Type I interferon protects cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). In this study, we utilized short hairpin RNA (shRNA) to deplete ISGs in SupT1 cells in order to identify ISGs that suppress the production of human immunodeficiency virus type 1 (HIV-1). Among the ISG candidates thus identified were interferon-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, that potently inhibit HIV-1 replication at least partially through interfering with virus entry. Further mutagenesis analysis shows that the intracellular region, rather than the N- and C-terminal extracellular domains, is essential for the antiviral activity of IFITM1. Altogether, these data suggest that the IFITM proteins serve as important components of the innate immune system to restrict HIV-1 infection.

SARS-CoV-2 mRNA vaccination induces robust humoral and cellular immunity in the circulation; however, it is currently unknown whether it elicits effective immune responses in the respiratory tract, particularly against variants of concern (VOCs), including Omicron. We compared the SARS-CoV-2 S–specific total and neutralizing antibody responses, and B and T cell immunity, in the bronchoalveolar lavage fluid (BAL) and blood of COVID-19–vaccinated individuals and hospitalized patients. Vaccinated individuals had significantly lower levels of neutralizing antibody against D614G, Delta (B.1.617.2), and Omicron BA.1.1 in the BAL compared with COVID-19 convalescents despite robust S-specific antibody responses in the blood. Furthermore, mRNA vaccination induced circulating S-specific B and T cell immunity, but in contrast to COVID-19 convalescents, these responses were absent in the BAL of vaccinated individuals. Using a mouse immunization model, we demonstrated that systemic mRNA vaccination alone induced weak respiratory mucosal neutralizing antibody responses, especially against SARS-CoV-2 Omicron BA.1.1 in mice; however, a combination of systemic mRNA vaccination plus mucosal adenovirus-S immunization induced strong neutralizing antibody responses not only against the ancestral virus but also the Omicron BA.1.1 variant. Together, our study supports the contention that the current COVID-19 vaccines are highly effective against severe disease development, likely through recruiting circulating B and T cell responses during reinfection, but offer limited protection against breakthrough infection, especially by the Omicron sublineage. Hence, mucosal booster vaccination is needed to establish robust sterilizing immunity in the respiratory tract against SARS-CoV-2, including infection by the Omicron sublineage and future VOCs.

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection. Many pathogenic viruses contain an envelope that must fuse with the cell membrane in order to gain entry and initiate infection. This process is mediated by one or more glycoproteins present on the surface of the virions, known as viral fusion proteins. Recently, a family of interferon-inducible transmembrane (IFITM) protein has been shown to block viral infection, including those of highly pathogenic viruses. Here we provide evidence that these IFITM proteins potently suppress membrane fusion induced by representatives of all three classes of viral fusion proteins. Interestingly, we found that the block is not at the steps of receptor binding or low pH that triggers conformational changes of viral fusion proteins required for membrane fusion. Rather, we discovered that the creation of hemifusion, an intermediate in which the outer membranes of the two lipid bilayers have merged but the inner membranes still remain intact is blocked by IFITM proteins. We further demonstrated that overexpression of IFITM proteins rigidify the cell membrane, thereby reducing membrane fluidity and fusion potential. Our study provides novel insight into the understanding of how IFITM proteins restrict viral entry and infection.

The waning efficacy of SARS-CoV-2 vaccines, combined with the continued emergence of variants resistant to vaccine-induced immunity, has reignited debate over the need for booster vaccine doses. To address this, we examined the neutralizing antibody response against the spike protein of five major SARS-CoV-2 variants, D614G, Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), in health care workers (HCWs) vaccinated with SARS-CoV-2 mRNA vaccines. Serum samples were collected before vaccination, 3 weeks after first vaccination, 1 month after second vaccination, and 6 months after second vaccination. Minimal neutralizing antibody titers were detected against Omicron pseudovirus at all four time points, including for most patients who had SARS-CoV-2 breakthrough infections. Neutralizing antibody titers against all other variant spike protein–bearing pseudoviruses declined markedly from 1 to 6 months after the second mRNA vaccine dose, although SARS-CoV-2 infection boosted vaccine responses. In addition, mRNA-1273–vaccinated HCWs exhibited about twofold higher neutralizing antibody titers than BNT162b2-vaccinated HCWs. Together, these results demonstrate possible waning of antibody-mediated protection against SARS-CoV-2 variants that is dependent on prior infection status and the mRNA vaccine received. They also show that the Omicron variant spike protein can almost completely escape from neutralizing antibodies elicited in recipients of only two mRNA vaccine doses.

Significance: It is currently unknown if SARS-CoV-2 can spread through cell–cell contacts, and if so, the underlying mechanisms and implications. In this work, we show, by using lentiviral pseudotyped virus, that the spike protein of SARS-CoV-2 mediates the viral cell-to-cell transmission, with an efficiency higher than that of SARS-CoV. We also find that cell–cell fusion contributes to cell-to-cell transmission, yet ACE2 is not absolutely required. While the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) differ in cell-free infectivity from wild type and from each other, these VOCs have similar cell-to-cell transmission capability and exhibit differential sensitivity to neutralization by vaccinee sera. Results from our study will contribute to a better understanding of SARS-CoV-2 spread and pathogenesis.