Functional impairment of "helpless" CD8 + memory T cells is transient and driven by prolonged but finite cognate antigen presentation.

Generation of functional CD8 + T cell memory typically requires engagement of CD4 + T cells. However, in certain scenarios, such as acutely-resolving viral infections, effector (T E ) and subsequent memory (T M ) CD8 + T cell formation appear impervious to a lack of CD4 + T cell help during priming. Nonetheless, such "helpless" CD8 + T M respond poorly to pathogen rechallenge. At present, the origin and long-term evolution of helpless CD8 + T cell memory remain incompletely understood. Here, we demonstrate that helpless CD8 + T E differentiation is largely normal but a multiplicity of helpless CD8 T M defects, consistent with impaired memory maturation, emerge as a consequence of prolonged yet finite exposure to cognate antigen. Importantly, these defects resolve over time leading to full restoration of CD8 + T M potential and recall capacity. Our findings provide a unified explanation for helpless CD8 + T cell memory and emphasize an unexpected CD8 + T M plasticity with implications for vaccination strategies and beyond.

Activation of protein kinase receptor (PKR) plays a pro-viral role in mammarenavirus-infected cells.

Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double-stranded RNA (dsRNA) sensor protein kinase receptor (PKR) pathway plays a critical role in the cell anti-viral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the anti-viral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro- and anti-viral activities.IMPORTANCEAs with many other viruses, the prototypic Old World mammarenavirus LCMV can interfere with the host cell innate immune response to infection, which includes the dsRNA sensor PKR pathway. A detailed understanding of LCMV-PKR interactions can provide novel insights about mammarenavirus-host cell interactions and facilitate the development of effective anti-viral strategies against human pathogenic mammarenaviruses. In the present work, we present evidence that LCMV multiplication is enhanced in PKR-deficient cells, but pharmacological inhibition of PKR activation unexpectedly resulted in severely restricted propagation of LCMV. Likewise, we document a robust PKR activation in LCMV-infected cells in the absence of detectable levels of dsRNA. Our findings have revealed a complex role of the PKR pathway during LCMV infection and uncovered the activation of PKR as a druggable target for the development of anti-viral drugs against human pathogenic mammarenaviruses.

Activation of Protein Kinase R (PKR) Plays a Pro-Viral Role in Mammarenavirus Infected Cells.

Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double strand (ds)RNA sensor protein kinase receptor (PKR) pathway plays a critical role in the cell antiviral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the antiviral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein (NP) resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro-and antiviral activities.

Chaperonin TRiC/CCT Participates in Mammarenavirus Multiplication in Human Cells via Interaction with the Viral Nucleoprotein.

The eukaryotic chaperonin containing tailless complex polypeptide 1 ring complex (CCT, also known as TCP-1 Ring Complex, TRiC/CCT) participates in the folding of 5% to 10% of the cellular proteome and has been involved in the life cycle of several viruses, including dengue, Zika, and influenza viruses, but the mechanisms by which the TRiC/CCT complex contributes to virus multiplication remain poorly understood. Here, we document that the nucleoprotein (NP) of the mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a substrate of the human TRiC/CCT complex, and that pharmacological inhibition of TRiC/CCT complex function, or RNAi-mediated knockdown of TRiC/CCT complex subunits, inhibited LCMV multiplication in human cells. We obtained evidence that the TRiC/CCT complex is required for the production of NP-containing virus-like particles (VLPs), and the activity of the virus ribonucleoprotein (vRNP) responsible for directing replication and transcription of the viral genome. Pharmacological inhibition of the TRIC/CCT complex also restricted multiplication of the live-attenuated vaccine candidates Candid#1 and ML29 of the hemorrhagic fever causing Junin (JUNV) and Lassa (LASV) mammarenaviruses, respectively. Our findings indicate that the TRiC/CCT complex is required for mammarenavirus multiplication and is an attractive candidate for the development of host directed antivirals against human-pathogenic mammarenaviruses. IMPORTANCE Host-directed antivirals have gained great interest as an antiviral strategy to counteract the rapid emergence of drug-resistant viruses. The chaperonin TRiC/CCT complex has been involved in the life cycle of several viruses, including dengue, Zika, and influenza viruses. Here, we have provided evidence that the chaperonin TRiC/CCT complex participates in mammarenavirus infection via its interaction with the viral NP. Importantly, pharmacological inhibition of TRiC/CCT function significantly inhibited multiplication of LCMV and the distantly related mammarenavirus JUNV in human cells. Our findings support that the TRiC/CCT complex is required for multiplication of mammarenaviruses and that the TRiC/CCT complex is an attractive host target for the development of antivirals against human-pathogenic mammarenaviruses.

Molecular Engineering of a Mammarenavirus with Unbreachable Attenuation.

Several mammarenaviruses cause severe hemorrhagic fever (HF) disease in humans and pose important public health problems in their regions of endemicity. There are no United States (US) Food and Drug Administration (FDA)-approved mammarenavirus vaccines, and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that has limited efficacy. Mammarenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome. Each genome segment contains two open reading frames (ORF) separated by a noncoding intergenic region (IGR). The large (L) segment encodes the RNA dependent RNA polymerase, L protein, and the Z matrix protein, whereas the small (S) segment encodes the surface glycoprotein precursor (GPC) and nucleoprotein (NP). In the present study, we document the generation of a recombinant form of the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) expressing a codon deoptimized (CD) GPC and containing the IGR of the S segment in both the S and L segments (rLCMV/IGR-CD). We show that rLCMV/IGR-CD is fully attenuated in C57BL/6 (B6) mice but able to provide complete protection upon a single administration against a lethal challenge with LCMV. Importantly, rLCMV/IGR-CD exhibited an unbreachable attenuation for its safe implementation as a live-attenuated vaccine (LAV). IMPORTANCE Several mammarenaviruses cause severe disease in humans and pose important public health problems in their regions of endemicity. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenaviral therapy is limited to an off-label use of ribavirin whose efficacy is controversial. Here, we describe the generation of recombinant version of the prototypic mammarenavirus lymphocytic choriomeningitis virus (rLCMV) combining the features of a codon deoptimized (CD) GPC and the noncoding intergenic region (IGR) of the S segment in both S and L genome segments, called rLCMV/IGR-CD. We present evidence that rLCMV/IGR-CD has excellent safety and protective efficacy features as live-attenuated vaccine (LAV). Importantly, rLCMV/IGR-CD prevents, in coinfected mice, the generation of LCMV reassortants with increased virulence. Our findings document a well-defined molecular strategy for the generation of mammarenavirus LAV candidates able to trigger long-term protective immunity, upon a single immunization, while exhibiting unique enhanced safety features, including unbreachable attenuation.

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.

Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase.

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

Inhibitors of anti-apoptotic Bcl-2 family proteins exhibit potent and broad-spectrum anti-mammarenavirus activity via cell cycle arrest at G0/G1 phase.

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their pro-apoptotic properties, but rather their ability of inducing cell arrest at G0/G1 phase. OLX and ABT-737 mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, siRNA-mediated knock down of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE: Anti-apoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors mediated cell cycle arrest at the G0/G1 phase, rather than their pro-apoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate anti-mammarenavirus activity in vivo , and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals.

Lassa Virus Vaccine Candidate ML29 Generates Truncated Viral RNAs Which Contribute to Interfering Activity and Attenuation.

Defective interfering particles (DIPs) are naturally occurring products during virus replication in infected cells. DIPs contain defective viral genomes (DVGs) and interfere with replication and propagation of their corresponding standard viral genomes by competing for viral and cellular resources, as well as promoting innate immune antiviral responses. Consequently, for many different viruses, including mammarenaviruses, DIPs play key roles in the outcome of infection. Due to their ability to broadly interfere with viral replication, DIPs are attractive tools for the development of a new generation of biologics to target genetically diverse and rapidly evolving viruses. Here, we provide evidence that in cells infected with the Lassa fever (LF) vaccine candidate ML29, a reassortant that carries the nucleoprotein (NP) and glycoprotein (GP) dominant antigens of the pathogenic Lassa virus (LASV) together with the L polymerase and Z matrix protein of the non-pathogenic genetically related Mopeia virus (MOPV), L-derived truncated RNA species are readily detected following infection at low multiplicity of infection (MOI) or in persistently-infected cells originally infected at high MOI. In the present study, we show that expression of green fluorescent protein (GFP) driven by a tri-segmented form of the mammarenavirus lymphocytic choriomeningitis virus (r3LCMV-GFP/GFP) was strongly inhibited in ML29-persistently infected cells, and that the magnitude of GFP suppression was dependent on the passage history of the ML29-persistently infected cells. In addition, we found that DIP-enriched ML29 was highly attenuated in immunocompetent CBA/J mice and in Hartley guinea pigs. Likewise, STAT-1-/- mice, a validated small animal model for human LF associated hearing loss sequelae, infected with DIP-enriched ML29 did not exhibit any hearing abnormalities throughout the observation period (62 days).

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism.

Neurological complications are common in patients with COVID-19. While SARS-CoV-2, the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood, and experimental models using human brain cells are urgently needed. Here we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found modest numbers of infected neurons and astrocytes, but greater infection of choroid plexus epithelial cells. We optimized a protocol to generate choroid plexus organoids from hiPSCs, which revealed productive SARS-CoV-2 infection that leads to increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our results provide evidence for SARS-CoV-2 neurotropism and support use of hiPSC-derived brain organoids as a platform to investigate the cellular susceptibility, disease mechanisms, and treatment strategies for SARS-CoV-2 infection.

Novel Dihydroorotate Dehydrogenase Inhibitors with Potent Interferon-Independent Antiviral Activity against Mammarenaviruses In Vitro.

Mammarenaviruses cause chronic infections in rodents, which are their predominant natural hosts. Human infection with some of these viruses causes high-consequence disease, posing significant issues in public health. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenavirus drugs are limited to an off-label use of ribavirin, which is only partially efficacious and associated with severe side effects. Dihydroorotate dehydrogenase (DHODH) inhibitors, which block de novo pyrimidine biosynthesis, have antiviral activity against viruses from different families, including Arenaviridae, the taxonomic home of mammarenaviruses. Here, we evaluate five novel DHODH inhibitors for their antiviral activity against mammarenaviruses. All tested DHODH inhibitors were potently active against lymphocytic choriomeningitis virus (LCMV) (half-maximal effective concentrations [EC50] in the low nanomolar range, selectivity index [SI] > 1000). The tested DHODH inhibitors did not affect virion cell entry or budding, but rather interfered with viral RNA synthesis. This interference resulted in a potent interferon-independent inhibition of mammarenavirus multiplication in vitro, including the highly virulent Lassa and Junín viruses.

Ebola-Specific CD8+ and CD4+ T-Cell Responses in Sierra Leonean Ebola Virus Survivors With or Without Post-Ebola Sequelae.

BACKGROUND: Ebola virus (EBOV) disease has killed thousands of West and Central Africans over the past several decades. Many who survive the acute disease later experience post-Ebola syndrome, a constellation of symptoms whose causative pathogenesis is unclear. METHODS: We investigated EBOV-specific CD8+ and CD4+ T-cell responses in 37 Sierra Leonean EBOV disease survivors with (n = 19) or without (n = 18) sequelae of arthralgia and ocular symptoms. Peripheral blood mononuclear cells were infected with recombinant vesicular stomatitis virus encoding EBOV antigens. We also studied the presence of EBOV-specific immunoglobulin G, antinuclear antibodies, anti-cyclic citrullinated peptide antibodies, rheumatoid factor, complement levels, and cytokine levels in these 2 groups. RESULTS: Survivors with sequelae had a significantly higher EBOV-specific CD8+ and CD4+ T-cell response. No differences in EBOV-specific immunoglobulin G, antinuclear antibody, or anti-cyclic citrullinated peptide antibody levels were found. Survivors with sequelae showed significantly higher rheumatoid factor levels. CONCLUSION: EBOV-specific CD8+ and CD4+ T-cell responses were significantly higher in Ebola survivors with post-Ebola syndrome. These findings suggest that pathogenesis may occur as an immune-mediated disease via virus-specific T-cell immune response or that persistent antigen exposure leads to increased and sustained T-cell responses.

Identification of Common CD8+ T Cell Epitopes from Lassa Fever Survivors in Nigeria and Sierra Leone.

Early and robust T cell responses have been associated with survival from Lassa fever (LF), but the Lassa virus-specific memory responses have not been well characterized. Regions within the virus surface glycoprotein (GPC) and nucleoprotein (NP) are the main targets of the Lassa virus-specific T cell responses, but, to date, only a few T cell epitopes within these proteins have been identified. We identified GPC and NP regions containing T cell epitopes and HLA haplotypes from LF survivors and used predictive HLA-binding algorithms to identify putative epitopes, which were then experimentally tested using autologous survivor samples. We identified 12 CD8-positive (CD8+) T cell epitopes, including epitopes common to both Nigerian and Sierra Leonean survivors. These data should be useful for the identification of dominant Lassa virus-specific T cell responses in Lassa fever survivors and vaccinated individuals as well as for designing vaccines that elicit cell-mediated immunity.IMPORTANCE The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine.

High crossreactivity of human T cell responses between Lassa virus lineages.

Lassa virus infects hundreds of thousands of people each year across rural West Africa, resulting in a high number of cases of Lassa fever (LF), a febrile disease associated with high morbidity and significant mortality. The lack of approved treatments or interventions underscores the need for an effective vaccine. At least four viral lineages circulate in defined regions throughout West Africa with substantial interlineage nucleotide and amino acid diversity. An effective vaccine should be designed to elicit Lassa virus specific humoral and cell mediated immunity across all lineages. Most current vaccine candidates use only lineage IV antigens encoded by Lassa viruses circulating around Sierra Leone, Liberia, and Guinea but not Nigeria where lineages I-III are found. As previous infection is known to protect against disease from subsequent exposure, we sought to determine whether LF survivors from Nigeria and Sierra Leone harbor memory T cells that respond to lineage IV antigens. Our results indicate a high degree of cross-reactivity of CD8+ T cells from Nigerian LF survivors to lineage IV antigens. In addition, we identified regions within the Lassa virus glycoprotein complex and nucleoprotein that contributed to these responses while T cell epitopes were not widely conserved across our study group. These data are important for current efforts to design effective and efficient vaccine candidates that can elicit protective immunity across all Lassa virus lineages.

A cell-based, infectious-free, platform to identify inhibitors of lassa virus ribonucleoprotein (vRNP) activity.

The mammarenavirus Lassa (LASV) is highly prevalent in West Africa where it infects several hundred thousand individuals annually resulting in a high number of Lassa fever (LF) cases, a febrile disease associated with high morbidity and significant mortality. Mounting evidence indicates that the worldwide-distributed prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. There are not Food and Drug Administration (FDA) licensed vaccines and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective and can cause significant side effects. Therefore, there is an unmet need for novel antiviral drugs to combat LASV. This task would be facilitated by the implementation of high throughput screens (HTS) to identify inhibitors of the activity of the virus ribonucleoprotein (vRNP) responsible for directing virus RNA genome replication and gene transcription. The use of live LASV for this purpose is jeopardized by the requirement of biosafety level 4 (BSL4) containment. We have developed a virus-free cell platform, where expression levels of reporter genes serve as accurate surrogates of vRNP activity, to develop cell-based assays compatible with HTS to identify inhibitors of LASV and LCMV mammarenavirus vRNP activities.

The ReFRAME library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors.

Several mammarenaviruses, chiefly Lassa virus (LASV) in Western Africa and Junín virus (JUNV) in the Argentine Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The lack of licensed mammarenavirus vaccines and partial efficacy of current anti-mammarenavirus therapy limited to an off-label use of the nucleoside analog ribavirin underscore an unmet need for novel therapeutics to combat human pathogenic mammarenavirus infections. This task can be facilitated by the implementation of "drug repurposing" strategies to reduce the time and resources required to advance identified antiviral drug candidates into the clinic. We screened a drug repurposing library of 11,968 compounds (Repurposing, Focused Rescue and Accelerated Medchem [ReFRAME]) and identified several potent inhibitors of LCMV multiplication that had also strong anti-viral activity against LASV and JUNV. Our findings indicate that enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis, the pro-viral MCL1 apoptosis regulator, BCL2 family member protein and the mitochondrial electron transport complex III, play critical roles in the completion of the mammarenavirus life cycle, suggesting they represent potential druggable targets to counter human pathogenic mammarenavirus infections.

Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays.

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000⁻300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

Analysis of CD8+ T cell response during the 2013-2016 Ebola epidemic in West Africa.

The recent Ebola epidemic exemplified the importance of understanding and controlling emerging infections. Despite the importance of T cells in clearing virus during acute infection, little is known about Ebola-specific CD8+ T cell responses. We investigated immune responses of individuals infected with Ebola virus (EBOV) during the 2013-2016 West Africa epidemic in Sierra Leone, where the majority of the >28,000 EBOV disease (EVD) cases occurred. We examined T cell memory responses to seven of the eight Ebola proteins (GP, sGP, NP, VP24, VP30, VP35, and VP40) and associated HLA expression in survivors. Of the 30 subjects included in our analysis, CD8+ T cells from 26 survivors responded to at least one EBOV antigen. A minority, 10 of 26 responders (38%), made CD8+ T cell responses to the viral GP or sGP. In contrast, 25 of the 26 responders (96%) made response to viral NP, 77% to VP24 (20 of 26), 69% to VP40 (18 of 26), 42% (11 of 26) to VP35, with no response to VP30. Individuals making CD8+ T cells to EBOV VP24, VP35, and VP40 also made CD8+ T cells to NP, but rarely to GP. We identified 34 CD8+ T cell epitopes for Ebola. Our data indicate the immunodominance of the EBOV NP-specific T cell response and suggest that its inclusion in a vaccine along with the EBOV GP would best mimic survivor responses and help boost cell-mediated immunity during vaccination.

BST-2 controls T cell proliferation and exhaustion by shaping the early distribution of a persistent viral infection.

The interferon inducible protein, BST-2 (or, tetherin), plays an important role in the innate antiviral defense system by inhibiting the release of many enveloped viruses. Consequently, viruses have evolved strategies to counteract the anti-viral activity of this protein. While the mechanisms by which BST-2 prevents viral dissemination have been defined, less is known about how this protein shapes the early viral distribution and immunological defense against pathogens during the establishment of persistence. Using the lymphocytic choriomeningitis virus (LCMV) model of infection, we sought insights into how the in vitro antiviral activity of this protein compared to the immunological defense mounted in vivo. We observed that BST-2 modestly reduced production of virion particles from cultured cells, which was associated with the ability of BST-2 to interfere with the virus budding process mediated by the LCMV Z protein. Moreover, LCMV does not encode a BST-2 antagonist, and viral propagation was not significantly restricted in cells that constitutively expressed BST-2. In contrast to this very modest effect in cultured cells, BST-2 played a crucial role in controlling LCMV in vivo. In BST-2 deficient mice, a persistent strain of LCMV was no longer confined to the splenic marginal zone at early times post-infection, which resulted in an altered distribution of LCMV-specific T cells, reduced T cell proliferation / function, delayed viral control in the serum, and persistence in the brain. These data demonstrate that BST-2 is important in shaping the anatomical distribution and adaptive immune response against a persistent viral infection in vivo.