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

Abstract

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 a dimeric lipopeptide fusion inhibitor that blocks this critical first step of infection for emerging coronaviruses and document that it completely prevents SARS-CoV-2 infection in ferrets. 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 non-toxic and thus readily translate into a safe and effective intranasal prophylactic approach to reduce transmission of SARS-CoV-2.

One-sentence summary: A dimeric form of a SARS-CoV-2-derived lipopeptide is a potent inhibitor of fusion and infection in vitro and transmission in vivo .

Figure 1:. Peptide-lipid conjugates that inhibit SARS-CoV-2 spike (S)-mediated fusion.

(A) The functional domains of SARS-CoV-2 S protein: receptor-binding domain (RBD) and heptad repeats (HRN and HRC) are indicated. (B) Sequence of the peptides that derive from the HRC domain of SARS-CoV-2 S. (C) Monomeric and dimeric forms of lipid tagged SARS-CoV-2 inhibitory peptides that were assessed in cell-cell fusion assays. (D) Cell-cell fusion assays with different inhibitory peptides. The percentage inhibition is shown for four different peptides used at increasing concentrations. Inhibitory concentrations 50% and 90% were calculated (dotted lines). Percent inhibition was calculated as the ratio of relative luminescence units in the presence of a specific concentration of inhibitor and the relative luminescence units in the absence of inhibitor and corrected for background luminescence. Data are means ± standard deviation (SD) from three separate experiments. The difference between the results for [SARS_HRC-PEG₄]₂-chol and SARS_HRC-PEG₄-chol lipopeptides are significant (Two-way ANOVA, **** p<0.0001). (E) Fusion inhibitory activity of [SARS_HRC-PEG₄]₂-chol peptide against SARS-CoV-2 S variants, MERS-CoV-2 S, and SARS-CoV S. Data are means ± standard deviation (SD) from three separate experiments.

Figure 2.. Inhibition of live SARS-CoV-2 entry by [SARS-CoV-2-HRC-peg₄]₂-chol peptide.

The percentage inhibition of infection is shown on VeroE6 and VeroE6-TMPRSS2 cells with increasing concentrations of [SARS-CoV-2-HRC-peg₄]₂-chol. A DMSO-diluted stock (A, as used in ferrets) and sucrose-diluted stock (B, potential formulation for human use) were tested side-by-side. Inhibitory concentrations 50% and 90% were calculated (dotted lines). (C) Inhibitory concentrations 50% and 90% of [SARS_HRC-PEG₄]₂-chol in live SARS-CoV-2 viral infection assays in VeroE6 cells with or without TMPRSS2 protease overexpression.

Figure 3.. SARS_HRC-PEG₄]₂-chol prevents SARS-CoV-2 transmission in vivo.

(a) Experimental design. (b) Viral loads detected in throat and nose swabs via RT-PCR. Viral loads are displayed as 40-Ct. Donor animals shown in grey, mock-treated animals in red, peptide-treated animals in green. 3/3 donor animals, 6/6 mock-treated animals and 0/6 lipopeptide-treated animals were productively infected. Symbols correspond to individual animals and are consistent throughout figures.

Figure 4.. [SARS_HRC-PEG₄]₂-chol-treated animals do not seroconvert.

Presence of neutralizing antibodies was determined in a live virus neutralization assay. Virus neutralizing antibodies are displayed as endpoint serum dilution factor to block SARS-CoV-2 replication. Donor animals shown in grey, mock-treated animals in red, peptide-treated animals in green. 3/3 donor animals, 6/6 mock-treated animals and 0/6 lipopeptide-treated animals seroconverted. Symbols correspond to individual animals and are consistent throughout figures.