Arenavirus Lymphocytic Choriomeningitis Virus Research Articles

Visualizing lymphocytic choriomeningitis virus infection in cells and living mice

SummaryMammarenavirus are a large family of enveloped negative-strand RNA viruses that include several agents responsible for severe hemorrhagic fevers. Until now, no FDA-licensed drug has been admitted for treating an arenavirus infection, and only few effective anti-arenavirus drugs have been tested in vivo. In this work, we designed a recombinant reporter arenavirus lymphocytic choriomeningitis virus that stably expressed nanoluciferase (LCMV-Nluc). The LCMV-Nluc was proved to share similar biological properties with wild-type LCMV and the Nluc intensity reliably reflected viral replication both in vitro and in vivo. Replication of the Nluc-encoding virus in living mice can be visualized by real-time bioluminescent imaging, and bioluminescence can be detected in a variety of organs of infected mice. This work provides a novel approach that enables real-time study of the arenavirus infection and is a convenient and valuable tool for screening of compounds that are active against arenaviruses in vitro and in living mice.

Abstract 3298: Propagation competence of a self-antigen-targeting arenavirus vector based cancer therapy determines antitumor efficacy in mouse melanoma

Abstract Breaking self-tolerance and inducing a robust immune response targeting tumor cell derived self-antigens by therapeutic vaccines has remained challenging. We have generated a replicating viral vector based on the arenavirus lymphocytic choriomeningitis virus expressing the melanoma tissue-specific self-antigen Trp2 (artLCMV-Trp2) and show that a single immunization of mice bearing established melanoma with this vector can break tolerance against this self antigen and induces high levels of tumor-infiltrating TRP2+ CD8+ T cells. These T cells express the effector cytokines IFNγ and TNFα, lack markers of T cell exhaustion, and their appearance coincides with marked inhibition of tumor growth and significant survival benefit. artLCMV-TRP2 induced a strong type I interferon response that resulted in transient type I-dependent Treg inhibition that was necessary for the generation of optimal antitumoral T cell responses. Moreover, artLCMV-TRP2 vaccination proved able to induce complete tumor remission when combined with infusion of exogenous TRP2-specific T cells into mice. In conclusion, replication competence is a key property of artLCMV-TRP2 that enabled effective anti-cancer immunity and therapeutic effect. Citation Format: Mette-Triin Purde, Fabienne Hartmann, Jovana Cupovic, Sarah Schmidt, David Bomze, Felix Stemeseder, Alexander Lercher, Lenka Besse, Fiamma Berner, Thomas Tüting, Andreas Bergthaler, Andrea Schietinger, Stefan Kochanek, Burkhard Ludewig, Klaus K. Orlinger, Tobias Bald, Sandra S. Ring, Lukas Flatz. Propagation competence of a self-antigen-targeting arenavirus vector based cancer therapy determines antitumor efficacy in mouse melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3298.

Screening and identification of Lassa virus endonuclease-targeting inhibitors from a fragment-based drug discovery library

Lassa virus (LASV) belongs to the Old World genus Mammarenavirus, family Arenaviridae, and order Bunyavirales. Arenavirus contains a segmented negative-sense RNA genome, which is in line with the bunyavirus and orthomyxoviruses. The segmented negative-sense RNA viruses utilize a cap-snatching strategy to provide primers cleavaged from the host capped mRNA for viral mRNA transcription. As a similar strategy and the conformational conservation shared with these viruses, the endonuclease (EN) would serve as an attractive target for developing broad-spectrum inhibitors. Using the LASV minigenome (MG) system, we screened a fragment-based drug discovery library and found that two hits, F1204 and F1781, inhibited LASV MG activity. Both hits also inhibited the prototype arenavirus Lymphocytic choriomeningitis virus (LCMV) MG activity. Furthermore, both hits effectively inhibited authentic LCMV and severe fever with thrombocytopenia syndrome virus (SFTSV) infections. Similarly, both hits could inhibit the activity of LASV, LCMV, and SFTSV EN. The combination of either compound with an arenavirus entry inhibitor had significant synergistic antiviral effects. Moreover, both hits were found to be capable of binding to LASV EN with a binding affinity at the micromolar level. These findings provide a basis for developing the hits as potential candidates for the treatment of segmented negative-sense RNA virus infections.

Activation of the STAT3 Signaling Pathway by the RNA-Dependent RNA Polymerase Protein of Arenavirus.

Arenaviruses cause chronic and asymptomatic infections in their natural host, rodents, and several arenaviruses cause severe hemorrhagic fever that has a high mortality in infected humans, seriously threatening public health. There are currently no FDA-licensed drugs available against arenaviruses; therefore, it is important to develop novel antiviral strategies to combat them, which would be facilitated by a detailed understanding of the interactions between the viruses and their hosts. To this end, we performed a transcriptomic analysis on cells infected with arenavirus lymphocytic choriomeningitis virus (LCMV), a neglected human pathogen with clinical significance, and found that the signal transducer and activator of transcription 3 (STAT3) signaling pathway was activated. A further investigation indicated that STAT3 could be activated by the RNA-dependent RNA polymerase L protein (Lp) of LCMV. Our functional analysis found that STAT3 cannot affect LCMV multiplication in A549 cells. We also found that STAT3 was activated by the Lp of Mopeia virus and Junin virus, suggesting that this activation may be conserved across certain arenaviruses. Our study explored the interactions between arenaviruses and STAT3, which may help us to better understand the molecular and cell biology of arenaviruses.

High-Throughput Screening of an FDA-Approved Drug Library Identifies Inhibitors against Arenaviruses and SARS-CoV-2.

Arenaviruses are a large family of enveloped negative-strand RNA viruses that include several causative agents of severe hemorrhagic fevers. Currently, there are no FDA-licensed drugs to treat arenavirus infection except for the off-labeled use of ribavirin. Here, we performed antiviral drug screening against the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) using an FDA-approved drug library. Five drug candidates were identified, including mycophenolic acid, benidipine hydrochloride, clofazimine, dabrafenib, and apatinib, for having strong anti-LCMV effects. Further analysis indicated that benidipine hydrochloride inhibited LCMV membrane fusion, and an adaptive mutation on the LCMV glycoprotein D414 site was found to antagonize the anti-LCMV activity of benidipine hydrochloride. Mycophenolic acid inhibited LCMV replication by depleting GTP production. We also found mycophenolic acid, clofazimine, dabrafenib, and apatinib can inhibit the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Owing to their FDA-approved status, these drug candidates can potentially be used rapidly in the clinical treatment of arenavirus and SARS-CoV-2 infection.

Abstract 4058: TheraT - a highly versatile arenavirus based vector platform for intravenous and intratumoral cancer immunotherapy

Abstract The unprecedented success of checkpoint blockade therapies clearly demonstrates the power of the immune system to fight cancer. Yet, only a minority of cancer patients respond with long-term control of the tumor or even cure, necessitating the development of other treatment modalities. Hookipa Pharma developed a novel attenuated, replication-competent viral vector platform (TheraT) that induces powerful cytotoxic T lymphocyte responses against foreign and self-antigens. In a preclinical model for human papilloma virus 16 (HPV16) associated cancer (TC-1), we evaluated immunogenicity and efficacy upon systemic and intratumoral application of TheraT vectors based on the arenaviruses lymphocytic choriomeningitis virus (LCMV) and pichinde virus (PICV). Both HB-201 (LCMV) and HB-202 (PICV) product candidates encode a non-oncogenic but highly antigenic E6/E7 fusion protein from HPV16. Independent of the route of administration, single administration of HB-201 or HB-202 induced potent peripheral E7-specific CD8+ T cell responses and led to efficient tumor growth control. Survival of TheraT treated animals was significantly longer compared to buffer treated animals. Similarly, single systemic as well as intratumoral application of HB-201 or HB-202 induced major infiltration of CD8+ T cells into the tumor microenvironment. Combination of intravenous HB-201 and anti-PD1 was well tolerated but did not further enhance efficacy in this model, implicating the presence of other immune evasion factors. In conclusion, replication-attenuated TheraT is safe, highly immunogenic and shows excellent therapeutic efficacy after single intravenous and intratumoral application. These data underline the potential and versatility of this novel vector platform. Mechanistic studies in various mouse tumor models are underway. Phase 1/2 clinical trial initiation of HB-201 monotherapy is planned for end of 2019 and preparations for an IND filing for a combination trial of HB-201 and HB-202 in H1 2020 have been initiated. Citation Format: Josipa Raguz, Sarah Schmidt, Theresa Kleisner, Manuel Zerbs, Goran Bekic, Sonja Feher, Daniel Oeler, Felix Stemeseder, Ursula Berka, Bettina Kiefmann, Sophie Schulha, Igor Matushansky, Henning Lauterbach, Klaus Orlinger. TheraT - a highly versatile arenavirus based vector platform for intravenous and intratumoral cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4058.

Arenavirus vectors for massive tumor self-antigen-specific CD8 T cell attack

Abstract Therapeutic vaccination regimens inducing high frequencies of clinically effective tumor-targeting CD8 T cells represent an unmet need in cancer immunotherapy. We exploit cutting-edge viral reverse genetics to harness the immunostimulatory properties of the arenaviruses Pichinde virus and lymphocytic choriomeningitis virus for therapeutic cancer vaccination. When administering to mice life-replicating vector formats of these two viruses, both delivering the same cancer testis self-antigen in a heterologous prime-boost regimen, tumor-specific CD8 T cell responses reached up to 50% of the circulating CD8 T cell pool. This massive CD8 T cell attack eliminated established solid tumors in a significant proportion of animals, accompanied by complete protection against tumor re-challenge. The magnitude of CD8 T cell responses was driven by alarmin signals and depended on the combined use of two genealogically distantly related arenaviruses. Combinations of closely related vectors or the repeated administration of the same vector were not inhibited by vector-neutralizing antibodies, but CD8 T cell immunodominance hierarchies favored vector backbone-targeted responses at the expense of self-reactive T cell specificities. These findings establish a powerful arenavirus-based tumor immunotherapy regimen exploiting key innate signaling pathways and reshuffling immunodominance hierarchies to break tumor self-directed tolerance

Identification of the dietary supplement capsaicin as an inhibitor of Lassa virus entry

The limited treatment options for the increasing occurrence of Lassa hemorrhagic fever in West Africa poses an urgent need for the discovery and development of novel therapeutics. Dietary supplements, especially natural products that are edible and safe for human use, are a good source of drug discovery with potential for uncovering novel applications. In this study, we tested 40 natural products of dietary supplements and identified capsaicin, a common dietary supplement abundant in chili peppers, as an inhibitor of Lassa virus (LASV) entry with EC50 of 6.9–10.0 μmol/L using an HIV based pseudovirus platform. Capsaicin inhibits the entry of five LASV strains but not against the Old World arenavirus lymphocytic choriomeningitis virus (LCMV), showing a preferential activity against LASV. Capsaicin inhibits LASV entry by blocking the pH dependent viral fusion through affecting the stable signal peptide (SSP)-GP2 transmembrane (GP2TM) region of the LASV surface glycoprotein. Mutational study revealed the key residues Ala25, Val431, Phe434 and Val435 in SSP-GP2TM region in capsaicin's antiviral effect. This study for the first time reveals a direct acting antiviral effect of capsaicin against the hemorrhagic fever causing LASV, providing detailed interaction hot spots in the unique SSP-GP2TM interface of LASV glycoprotein that is crucial in fusion inhibition, and offering a new strategy in discovering and developing antivirals from natural products that are safe for human use.

Live-attenuated LCMV-based vector for active immunotherapy of HPV16+ cancer.

e14303 Background: Active immunization against cancer requires the induction of exquisitely potent tumor-specific CD8+ T cell (CTL) responses, which can be repeatedly boosted and reactivated. Hookipa Pharma engineered a replication-attenuated viral vector platform (TheraT) based on the arenavirus lymphocytic choriomeningitis virus (LCMV), which meets these aforementioned criteria. Here, we present a preclinical data package of Hookipa´s lead immunotherapy product HB-201, targeting HPV16-driven cancer. Methods: TheraT-E7E6 encodes a highly immunogenic, non-oncogenic version of the human papilloma virus 16 (HPV16) oncoproteins E7 and E6. Results: Attenuation and safety of TheraT-E7E6 were demonstrated by i) rapid viral clearance after systemic administration of the vector and ii) reduced neurovirulence in mice.TheraT-E7E6 can be administered intravenously as systemic therapy or intratumorally as local therapy with an abscopal-like effect. Treatment was shown to induce substantial CD8+ T cell expansion and high frequencies of E7- and E6-specific CTL responses with a balanced effector / central memory profile. These responses were boosted and reactivated upon TheraT-E7E6 re-administration. TheraT-E7E6 eradicated palpable tumors in a syngeneic mouse TC-1 tumor model of HPV-driven cancer. Even in mice with large tumors (~300mm3) TheraT-E7E6 afforded significant tumor control and improved survival, with high frequencies of E7-specific CTLs persisting for several weeks. Animals which cleared the tumor after TheraT therapy were long term protected from tumor re-challenge. Furthermore, TheraT is efficacious in checkpoint inhibitory refractory models and can also synergize in combination with checkpoint inhibitors (CPIs). Conclusions: In conclusion, replication-attenuated TheraT-E7E6 is safe, highly immunogenic and shows excellent therapeutic efficacy as mono- or combination therapy in a preclinical model of HPV-induced cancer. Clinical trials for the treatment of HPV16+ cancers will be initiated in 2019.

Abstract B131: Live-attenuated LCMV-based vector for active immunotherapy of HPV16+ cancer

Abstract Active immunization against cancer requires the induction of exquisitely potent tumor-specific CD8+ T-cell (CTL) responses, which can be repeatedly boosted and reactivated. Hookipa Pharma engineered a replication-attenuated viral vector platform (TheraT®) based on the arenavirus lymphocytic choriomeningitis virus (LCMV), which meets these aforementioned criteria. Here, we present a preclinical data package of Hookipa´s lead immunotherapy product HB-201, targeting HPV16-driven head and neck cancer. It encodes a non-oncogenic version of the human papilloma virus 16 (HPV16) oncoproteins E7 and E6 (TheraT®-E7E6). Attenuation and safety of TheraT®-E7E6 were demonstrated by i) rapid viral clearance after systemic administration of the vector and ii) reduced neurovirulence in mice. TheraT®-E7E6 treatment was shown to induce substantial CD8+ T-cell expansion and high frequencies of E7- and E6-specific CTL responses with a balanced effector / central memory profile. These responses were boosted and reactivated upon TheraT®-E7E6 re-administration. TheraT®-E7E6 eradicated palpable TC-1 tumors, a syngeneic mouse tumor model of HPV-driven cancer. Even in mice with large tumors (~300mm3), TheraT®-E7E6 afforded significant tumor control and improved survival, with high frequencies of E7-specific CTLs persisting for several weeks. Furthermore, the efficacy of TheraT®-E7E6 can be potentiated in combination therapies, e.g., with PD-1 checkpoint inhibitors. In conclusion, replication-attenuated TheraT®-E7E6 is safe, highly immunogenic and shows excellent therapeutic efficacy as mono- or combination therapy in a preclinical model of HPV-induced cancer. Clinical trials for the treatment of HPV16+ cancers will be initiated in 2019. Citation Format: Sarah Schmidt, Ahmed ElGazzar, Weldy V. Bonilla, Mindaugas Pauzuolis, Andrea Reiter, Theresa Kleissner, Daniel Oeler, Felix Stemeseder, Ursula Berka, Bettina Kiefmann, Sophie Schulha, Igor Matushansky, Doron Merkler, Daniel Pinschewer, Klaus Orlinger. Live-attenuated LCMV-based vector for active immunotherapy of HPV16+ cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B131.

Mining a Kröhnke Pyridine Library for Anti-Arenavirus Activity.

Several arenaviruses cause hemorrhagic fever (HF) disease in humans and represent important public health problems in their endemic regions. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus is a neglected human pathogen of clinical significance. There are no licensed arenavirus vaccines, and current antiarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel therapeutics to combat human pathogenic arenaviruses, a task that will be facilitated by the identification of compounds with antiarenaviral activity that could serve as probes to identify arenavirus-host interactions suitable for targeting, as well as lead compounds to develop future antiarenaviral drugs. Screening of a combinatorial library of Krönhke pyridines identified compound KP-146 [(5-(5-(2,3-dihydrobenzo[ b][1,4] dioxin-6-yl)-4'-methoxy-[1,1'-biphenyl]-3-yl)thiophene-2-carboxamide] as having strong anti-lymphocytic choriomeningitis virus (LCMV) activity in cultured cells. KP-146 did not inhibit LCMV cell entry but rather interfered with the activity of the LCMV ribonucleoprotein (vRNP) responsible for directing virus RNA replication and gene transcription, as well as with the budding process mediated by the LCMV matrix Z protein. LCMV variants with increased resistance to KP-146 did not emerge after serial passages in the presence of KP-146. Our findings support the consideration of Kröhnke pyridine scaffold as a valuable source to identify compounds that could serve as tools to dissect arenavirus-host interactions, as well as lead candidate structures to develop antiarenaviral drugs.

Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein.

RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.

A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV.IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.

Spatiotemporally restricted arenavirus replication induces immune surveillance and type I interferon-dependent tumour regression

Immune-mediated effector molecules can limit cancer growth, but lack of sustained immune activation in the tumour microenvironment restricts antitumour immunity. New therapeutic approaches that induce a strong and prolonged immune activation would represent a major immunotherapeutic advance. Here we show that the arenaviruses lymphocytic choriomeningitis virus (LCMV) and the clinically used Junin virus vaccine (Candid#1) preferentially replicate in tumour cells in a variety of murine and human cancer models. Viral replication leads to prolonged local immune activation, rapid regression of localized and metastatic cancers, and long-term disease control. Mechanistically, LCMV induces antitumour immunity, which depends on the recruitment of interferon-producing Ly6C+ monocytes and additionally enhances tumour-specific CD8+ T cells. In comparison with other clinically evaluated oncolytic viruses and to PD-1 blockade, LCMV treatment shows promising antitumoural benefits. In conclusion, therapeutically administered arenavirus replicates in cancer cells and induces tumour regression by enhancing local immune responses.

Development of live-attenuated arenavirus vaccines based on codon deoptimization of the viral glycoprotein

Several arenaviruses, chiefly Lassa (LASV) in West Africa, cause hemorrhagic fever (HF) disease in humans and pose important public health problems in their endemic regions. To date, there are no FDA-approved arenavirus vaccines and current anti-arenaviral therapy is limited to the use of ribavirin that has very limited efficacy. In this work we document that a recombinant prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) with a codon deoptimized (CD) surface glycoprotein (GP), rLCMV/CD, exhibited wild type (WT)-like growth properties in cultured cells despite barely detectable GP expression levels in rLCMV/CD-infected cells. Importantly, rLCMV/CD was highly attenuated in vivo but able to induce complete protection against a subsequent lethal challenge with rLCMV/WT. Our findings support the feasibility of implementing an arenavirus GP CD-based approach for the development of safe and effective live-attenuated vaccines (LAVs) to combat diseases caused by human pathogenic arenaviruses.

The High Degree of Sequence Plasticity of the Arenavirus Noncoding Intergenic Region (IGR) Enables the Use of a Nonviral Universal Synthetic IGR To Attenuate Arenaviruses.

Hemorrhagic fever arenaviruses (HFAs) pose important public health problems in regions where they are endemic. Concerns about human-pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and because current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. We have recently shown that the noncoding intergenic region (IGR) present in each arenavirus genome segment, the S and L segments (S-IGR and L-IGR, respectively), plays important roles in the control of virus protein expression and that this knowledge could be harnessed for the development of live-attenuated vaccine strains to combat HFAs. In this study, we further investigated the sequence plasticity of the arenavirus IGR. We demonstrate that recombinants of the prototypic arenavirus lymphocytic choriomeningitis virus (rLCMVs), whose S-IGRs were replaced by the S-IGR of Lassa virus (LASV) or an entirely nonviral S-IGR-like sequence (Ssyn), are viable, indicating that the function of S-IGR tolerates a high degree of sequence plasticity. In addition, rLCMVs whose L-IGRs were replaced by Ssyn or S-IGRs of the very distantly related reptarenavirus Golden Gate virus (GGV) were viable and severely attenuated in vivo but able to elicit protective immunity against a lethal challenge with wild-type LCMV. Our findings indicate that replacement of L-IGR by a nonviral Ssyn could serve as a universal molecular determinant of arenavirus attenuation. Hemorrhagic fever arenaviruses (HFAs) cause high rates of morbidity and mortality and pose important public health problems in regions where they are endemic. Implementation of live-attenuated vaccines (LAVs) will represent a major step to combat HFAs. Here we document that the arenavirus noncoding intergenic region (IGR) has a high degree of plasticity compatible with virus viability. This observation led us to generate recombinant LCMVs containing nonviral synthetic IGRs. These rLCMVs were severely attenuated in vivo but able to elicit protective immunity against a lethal challenge with wild-type LCMV. These nonviral synthetic IGRs can be used as universal molecular determinants of arenavirus attenuation for the rapid development of safe and effective, as well as stable, LAVs to combat HFA.

Tropism of CPMV to Professional Antigen Presenting Cells Enables a Platform to Eliminate Chronic Infections.

Chronic viral infections (e.g., HIV, HBV, HCV) represent a significant source of morbidity and mortality with over 500 million people infected worldwide. Dendritic cells (DCs) and macrophages are key cell types for productive viral replication and persistent systemic infection. We demonstrate that the plant virus cowpea mosaic virus (CPMV) displays tropism for such antigen presenting cells in both mice and humans, thus making it an ideal candidate for targeted drug delivery toward viral infections. Furthermore, we show inhibition of a key host protein for viral infection, site-1 protease (S1P), using the small molecule PF-429242 in the model pathogen arenavirus lymphocytic choriomeningitis virus (LCMV) limits viral growth. By packaging PF-429242 in CPMV, we are able to control drug release and efficiently deliver the drug. This sets the groundwork for utilizing the natural tropism of CPMV for a therapeutic approach that specifically targets cell types most commonly subverted by chronic viruses.

General Molecular Strategy for Development of Arenavirus Live-Attenuated Vaccines.

Hemorrhagic fever arenaviruses (HFA) pose important public health problems in regions where they are endemic. Thus, Lassa virus (LASV) infects several hundred thousand individuals yearly in West Africa, causing a large number of Lassa fever cases associated with high morbidity and mortality. Concerns about human-pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and because current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. The Mopeia virus (MOPV)/LASV reassortant (ML29) is a LASV candidate live-attenuated vaccine (LAV) that has shown promising results in animal models. Nevertheless, the mechanism of ML29 attenuation remains unknown, which raises concerns about the phenotypic stability of ML29 in response to additional mutations. Development of LAVs based on well-defined molecular mechanisms of attenuation will represent a major step in combatting HFA. We used the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) to develop a general molecular strategy for arenavirus attenuation. Our approach involved replacement of the noncoding intergenic region (IGR) of the L genome segment with the IGR of the S genome segment to generate a recombinant LCMV, rLCMV(IGR/S-S), that was highly attenuated in vivo but induced protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV(IGR/S-S) was associated with a stable reorganization of the control of viral gene expression. This strategy can facilitate the rapid development of LAVs with the antigenic composition of the parental HFA and a mechanism of attenuation that minimizes concerns about increased virulence that could be caused by genetic changes in the LAV. Hemorrhagic fever arenaviruses (HFA) cause high morbidity and mortality, and pose important public health problems in the regions where they are endemic. Implementation of live-attenuated vaccines (LAV) will represent a major step in combatting HFA. Here we have used the prototypic arenavirus LCMV to document a general molecular strategy for arenavirus attenuation that can facilitate the rapid development of safe and effective, as well as stable, LAV to combat HFA.

Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication.

Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed arenavirus vaccines and current antiarenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name: N-[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong anti-LCMV activity in the absence of cell toxicity. Mechanism-of-action studies revealed that F3406 inhibited LCMV cell entry by specifically interfering with the pH-dependent fusion in the endosome compartment that is mediated by LCMV glycoprotein GP2 and required to release the virus ribonucleoprotein into the cell cytoplasm to initiate transcription and replication of the virus genome. We identified residue M437 within the transmembrane domain of GP2 as critical for virus susceptibility to F3406. Hemorrhagic fever arenaviruses (HFA) are important human pathogens that cause high morbidity and mortality in areas where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Concerns posed by arenavirus infections are aggravated by the lack of U.S. Food and Drug Administration-licensed arenavirus vaccines and current antiarenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. Here we describe a novel recombinant LCMV and its use to develop a cell-based assay suitable for HTS to rapidly identify inhibitors arenavirus multiplication. The concepts and experimental strategies we describe in this work provide the bases for the rapid identification and characterization of novel anti-HFA therapeutics.

Arenavirus Genome Rearrangement for the Development of Live Attenuated Vaccines.

Several members of the Arenaviridae family cause hemorrhagic fever disease in humans and pose serious public health problems in their geographic regions of endemicity as well as a credible biodefense threat. To date, there have been no FDA-approved arenavirus vaccines, and current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. Arenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome. Each genome segment uses an ambisense coding strategy to direct the synthesis of two viral polypeptides in opposite orientations, separated by a noncoding intergenic region. Here we have used minigenome-based approaches to evaluate expression levels of reporter genes from the nucleoprotein (NP) and glycoprotein precursor (GPC) loci within the S segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). We found that reporter genes are expressed to higher levels from the NP than from the GPC locus. Differences in reporter gene expression levels from the NP and GPC loci were confirmed with recombinant trisegmented LCM viruses. We then used reverse genetics to rescue a recombinant LCMV (rLCMV) containing a translocated viral S segment (rLCMV/TransS), where the viral NP and GPC open reading frames replaced one another. The rLCMV/TransS showed slower growth kinetics in cultured cells and was highly attenuated in vivo in a mouse model of lethal LCMV infection, but immunization with rLCMV/TransS conferred complete protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV/TransS was associated with reduced NP expression levels. These results open a new avenue for the development of arenavirus live attenuated vaccines based on rearrangement of their viral genome. Several arenaviruses cause severe hemorrhagic fever in humans and also pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections and antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and associated with side effects. Here we describe, for the first time, the generation of a recombinant LCMV where the viral protein products encoded by the S RNA segment (NP and GPC) were swapped to generate rLCMV/TransS. rLCMV/TransS exhibited reduced viral multiplication in cultured cells and was highly attenuated in vivo while conferring protection, upon a single immunization dose, against a lethal challenge with wild-type LCMV. Our studies provide a proof of concept for the rational development of safe and protective live attenuated vaccine candidates based on genome reorganization for the treatment of pathogenic arenavirus infections in humans.