An mRNA-LNP-based Lassa virus vaccine induces protective immunity in mice.

The mammarenavirus Lassa virus (LASV) causes the life-threatening hemorrhagic fever disease, Lassa fever. The lack of licensed medical countermeasures against LASV underscores the urgent need for the development of novel LASV vaccines, which has been hampered by the requirement for a biosafety level 4 facility to handle live LASV. Here, we investigated the efficacy of mRNA-lipid nanoparticle (mRNA-LNP)-based vaccines expressing the LASV glycoprotein precursor (LASgpc) or nucleoprotein (LCMnp) of the prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), in mice. Two doses of LASgpc- or LCMnp-mRNA-LNP administered intravenously (i.v.) protected C57BL/6 mice from a lethal challenge with a recombinant (r) LCMV expressing a modified LASgpc (rLCMV/LASgpc2m) inoculated intracranially. Intramuscular (i.m.) immunization with two doses of LASgpc- or LCMnp-mRNA-LNP significantly reduced the viral load in C57BL/6 mice inoculated i.v. with rLCMV/LASgpc2m. High levels of viremia and lethality were observed in CBA mice inoculated i.v. with rLCMV/LASgpc2m, which were abrogated by i.m. immunization with two doses of LASgpc-mRNA-LNP. The protective efficacy of two i.m. doses of LCMnp-mRNA-LNP was confirmed in a lethal hemorrhagic disease model of FVB mice i.v. inoculated with wild-type rLCMV. In all conditions tested, negligible and high levels of LASgpc- and LCMnp-specific antibodies were detected in mRNA-LNP-immunized mice, respectively, but robust LASgpc- and LCMnp-specific CD8+ T cell responses were induced. Accordingly, plasma from LASgpc-mRNA-LNP-immunized mice did not exhibit neutralizing activity. Our findings and surrogate mouse models of LASV infection, which can be studied at a reduced biocontainment level, provide a critical foundation for the rapid development of mRNA-LNP-based LASV vaccines.IMPORTANCELassa virus (LASV) is a highly pathogenic mammarenavirus responsible for several hundred thousand infections annually in West African countries, causing a high number of lethal Lassa fever (LF) cases. Despite its significant impact on human health, clinically approved, safe, and effective medical countermeasures against LF are not available. The requirement of a biosafety level 4 facility to handle live LASV has been one of the main obstacles to the research and development of LASV countermeasures. Here, we report that two doses of mRNA-lipid nanoparticle-based vaccines expressing the LASV glycoprotein precursor (LASgpc) or nucleoprotein (LCMnp) of lymphocytic choriomeningitis virus (LCMV), a mammarenavirus genetically closely related to LASV, conferred protection to recombinant LCMV-based surrogate mouse models of lethal LASV infection. Notably, robust LASgpc- and LCMnp-specific CD8+ T cell responses were detected in mRNA-LNP-immunized mice, whereas no virus-neutralizing activity was observed.

Phenothiazines inhibit SARS-CoV-2 cell entry via a blockade of spike protein binding to neuropilin-1.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells using angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP-1) as the primary receptor and entry co-factor, respectively. Cell entry is the first and major step in initiation of the viral life cycle, representing an ideal target for antiviral interventions. In this study, we used a recombinant replication-deficient vesicular stomatitis virus-based pseudovirus bearing the spike protein of SARS-CoV-2 (SARS2-S) to screen a US Food and Drug Administration-approved drug library and identify inhibitors of SARS-CoV-2 cell entry. The screen identified 24 compounds as primary hits, and the largest therapeutic target group formed by these primary hits was composed of seven dopamine receptor D2 (DRD2) antagonists. Cell-based and biochemical assays revealed that the DRD2 antagonists inhibited both fusion activity and the binding of SARS2-S to NRP-1, but not its binding to ACE2. On the basis of structural similarity to the seven identified DRD2 antagonists, which included six phenothiazines, we examined the anti-SARS-CoV-2 activity of an additional 15 phenothiazines and found that all the tested phenothiazines shared an ability to inhibit SARS2-S-mediated cell entry. One of the phenothiazines, alimemazine, which had the lowest 50% effective concentration of the tested phenothiazines, exhibited a clear inhibitory effect on SARS2-S-NRP-1 binding and SARS-CoV-2 multiplication in cultured cells but not in a mouse infection model. Our findings provide a basis for the development of novel anti-SARS-CoV-2 therapeutics that interfere with SARS2-S binding to NRP-1.

The Pan-ErbB tyrosine kinase inhibitor afatinib inhibits multiple steps of the mammarenavirus life cycle.

The mammarenavirus Lassa virus (LASV) causes a life-threatening acute febrile disease, Lassa fever (LF). To date, no US Food and Drug Administration (FDA)-licensed medical countermeasures against LASV are available. This underscores the need for the development of novel anti-LASV drugs. Here, we screen an FDA-approved drug library to identify novel anti-LASV drug candidates using an infectious-free cell line expressing a functional LASV ribonucleoprotein (vRNP), where levels of vRNP-directed reporter gene expression serve as a surrogate for vRNP activity. Our screen identified the pan-ErbB tyrosine kinase inhibitor afatinib as a potent inhibitor of LASV vRNP activity. Afatinib inhibited multiplication of lymphocytic choriomeningitis virus (LCMV) a mammarenavirus closely related to LASV. Cell-based assays revealed that afatinib inhibited multiple steps of the LASV and LCMV life cycles. Afatinib also inhibited multiplication of Junín virus vaccine strain Candid#1, indicating that afatinib can have antiviral activity against a broad range of human pathogenic mammarenaviruses.

A small stem-loop-forming region within the 3'-UTR of a nonpolyadenylated LCMV mRNA promotes translation.

Mammalian arenavirus (mammarenavirus) mRNAs are characterized by 5'-capped and 3'-nonpolyadenylated untranslated regions (UTRs). We previously reported that the nonpolyadenylated 3'-UTR of viral mRNA (vmRNA), which is derived from the noncoding intergenic region (IGR), regulates viral protein levels at the posttranscriptional level. This finding provided the basis for the development of novel live-attenuated vaccines (LAVs) against human pathogenic mammarenaviruses. Detailed information about the roles of specific vmRNA 3'-UTR sequences in controlling translation efficiency will help in understanding the mechanism underlying attenuation by IGR manipulations. Here, we characterize the roles of cis-acting mRNA regulatory sequences of a prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), in modulating translational efficiency. Using in vitro transcribed RNA mimics encoding a reporter gene, we demonstrate that the 3'-UTR of nucleoprotein (NP) mRNA without a poly(A) tail promotes translation in a poly(A)-binding protein-independent manner. Comparison with the 3'-UTR of glycoprotein precursor mRNA, which is translated less efficiently, revealed that a 10-nucleotide sequence proximal to the NP open reading frame is essential for promoting translation. Modification of this 10-nucleotide sequence also impacted reporter gene expression in recombinant LCMV. Our findings will enable rational design of the 10-nucleotide sequence to further improve our mammarenavirus LAV candidates and to develop a novel LCMV vector capable of controlling foreign gene expression.

Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), employs host-cell angiotensin-converting enzyme 2 (ACE2) for cell entry. Genetic analyses of ACE2 have identified several single-nucleotide polymorphisms (SNPs) specific to different human populations. Molecular dynamics simulations have indicated that several of these SNPs could affect interactions between SARS-CoV-2 and ACE2, thereby providing a partial explanation for the regional differences observed in SARS-CoV-2 infectivity and severity. However, the significance of population-specific ACE2 SNPs in SARS-CoV-2 infectivity is unknown, as no in vitro validation studies have been performed. Here, we analyzed the impact of eight SNPs found in specific populations on receptor binding and cell entry in vitro. Except for a SNP causing a nonsense mutation that reduced ACE2 expression, none of the selected SNPs markedly altered the interaction between ACE2 and the SARS-CoV-2 spike protein (SARS-2-S), which is responsible for receptor recognition and cell entry, or the efficiency of viral cell entry mediated by SARS-2-S. Our findings indicate that ACE2 polymorphisms have limited impact on the ACE2-dependent cell entry of SARS-CoV-2 and underscore the importance of future studies on the involvement of population-specific SNPs of other host genes in susceptibility toward SARS-CoV-2 infection.