Inhibition of Measles Viral Fusion Is Enhanced by Targeting Multiple Domains of the Fusion Protein

Measles virus (MeV) infection remains a significant public health threat despite ongoing global efforts to increase vaccine coverage. As eradication of MeV stalls, and vulnerable populations expand, effective antivirals against MeV are in high demand. Here, we describe the development of an antiviral peptide that targets the MeV fusion (F) protein. This antiviral peptide construct is composed of a carbobenzoxy-d-Phe-l-Phe-Gly (fusion inhibitor peptide; FIP) conjugated to a lipidated MeV F C-terminal heptad repeat (HRC) domain derivative. Initial in vitro testing showed high antiviral potency and specific targeting of MeV F-associated cell plasma membranes, with minimal cytotoxicity. The FIP and HRC-derived peptide conjugates showed synergistic antiviral activities when administered individually. However, their chemical conjugation resulted in markedly increased antiviral potency. In vitro mechanistic experiments revealed that the FIP-HRC lipid conjugate exerted its antiviral activity predominantly through stabilization of the prefusion F, while HRC-derived peptides alone act predominantly on the F protein after its activation. Coupled with in vivo experiments showing effective prevention of MeV infection in cotton rats, FIP-HRC lipid conjugates show promise as potential MeV antivirals via specific targeting and stabilization of the prefusion MeV F structure

Engineering Protease-Resistant Peptides to Inhibit Human Parainfluenza Viral Respiratory Infection

The lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. A viral fusion protein (F), once activated in proximity to a target cell, undergoes a series of conformational changes that first extend the trimer subunits to allow insertion of the hydrophobic domains into the target cell membrane and then refold the trimer into a stable postfusion state, driving the merger of the viral and host cell membranes. Lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F inhibit infection by interfering with the structural transitions of the trimeric F assembly. Clinical application of this strategy, however, requires improving the in vivo stability of antiviral peptides. We show that the HRC peptide backbone can be modified via partial replacement of α-amino acid residues with β-amino acid residues to generate α/β-peptides that retain antiviral activity but are poor protease substrates. Relative to a conventional α-lipopeptide, our best α/β-lipopeptide exhibits improved persistence in vivo and improved anti-HPIV3 antiviral activity in animals

Potency of Fusion-Inhibitory Lipopeptides against SARS-CoV-2 Variants of Concern

SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, with waves resulting from new variants that evade immunity generated by vaccines and previous strains and escape available monoclonal antibody therapy. Fusion-inhibitory peptides may provide an intervention strategy that is not similarly affected by this viral evolution.The ability of SARS-CoV-2 to evolve in response to selective pressures poses a challenge to vaccine and antiviral efficacy. The S1 subunit of the spike (S) protein contains the receptor-binding domain and is therefore under selective pressure to evade neutralizing antibodies elicited by vaccination or infection. In contrast, the S2 subunit of S is only transiently exposed after receptor binding, which makes it a less efficient target for antibodies. As a result, S2 has a lower mutational frequency than S1. We recently described monomeric and dimeric SARS-CoV-2 fusion-inhibitory lipopeptides that block viral infection by interfering with S2 conformational rearrangements during viral entry. Importantly, a dimeric lipopeptide was shown to block SARS-CoV-2 transmission between ferrets in vivo. Because the S2 subunit is relatively conserved in newly emerging SARS-CoV-2 variants of concern (VOCs), we hypothesize that fusion-inhibitory lipopeptides are cross-protective against infection with VOCs. Here, we directly compared the in vitro efficacies of two fusion-inhibitory lipopeptides against VOC, in comparison with a set of seven postvaccination sera (two doses) and a commercial monoclonal antibody preparation. For the beta, delta, and omicron VOCs, it has been reported that convalescent and postvaccination sera are less potent in virus neutralization assays. Both fusion-inhibitory lipopeptides were equally effective against all five VOCs compared to ancestral virus, whereas postvaccination sera and therapeutic monoclonal antibody lost potency to newer VOCs, in particular to omicron BA.1 and BA.2. The neutralizing activity of the lipopeptides is consistent, and they can be expected to neutralize future VOCs based on their mechanism of action. IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, with waves resulting from new variants that evade immunity generated by vaccines and previous strains and escape available monoclonal antibody therapy. Fusion-inhibitory peptides may provide an intervention strategy that is not similarly affected by this viral evolution

Nebulized fusion inhibitory peptide protects cynomolgus macaques from measles virus infection

Abstract Measles is the most contagious airborne viral infection and the leading cause of child death among vaccine-preventable diseases. We show here that aerosolized lipopeptide fusion inhibitors, derived from heptad-repeat regions of the measles virus (MeV) fusion protein, block respiratory MeV infection in a non-human primate model, the cynomolgus macaque. We used a custom-designed mesh nebulizer to ensure efficient aerosol delivery of peptides to the respiratory tract and demonstrated the absence of adverse effects and lung pathology in macaques. The nebulized peptide efficiently prevented MeV infection, resulting in the complete absence of MeV RNA, MeV-infected cells, and MeV-specific humoral responses in treated animals. This strategy provides an additional shield which complements vaccination to fight against respiratory infection, presenting a proof-of-concept for the aerosol delivery of fusion inhibitory peptides to protect against measles and other airborne viruses, including SARS-CoV-2, in case of high-risk exposure, that can be readily translated to human trials

Intranasal fusion inhibitory lipopeptide prevents direct-contact SARS-CoV-2 transmission in ferrets

Containment of the COVID-19 pandemic requires reducing viral transmission. SARS-CoV-2 infection is initiated by membrane fusion between the viral and host cell membranes, mediated by the viral spike protein. We have designed lipopeptide fusion inhibitors that block this critical first step of infection, and based on in vitro efficacy and in vivo biodistribution selected a dimeric form for evaluation in an animal model. Daily intranasal administration to ferrets completely prevented SARS-CoV-2 direct-contact transmission during 24-hour co-housing with infected animals, under stringent conditions that resulted in infection of 100% of untreated animals. These lipopeptides are highly stable and thus may readily translate into safe and effective intranasal prophylaxis to reduce transmission of SARS-CoV-2