviral activities; as such, it informs future studies evaluating the efficacy of BNM-III-170 in reducing the viral reservoir.

Stoichiometry of HIV-1 envelope glycoprotein protomers with changes that stabilize or destabilize the pretriggered conformation

mBio · 2025-10-24

). CD4 binding induces Env to make transitions from its pretriggered conformation (PTC) to more "open" conformations that are sensitive to inhibition by antibodies, CD4-mimetic compounds (CD4mcs), and exposure to cold. Changes in functional membrane Envs that either stabilize or destabilize the PTC have been identified. Here, we investigate the stoichiometric requirements for the PTC-stabilizing and -destabilizing changes in the Env protomers. To this end, we generated viruses bearing Envs with mixed protomers exhibiting different degrees of PTC stability and determined the sensitivity of the viruses to cold (0°C) and, in some cases, to a CD4mc. The number of stabilized Env protomers required to achieve stabilization of the PTC was inversely related to the degree of PTC stabilization that resulted from the introduced Env change. For strongly stabilizing Env changes, modification of a single protomer was sufficient to achieve PTC stabilization; apparently, with adequate stability, the modified protomer can constrain the conformation of the other two protomers to maintain the PTC. Weakly stabilizing Env changes needed to be present in all three protomers to achieve efficient stabilization of the PTC. In many cases, the PTC was disrupted when destabilizing changes were present in only a single protomer. These complementary results suggest that conformational symmetry among the protomers of the functional Env trimer is conducive to the integrity of the PTC.IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer consists of three protomers. In response to receptor binding, the flexible Env changes its conformation to mediate virus entry into host cells. The shape-shifting ability of Env also contributes to HIV-1's capacity to evade the host immune system. The pretriggered (state 1) conformation (PTC) of Env is an important target for virus entry inhibitors and host antibodies but is unstable and therefore incompletely characterized. Changes in Env amino acids that either stabilize or destabilize the PTC have been identified. Here, we define how many Env protomers need to be modified by these changes to achieve stabilization or destabilization of the PTC. These results can guide the placement of changes in the HIV-1 Env protomers to control the movement of the Env trimer from the PTC, allowing better characterization of this elusive conformation and testing of its utility in vaccines.

The asymmetric opening of HIV-1 Env by a potent CD4 mimetic enables anti-coreceptor binding site antibodies to mediate ADCC

Nature Communications · 2025-12-01 · 4 citations

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. When administrated along with the anti-CoRBS 17b, CJF-III-288 delayed viral rebound after ART interruption in HIV-1-infected humanized mice, demonstrating potential for eliciting ADCC in vivo. Structural and conformational analyses reveal that CJF-III-288, in combination with this anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation. This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

The membrane-proximal external region of human immunodeficiency virus (HIV-1) envelope glycoprotein trimers in A18-lipid nanodiscs

Communications Biology · 2025-03-15 · 3 citations

Abstract During human immunodeficiency virus (HIV-1) entry, the metastable pretriggered envelope glycoprotein (Env) trimer ((gp120/gp41) 3 ) opens asymmetrically. We present cryo-EM structures of cleaved asymmetric Env trimers in amphipol-lipid nanodiscs. The gp41 membrane-proximal external region (MPER) could be traced in Env protomers that remained close to the nanodisc despite Env tilting. The MPER interacts with the gp120 C-termini and gp41 α9 helices at the base of the Env trimer. MPER conformation is coupled with the tilt angles of the α9 helices, the helicity of the gp41 heptad repeat (HR1 N ) regions, and the opening angles between the protomers of the asymmetric trimers. Our structural models explain the stabilizing effects of MPER integrity and Env proteolytic maturation on the pretriggered Env conformation. Superimposed on the asymmetry of the Env protomers, variation in the glycans at the trimer apex creates substantial structural heterogeneity in the V2 quaternary epitopes of difficult-to-elicit broadly neutralizing antibodies.

Characterization of full-length and cytoplasmic tail-truncated envelope glycoproteins incorporated into human immunodeficiency virus (HIV-1) virions and virus-like particles

Journal of Virology · 2025-11-26 · 1 citations

]. Env cleavage stabilizes the pretriggered Env conformation (PTC), the major target for broadly neutralizing antibodies. Although the mature Env is relatively enriched in virions and virus-like particles (VLPs), conformationally flexible uncleaved Envs typically contaminate preparations of these particles. In non-permissive cells, the long ~149-residue gp41 cytoplasmic tail (CT) is necessary for Env incorporation into virions. In a minority of HIV-1 strains, the gp41 CT is clipped in virions by the viral protease. Here, we compare Envs with CT truncations and CT alterations that increase or decrease protease clipping in permissive cells. Changes in protease clipping affected amphotericin B sensitivity but did not alter other viral phenotypes. By contrast, a corresponding CT truncation (L748STOP) increased cell-surface and virion Env levels, cell-cell fusion, and virus infectivity and cytotoxicity. Notably, in diverse HIV-1 strains, the ratio of cleaved/uncleaved Envs in preparations of virions and extracellular vesicles was increased by this CT truncation. ESCRT and ALIX-binding region (EABR) vesicles incorporated significantly more uncleaved CT-truncated Env than HIV-1 VLPs. Env CT deletion/truncation did not qualitatively alter the viral neutralization profile; however, increased antibody concentrations were required to neutralize viruses with the higher levels of cleaved Env that resulted from CT truncation. Specific CT truncations provide a means of enriching the PTC and limiting the incorporation of nonfunctional and conformationally heterogeneous uncleaved Envs into preparations of virions and VLPs. IMPORTANCE: The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates entry of the virus into host cells. The pretriggered conformation (PTC) of Env is the major target for protective broadly neutralizing antibodies, but the PTC is unstable and therefore difficult to study. The cleavage of the flexible Env precursor stabilizes the PTC. Therefore, the presence of uncleaved Env compromises the purity of the PTC in Env preparations. We found that certain truncations of the Env cytoplasmic tail resulted in improved ratios of cleaved:uncleaved Env in preparations of HIV-1 viruses or virus-like particles. In some contexts, cytoplasmic tail truncation increased the level of Env in virus preparations. Although higher concentrations of antibodies were required to neutralize these viruses, Envs with specific truncations of the cytoplasmic tail retained the PTC. Thus, cytoplasmic tail truncation could assist efforts to purify and characterize the Env PTC on the viral membrane.

Characterization of full-length and cytoplasmic tail-truncated envelope glycoproteins incorporated into human immunodeficiency virus (HIV-1) virions and virus-like particles

bioRxiv (Cold Spring Harbor Laboratory) · 2025-07-10 · 2 citations

ABSTRACT During transport to the surface of infected cells, the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is cleaved to produce the mature functional Env trimer ((gp120/gp41) 3 ). Env cleavage stabilizes the pretriggered Env conformation (PTC), the major target for broadly neutralizing antibodies. Although the mature Env is relatively enriched in virions and virus-like particles (VLPs), conformationally flexible uncleaved Envs typically contaminate preparations of these particles. In non-permissive cells, the long ∼149-residue gp41 cytoplasmic tail (CT) is necessary for Env incorporation into virions. In a minority of HIV-1 strains, the gp41 CT is clipped in virions by the viral protease. Here, we compare Envs with CT truncations and CT alterations that increase or decrease protease clipping in permissive cells. Changes in protease clipping affected amphotericin B sensitivity, but did not alter other viral phenotypes. By contrast, a corresponding CT truncation (L748STOP) increased cell-surface and virion Env levels, cell-cell fusion, and virus infectivity and cytotoxicity. Notably, in diverse HIV-1 strains, the ratio of cleaved/uncleaved Envs in preparations of virions and extracellular vesicles was increased by this CT truncation. ESCRT and ALIX-binding region (EABR) vesicles incorporated significantly more uncleaved CT-truncated Env than HIV-1 VLPs. Env CT deletion/truncation did not qualitatively alter the viral neutralization profile; however, increased antibody concentrations were required to neutralize viruses with the higher levels of cleaved Env that resulted from CT truncation. Specific CT truncations provide a means of enriching the PTC and limiting the incorporation of nonfunctional and conformationally heterogeneous uncleaved Envs into preparations of virions and VLPs. IMPORTANCE The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates entry of the virus into host cells. The pretriggered conformation (PTC) of Env is the major target for protective broadly neutralizing antibodies, but the PTC is unstable and therefore difficult to study. The cleavage of the flexible Env precursor stabilizes the PTC. Therefore, the presence of uncleaved Env compromises the purity of the PTC in Env preparations. We found that certain truncations of the Env cytoplasmic tail resulted in improved ratios of cleaved:uncleaved Env in preparations of HIV-1 viruses or virus-like particles. In some contexts, cytoplasmic tail truncation increased the level of Env in virus preparations. Although higher concentrations of antibodies were required to neutralize these viruses, Envs with specific truncations of the cytoplasmic tail retained the PTC. Thus, cytoplasmic tail truncation could assist efforts to purify and characterize the Env PTC on the viral membrane.

Inhibition of human immunodeficiency virus (HIV-1) infectivity by expression of poorly or broadly neutralizing antibodies against Env in virus-producing cells

Journal of Virology · 2024-01-30 · 1 citations

The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein precursor (gp160) trimerizes, is modified by high-mannose glycans in the endoplasmic reticulum, and is transported via Golgi and non-Golgi secretory pathways to the infected cell surface. In the Golgi, gp160 is partially modified by complex carbohydrates and proteolytically cleaved to produce the mature functional Env trimer, which is preferentially incorporated into virions. Broadly neutralizing antibodies (bNAbs) generally recognize the cleaved Env trimer, whereas poorly neutralizing antibodies (pNAbs) bind the conformationally flexible gp160. We found that expression of bNAbs, pNAbs, or soluble/membrane forms of the receptor, CD4, in cells producing HIV-1 all decreased viral infectivity. Four patterns of co-expressed ligand:Env were observed: (i) ligands (CD4, soluble CD4-Ig, and some pNAbs) that specifically recognize the CD4-bound Env conformation resulted in uncleaved Envs lacking complex glycans that were not incorporated into virions; (ii) other pNAbs produced Envs with some complex carbohydrates and severe defects in cleavage, which were relieved by brefeldin A treatment; (iii) bNAbs that recognize gp160 as well as mature Envs resulted in Envs with some complex carbohydrates and moderate decreases in virion Env cleavage; and (iv) bNAbs that preferentially recognize mature Envs produced cleaved Envs with complex glycans in cells and on virions. The low infectivity observed upon co-expression of pNAbs or CD4 could be explained by disruption of Env trafficking, reducing the level of Env and/or increasing the fraction of uncleaved Env on virions. In addition to bNAb effects on virion Env cleavage, the secreted bNAbs neutralized the co-expressed viruses.IMPORTANCEThe Env trimers on the HIV-1 mediate virus entry into host cells. Env is synthesized in infected cells, modified by complex sugars, and cleaved to form a mature, functional Env, which is incorporated into virus particles. Env elicits antibodies in infected individuals, some of which can neutralize the virus. We found that antibodies co-expressed in the virus-producing cell can disrupt Env transit to the proper compartment for cleavage and sugar modification and, in some cases, block incorporation into viruses. These studies provide insights into the processes by which Env becomes functional in the virus-producing cell and may assist attempts to interfere with these events to inhibit HIV-1 infection.

Structure and Function of Human Pathogenic Retroviruses

2024-10-29

The human retroviruses represent an emerging class of complex pathogens involved in a wide variety of maladies including leukemias and lymphomas, diseases of the central nervous system, and immune function impairment. Four different types of human retroviruses have been isolated: HTLV-I, the etiological agent of a malignant T-cell leukemia/lymphoma (ATLL); HTLV-II, a virus associated with a more benign form of T-cell leukemia; HIV-1 (previously called HTLV-III or LAV-1), the etiological agent of acquired immune deficiency syndrome and related disorders; and HIV-2 (also called LAV-2), a virus that has recently been isolated from people living in West Africa. Salient features of the pathogenesis and structure of these viruses are summarized in this chapter.

Inducible cell lines producing replication-defective human immunodeficiency virus particles containing envelope glycoproteins stabilized in a pretriggered conformation

Journal of Virology · 2024-11-07 · 2 citations

ABSTRACT During the process by which human immunodeficiency virus (HIV-1) enters cells, the envelope glycoprotein (Env) trimer on the virion surface engages host cell receptors. Binding to the receptor CD4 induces Env to undergo transitions from a pretriggered, “closed” (State-1) conformation to more “open” (State 2/3) conformations. Most broadly neutralizing antibodies (bNAbs), which are difficult to elicit, recognize the pretriggered (State-1) conformation. More open Env conformations are recognized by poorly neutralizing antibodies (pNAbs), which are readily elicited during natural infection and vaccination with current Env immunogens. Env heterogeneity likely contributes to HIV-1 persistence by skewing antibody responses away from the pretriggered conformation. The conformationally flexible gp160 Env precursor on the infected cell or virion surface potentially presents multiple pNAb epitopes to the host immune system. Although proteolytic cleavage to produce the functional, mature Env trimer [(gp120/gp41) 3 ] stabilizes State-1, many primary HIV-1 Envs spontaneously sample more open conformations. Here, we establish inducible cell lines that produce replication-defective HIV-1 particles with Env trimers stabilized in a pretriggered conformation. The mature Env is enriched on virus-like particles (VLPs). Using complementary approaches, we estimate an average of 25–50 Env trimers on each VLP. The stabilizing changes in Env limit the natural conformational heterogeneity of the VLP Env trimers, allowing recognition by bNAbs but not pNAbs. These defective VLPs provide a more homogeneous source of pretriggered Env trimers in a native membrane environment. Thus, these VLPs may facilitate the characterization of this functionally important Env conformation and its interaction with the immune system. IMPORTANCE A major impediment to the development of an effective HIV/AIDS vaccine is the inefficiency with which human immunodeficiency virus (HIV-1) envelope glycoproteins elicit antibodies that neutralize multiple virus strains. Neutralizing antibodies recognize a particular shape of the envelope glycoproteins that resides on the viral membrane before the virus engages the host cell. Here, we report the creation of stable cell lines that inducibly produce non-infectious HIV-like particles. The normally flexible envelope glycoprotein spikes on these virus-like particles have been stabilized in a conformation that is recognized by broadly neutralizing antibodies. These virus-like particles allow the study of the envelope glycoprotein conformation, its modification by sugars, and its ability to elicit desired neutralizing antibodies.

Conformations of membrane human immunodeficiency virus (HIV-1) envelope glycoproteins solubilized in Amphipol A18 lipid-nanodiscs

Journal of Virology · 2024-09-09 · 6 citations

Upon binding to the host cell receptor, CD4, the pretriggered (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer undergoes transitions to downstream conformations important for virus entry. State 1 is targeted by most broadly neutralizing antibodies (bNAbs), whereas downstream conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. Extraction of Env from the membranes of viruses or Env-expressing cells disrupts the metastable State-1 Env conformation, even when detergent-free approaches like styrene-maleic acid lipid nanoparticles (SMALPs) are used. Here, we combine three strategies to solubilize and purify mature membrane Envs that are antigenically native (i.e., recognized by bNAbs and not pNAbs): (1) solubilization of Env with a novel amphipathic copolymer, Amphipol A18; (2) use of stabilized pretriggered Env mutants; and (3) addition of the State-1-stabilizing entry inhibitor, BMS-806. Amphipol A18 was superior to the other amphipathic copolymers tested (SMA and AASTY 11-50) for preserving a native Env conformation. A native antigenic profile of A18 Env-lipid-nanodiscs was maintained for at least 7 days at 4°C and 2 days at 37°C in the presence of BMS-806 and was also maintained for at least 1 h at 37°C in a variety of adjuvants. The damaging effects of a single cycle of freeze-thawing on the antigenic profile of the A18 Env-lipid-nanodiscs could be prevented by the addition of 10% sucrose or 10% glycerol. These results underscore the importance of the membrane environment to the maintenance of a pretriggered (State-1) Env conformation and provide strategies for the preparation of lipid-nanodiscs containing native membrane Envs.IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins like Env rely on purification procedures that maintain their natural conformation. In this study, we show that an amphipathic copolymer A18 can directly extract HIV-1 Env from a membrane without the use of detergents. A18 promotes the formation of nanodiscs that contain Env and membrane lipids. Env in A18-lipid nanodiscs largely preserves features recognized by broadly neutralizing antibodies (bNAbs) and conceals features potentially recognized by poorly neutralizing antibodies (pNAbs). 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 for future studies of HIV-1 Env structure, interaction with receptors and antibodies, and immunogenicity.