Prevention of Respiratory Syncytial Virus Infection in Healthy Adults by a Single Immunization of Ad26.RSV.preF in a Human Challenge Study.

BACKGROUND: Respiratory syncytial virus (RSV) is a significant cause of severe lower respiratory tract disease in children and older adults, but has no approved vaccine. This study assessed the potential of Ad26.RSV.preF to protect against RSV infection and disease in an RSV human challenge model. METHODS: In this double-blind, placebo-controlled study, healthy adults aged 18-50 years were randomized 1:1 to receive 1 × 1011 vp Ad26.RSV.preF or placebo intramuscularly. Twenty-eight days postimmunization, volunteers were challenged intranasally with RSV-A (Memphis 37b). Assessments included viral load (VL), RSV infections, clinical symptom score (CSS), safety, and immunogenicity. RESULTS: Postchallenge, VL, RSV infections, and disease severity were lower in Ad26.RSV.preF (n = 27) vs placebo (n = 26) recipients: median VL area under the curve (AUC) quantitative real-time polymerase chain reaction: 0.0 vs 236.0 (P = .012; predefined primary endpoint); median VL-AUC quantitative culture: 0.0 vs 109; RSV infections 11 (40.7%) vs 17 (65.4%); median RSV AUC-CSS 35 vs 167, respectively. From baseline to 28 days postimmunization, geometric mean fold increases in RSV A2 neutralizing antibody titers of 5.8 and 0.9 were observed in Ad26.RSV.preF and placebo, respectively. Ad26.RSV.preF was well tolerated. CONCLUSIONS: Ad26.RSV.preF demonstrated protection from RSV infection through immunization in a human challenge model, and therefore could potentially protect against natural RSV infection and disease. CLINICAL TRIALS REGISTRATION: NCT03334695; CR108398, 2017-003194-33 (EudraCT); VAC18193RSV2002.

Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV-1 by Peptide Triazole Thiols.

We investigated the mode of action underlying lytic inactivation of HIV-1 virions by peptide triazole thiol (PTT), in particular the relationship between gp120 disulfides and the C-terminal cysteine-SH required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking and PTTs with serially truncated linkers between pharmacophore isoleucine-ferrocenyltriazole-proline-tryptophan and cysteine-SH were synthesized. PTT variants showed loss of lytic activity but not binding and infection inhibition upon SH blockade. A disproportionate loss of lysis activity vs binding and infection inhibition was observed upon linker truncation. Molecular docking of PTT onto gp120 argued that, with sufficient linker length, the peptide SH could approach and disrupt several alternative gp120 disulfides. Inhibition of lysis by gp120 mAb 2G12, which binds at the base of the V3 loop, as well as disulfide mutational effects, argued that PTT-induced disruption of the gp120 disulfide cluster at the base of the V3 loop is an important step in lytic inactivation of HIV-1. Further, PTT-induced lysis was enhanced after treating virus with reducing agents dithiothreitol and tris (2-carboxyethyl)phosphine. Overall, the results are consistent with the view that the binding of PTT positions the peptide SH group to interfere with conserved disulfides clustered proximal to the CD4 binding site in gp120, leading to disulfide exchange in gp120 and possibly gp41, rearrangement of the Env spike, and ultimately disruption of the viral membrane. The dependence of lysis activity on thiol-disulfide interaction may be related to intrinsic disulfide exchange susceptibility in gp120 that has been reported previously to play a role in HIV-1 cell 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.

Covalent conjugation of a peptide triazole to HIV-1 gp120 enables intramolecular binding site occupancy.

The HIV-1 gp120 glycoprotein is the main viral surface protein responsible for initiation of the entry process and, as such, can be targeted for the development of entry inhibitors. We previously identified a class of broadly active peptide triazole (PT) dual antagonists that inhibit gp120 interactions at both its target receptor and coreceptor binding sites, induce shedding of gp120 from virus particles prior to host-cell encounter, and consequently can prevent viral entry and infection. However, our understanding of the conformational alterations in gp120 by which PT elicits its dual receptor antagonism and virus inactivation functions is limited. Here, we used a recently developed computational model of the PT-gp120 complex as a blueprint to design a covalently conjugated PT-gp120 recombinant protein. Initially, a single-cysteine gp120 mutant, E275CYU-2, was expressed and characterized. This variant retains excellent binding affinity for peptide triazoles, for sCD4 and other CD4 binding site (CD4bs) ligands, and for a CD4-induced (CD4i) ligand that binds the coreceptor recognition site. In parallel, we synthesized a PEGylated and biotinylated peptide triazole variant that retained gp120 binding activity. An N-terminally maleimido variant of this PEGylated PT, denoted AE21, was conjugated to E275C gp120 to produce the AE21-E275C covalent conjugate. Surface plasmon resonance interaction analysis revealed that the PT-gp120 conjugate exhibited suppressed binding of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope protein. Similar to the noncovalent PT-gp120 complex, the covalent conjugate was able to bind the conformationally dependent mAb 2G12. The results argue that the PT-gp120 conjugate is structurally organized, with an intramolecular interaction between the PT and gp120 domains, and that this structured state embodies a conformationally entrapped gp120 with an altered bridging sheet but intact 2G12 epitope. The similarities of the PT-gp120 conjugate to the noncovalent PT-gp120 complex support the orientation of binding of PT to gp120 predicted in the molecular dynamics simulation model of the PT-gp120 noncovalent complex. The conformationally stabilized covalent conjugate can be used to expand the structural definition of the PT-induced "off" state of gp120, for example, by high-resolution structural analysis. Such structures could provide a guide for improving the subsequent structure-based design of inhibitors with the peptide triazole mode of action.