Irreversible Inactivation of SARS-CoV-2 by Lectin Engagement with Two Glycan Clusters on the Spike Protein.

Host cell infection by SARS-CoV-2, similar to that by HIV-1, is driven by a conformationally metastable and highly glycosylated surface entry protein complex, and infection by these viruses has been shown to be inhibited by the mannose-specific lectins cyanovirin-N (CV-N) and griffithsin (GRFT). We discovered in this study that CV-N not only inhibits SARS-CoV-2 infection but also leads to irreversibly inactivated pseudovirus particles. The irreversibility effect was revealed by the observation that pseudoviruses first treated with CV-N and then washed to remove all soluble lectin did not recover infectivity. The infection inhibition of SARS-CoV-2 pseudovirus mutants with single-site glycan mutations in spike suggested that two glycan clusters in S1 are important for both CV-N and GRFT inhibition: one cluster associated with the RBD (receptor binding domain) and the second with the S1/S2 cleavage site. We observed lectin antiviral effects with several SARS-CoV-2 pseudovirus variants, including the recently emerged omicron, as well as a fully infectious coronavirus, therein reflecting the breadth of lectin antiviral function and the potential for pan-coronavirus inactivation. Mechanistically, observations made in this work indicate that multivalent lectin interaction with S1 glycans is likely a driver of the lectin infection inhibition and irreversible inactivation effect and suggest the possibility that lectin inactivation is caused by an irreversible conformational effect on spike. Overall, lectins' irreversible inactivation of SARS-CoV-2, taken with their breadth of function, reflects the therapeutic potential of multivalent lectins targeting the vulnerable metastable spike before host cell encounter.

Identification of a glycan cluster in gp120 essential for irreversible HIV-1 lytic inactivation by a lectin-based recombinantly engineered protein conjugate.

We previously discovered a class of recombinant lectin conjugates, denoted lectin DLIs ('dual-acting lytic inhibitors') that bind to the HIV-1 envelope (Env) protein trimer and cause both lytic inactivation of HIV-1 virions and cytotoxicity of Env-expressing cells. To facilitate mechanistic investigation of DLI function, we derived the simplified prototype microvirin (MVN)-DLI, containing an MVN domain that binds high-mannose glycans in Env, connected to a DKWASLWNW sequence (denoted 'Trp3') derived from the membrane-associated region of gp41. The relatively much stronger affinity of the lectin component than Trp3 argues that the lectin functions to capture Env to enable Trp3 engagement and consequent Env membrane disruption and virolysis. The relatively simplified engagement pattern of MVN with Env opened up the opportunity, pursued here, to use recombinant glycan knockout gp120 variants to identify the precise Env binding site for MVN that drives DLI engagement and lysis. Using mutagenesis combined with a series of biophysical and virological experiments, we identified a restricted set of residues, N262, N332 and N448, all localized in a cluster on the outer domain of gp120, as the essential epitope for MVN binding. By generating these mutations in the corresponding HIV-1 virus, we established that the engagement of this glycan cluster with the lectin domain of MVN*-DLI is the trigger for DLI-derived virus and cell inactivation. Beyond defining the initial encounter step for lytic inactivation, this study provides a guide to further elucidate DLI mechanism, including the stoichiometry of Env trimer required for function, and downstream DLI optimization.

Pharmacokinetic stability of macrocyclic peptide triazole HIV-1 inactivators alone and in liposomes.

Previously, we reported the discovery of macrocyclic peptide triazoles (cPTs) that bind to HIV-1 Env gp120, inhibit virus cell infection with nanomolar potencies, and cause irreversible virion inactivation. Given the appealing virus-killing activity of cPTs and resistance to protease cleavage observed in vitro, we here investigated in vivo pharmacokinetics of the cPT AAR029b. AAR029b was investigated both alone and encapsulated in a PEGylated liposome formulation that was designed to slowly release inhibitor. Pharmacokinetic analysis in rats showed that the half-life of FITC-AAR029b was substantial both alone and liposome-encapsulated, 2.92 and 8.87 hours, respectively. Importantly, liposome-encapsulated FITC-AAR029b exhibited a 15-fold reduced clearance rate from serum compared with the free FITC-cPT. This work thus demonstrated both the in vivo stability of cPT alone and the extent of pharmacokinetic enhancement via liposome encapsulation. The results obtained open the way to further develop cPTs as long-acting HIV-1 inactivators against HIV-1 infection.

Mechanism of multivalent nanoparticle encounter with HIV-1 for potency enhancement of peptide triazole virus inactivation.

Entry of HIV-1 into host cells remains a compelling yet elusive target for developing agents to prevent infection. A peptide triazole (PT) class of entry inhibitor has previously been shown to bind to HIV-1 gp120, suppress interactions of the Env protein at host cell receptor binding sites, inhibit cell infection, and cause envelope spike protein breakdown, including gp120 shedding and, for some variants, virus membrane lysis. We found that gold nanoparticle-conjugated forms of peptide triazoles (AuNP-PT) exhibit substantially more potent antiviral effects against HIV-1 than corresponding peptide triazoles alone. Here, we sought to reveal the mechanism of potency enhancement underlying nanoparticle conjugate function. We found that altering the physical properties of the nanoparticle conjugate, by increasing the AuNP diameter and/or the density of PT conjugated on the AuNP surface, enhanced potency of infection inhibition to impressive picomolar levels. Further, compared with unconjugated PT, AuNP-PT was less susceptible to reduction of antiviral potency when the density of PT-competent Env spikes on the virus was reduced by incorporating a peptide-resistant mutant gp120. We conclude that potency enhancement of virolytic activity and corresponding irreversible HIV-1 inactivation of PTs upon AuNP conjugation derives from multivalent contact between the nanoconjugates and metastable Env spikes on the HIV-1 virus. The findings reveal that multispike engagement can exploit the metastability built into virus the envelope to irreversibly inactivate HIV-1 and provide a conceptual platform to design nanoparticle-based antiviral agents for HIV-1 specifically and putatively for metastable enveloped viruses generally.

Cross-Sectional Survey of Factors Contributing to COVID-19 Testing Hesitancy Among US Adults at Risk of Severe Outcomes from COVID-19.

INTRODUCTION: The United States Centers for Disease Control and Prevention (CDC) advises testing individuals for COVID-19 after exposure or if they display symptoms. However, a deeper understanding of demographic factors associated with testing hesitancy is necessary. METHODS: A US nationwide cross-sectional survey of adults with risk factors for developing severe COVID-19 ("high-risk" individuals) was conducted from August 18-September 5, 2023. Objectives included characterizing demographics and attitudes associated with COVID-19 testing. Inverse propensity weighting was used to weight the data to accurately reflect the high-risk adult US population as reflected in IQVIA medical claims data. We describe here the weighted results modeled to characterize demographic factors driving hesitancy. RESULTS: In the weighted sample of 5019 respondents at high risk for severe COVID-19, 58.2% were female, 37.8% were ≥ 65 years old, 77.1% were White, and 13.9% had a postgraduate degree. Overall, 67% were Non-testers (who indicated that they were unlikely or unsure of their likelihood of being tested within the next 6 months); these respondents were significantly more likely than Testers (who indicated a higher probability of testing within 6 months) to be female (60.2 vs. 54.1%; odds ratio [OR] [95% confidence interval (CI)], 1.3 [1.1‒1.4]), aged ≥ 65 years old (41.5 vs. 30.3%; OR [95% CI] compared with ages 18‒34 years, 0.6 [0.5‒0.7]), White (82.1 vs. 66.8%; OR [95% CI], 1.4 [1.1‒1.8]), and to identify as politically conservative (40.9 vs. 18.1%; OR [95% CI], 2.6 [2.3‒2.9]). In contrast, Testers were significantly more likely than Non-testers to have previous experience with COVID-19 testing, infection, or vaccination; greater knowledge regarding COVID-19 and testing; greater healthcare engagement; and concerns about COVID-19. CONCLUSIONS: Older, female, White, rural-dwelling, and politically conservative high-risk adults are the most likely individuals to experience COVID-19 testing hesitancy. Understanding these demographic factors will help guide strategies to improve US testing rates.

HIV-1 Env-Dependent Cell Killing by Bifunctional Small-Molecule/Peptide Conjugates.

A strategy has been established for the synthesis of a family of bifunctional HIV-1 inhibitor covalent conjugates with the potential to bind simultaneously to both the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein trimeric complex (Env). One component of the conjugates is derived from BNM-III-170, a small-molecule CD4 mimic that binds to gp120. The second component, comprised of the peptide DKWASLWNW ("Trp3"), was derived from the N-terminus of the HIV-1 gp41 Membrane Proximal External Region (MPER) and found previously to bind to the gp41 subunit of Env. The resulting bifunctional conjugates were shown to inhibit virus cell infection with low micromolar potency and to induce lysis of the HIV-1 virion. Crucially, virolysis was found to be dependent on the covalent linkage of the BNM-III-170 and Trp3 domains, as coadministration of a mixture of the un-cross-linked components proved to be nonlytic. However, a significant magnitude of lytic activity was observed in Env-negative and other control pseudoviruses, suggesting parallel mechanisms of action of the conjugates involving Env interaction and direct membrane disruption. Computational modeling suggested strong membrane-binding activity of BNM-III-170, which may underly the nonspecific virolytic effects of the conjugates. To investigate the scope of the membrane effect, cell-based cytotoxicity and membrane permeability assays were performed employing flow cytometry. Here, we observed a dose-dependent and specific cytotoxic effect on HIV-1 Env-expressing cells by the small-molecule bifunctional inhibitor. Most importantly, Env-negative cells were not susceptible to the cytotoxic effect upon exposure to this construct at concentrations where cell-killing effects were observed for Env-positive cells. Computational structural modeling supports a mechanism in which the bifunctional inhibitors bind to the gp120 and gp41 subunits in tandem in open-state Env trimers and induce relative motion of the gp120 subunits consistent with models of Env inactivation. This observation supports the idea that the cell-killing effect of the small-molecule bifunctional inhibitor is due to specific Env conformational triggering. This work lays important groundwork to advance a small-molecule bifunctional inhibitor approach for eliminating Env-expressing infected cells and the eradication of HIV-1.