Deep mutational scanning reveals functional constraints and antibody-escape potential of Lassa virus glycoprotein complex.

Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of the Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we used pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affected cell entry and antibody neutralization. Our experiments defined functional constraints throughout GPC. We quantified how GPC mutations affected neutralization with a panel of monoclonal antibodies. All antibodies tested were escaped by mutations that existed among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid the design of therapeutics and vaccines.

Potent immunogenicity and protective efficacy of a multi-pathogen vaccination targeting Ebola, Sudan, Marburg and Lassa viruse.

Viral haemorrhagic fevers (VHF) pose a significant threat to human health. In recent years, VHF outbreaks caused by Ebola, Marburg and Lassa viruses have caused substantial morbidity and mortality in West and Central Africa. In 2022, an Ebola disease outbreak in Uganda caused by Sudan virus resulted in 164 cases with 55 deaths. In 2023, a Marburg disease outbreak was confirmed in Equatorial Guinea and Tanzania resulting in over 49 confirmed or suspected cases; 41 of which were fatal. There are no clearly defined correlates of protection against these VHF, impeding targeted vaccine development. Any vaccine developed should therefore induce strong and preferably long-lasting humoral and cellular immunity against these viruses. Ideally this immunity should also cross-protect against viral variants, which are known to circulate in animal reservoirs and cause human disease. We have utilized two viral vectored vaccine platforms, an adenovirus (ChAdOx1) and Modified Vaccinia Ankara (MVA), to develop a multi-pathogen vaccine regime against three filoviruses (Ebola virus, Sudan virus, Marburg virus) and an arenavirus (Lassa virus). These platform technologies have consistently demonstrated the capability to induce robust cellular and humoral antigen-specific immunity in humans, most recently in the rollout of the licensed ChAdOx1-nCoV19/AZD1222. Here, we show that our multi-pathogen vaccines elicit strong cellular and humoral immunity, induce a diverse range of chemokines and cytokines, and most importantly, confers protection after lethal Ebola virus, Sudan virus and Marburg virus challenges in a small animal model.

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.

Rapid protection induced by a single-shot Lassa vaccine in male cynomolgus monkeys.

Lassa fever hits West African countries annually in the absence of licensed vaccine to limit the burden of this viral hemorrhagic fever. We previously developed MeV-NP, a single-shot vaccine protecting cynomolgus monkeys against divergent strains one month or more than a year before Lassa virus infection. Given the limited dissemination area during outbreaks and the risk of nosocomial transmission, a vaccine inducing rapid protection could be useful to protect exposed people during outbreaks in the absence of preventive vaccination. Here, we test whether the time to protection can be reduced after immunization by challenging measles virus pre-immune male cynomolgus monkeys sixteen or eight days after a single shot of MeV-NP. None of the immunized monkeys develop disease and they rapidly control viral replication. Animals immunized eight days before the challenge are the best controllers, producing a strong CD8 T-cell response against the viral glycoprotein. A group of animals was also vaccinated one hour after the challenge, but was not protected and succumbed to the disease as the control animals. This study demonstrates that MeV-NP can induce a rapid protective immune response against Lassa fever in the presence of MeV pre-existing immunity but can likely not be used as therapeutic vaccine.

Construction and Immunological Evaluation of an Adenoviral Vector-Based Vaccine Candidate for Lassa Fever.

Lassa virus (LASV) is a rodent-borne arenavirus circulating in West African regions that causes Lassa fever (LF). LF is normally asymptomatic at the initial infection stage, but can progress to severe disease with multiorgan collapse and hemorrhagic fever. To date, the therapeutic choices are limited, and there is no approved vaccine for avoiding LASV infection. Adenoviral vector-based vaccines represent an effective countermeasure against LASV because of their safety and adequate immunogenicity, as demonstrated in use against other emerging viral infections. Here, we constructed and characterized a novel Ad5 (E1-, E3-) vectored vaccine containing the glycoprotein precursor (GPC) of LASV. Ad5-GPCLASV elicited both humoral and cellular immune responses in BALB/c mice. Moreover, a bioluminescent imaging-based BALB/c mouse model infected with GPC-bearing and luciferase-expressing replication-incompetent LASV pseudovirus was utilized to evaluate the vaccine efficacy. The bioluminescence intensity of immunized mice was significantly lower than that of control mice after being inoculated with LASV pseudovirus. This study suggests that Ad5-GPCLASV represents a potential vaccine candidate against LF.

Multivalent DNA Vaccines as A Strategy to Combat Multiple Concurrent Epidemics: Mosquito-Borne and Hemorrhagic Fever Viruses.

The emergence of multiple concurrent infectious diseases localized in the world creates a complex burden on global public health systems. Outbreaks of Ebola, Lassa, and Marburg viruses in overlapping regions of central and West Africa and the co-circulation of Zika, Dengue, and Chikungunya viruses in areas with A. aegypti mosquitos highlight the need for a rapidly deployable, safe, and versatile vaccine platform readily available to respond. The DNA vaccine platform stands out as such an application. Here, we present proof-of-concept studies from mice, guinea pigs, and nonhuman primates for two multivalent DNA vaccines delivered using in vivo electroporation (EP) targeting mosquito-borne (MMBV) and hemorrhagic fever (MHFV) viruses. Immunization with MMBV or MHFV vaccines via intradermal EP delivery generated robust cellular and humoral immune responses against all target viral antigens in all species. MMBV vaccine generated antigen-specific binding antibodies and IFNγ-secreting lymphocytes detected in NHPs up to six months post final immunization, suggesting induction of long-term immune memory. Serum from MHFV vaccinated NHPs demonstrated neutralizing activity in Ebola, Lassa, and Marburg pseudovirus assays indicating the potential to offer protection. Together, these data strongly support and demonstrate the versatility of DNA vaccines as a multivalent vaccine development platform for emerging infectious diseases.

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).

Systemic viral spreading and defective host responses are associated with fatal Lassa fever in macaques.

Lassa virus (LASV) is endemic in West Africa and induces a viral hemorrhagic fever (VHF) with up to 30% lethality among clinical cases. The mechanisms involved in control of Lassa fever or, in contrast, the ensuing catastrophic illness and death are poorly understood. We used the cynomolgus monkey model to reproduce the human disease with asymptomatic to mild or fatal disease. After initial replication at the inoculation site, LASV reached the secondary lymphoid organs. LASV did not spread further in nonfatal disease and was rapidly controlled by balanced innate and T-cell responses. Systemic viral dissemination occurred during severe disease. Massive replication, a cytokine/chemokine storm, defective T-cell responses, and multiorgan failure were observed. Clinical, biological, immunological, and transcriptomic parameters resembled those observed during septic-shock syndrome, suggesting that similar pathogenesis is induced during Lassa fever. The outcome appears to be determined early, as differentially expressed genes in PBMCs were associated with fatal and non-fatal Lassa fever outcome very early after infection. These results provide a full characterization and important insights into Lassa fever pathogenesis and could help to develop early diagnostic tools.

Lassa Fever among Children in Eastern Province, Sierra Leone: A 7-year Retrospective Analysis (2012-2018).

Pediatric Lassa fever (LF) usually presents as a nonspecific febrile illness, similar to other endemic diseases in countries like Sierra Leone, where LF is considered to be hyperendemic. The nonspecificity of presentation and lack of research have made it difficult to fully understand best practices for pediatric management. We aim to describe clinical characteristics of hospitalized pediatric patients suspected or diagnosed with LF and assess factors associated with hospital outcomes among those with LF antigen-positive results. We conducted a 7-year retrospective cohort study using routine data for all children younger than 18 years admitted at the Kenema Government Hospital's LF ward. A total of 292 children with suspected or confirmed LF were analyzed. Overall, mortality was high (21%). Children with antigen-positive results had a high case fatality rate of 63% (P < 0.01). In univariate analyses, children who presented with unexplained bleeding (odds ratio [OR]: 3.58; 95% CI: 1.08-11.86; P = 0.040) and confusion (altered sensorium) (OR: 5.37; 95% CI: 1.34-21.48; P = 0.020) had increased odds of death. Abnormal serum levels of alanine aminotransferase (P = 0.001), creatinine (P = 0.004), and potassium (P = 0.003) were associated with increased likelihood of death in these children. Treatment with ribavirin was not significantly associated with survival (P = 0.916). Our findings provide insights into current pediatric LF clinical presentation and management. More evidence-based, high-quality research in creating predictive algorithms of antigen-positivity and hospital outcomes is needed in the management of pediatric LF.

Innate Immune Responses to Acute Viral Infection During Pregnancy.

Immunological adaptations in pregnancy allow maternal tolerance of the semi-allogeneic fetus but also increase maternal susceptibility to infection. At implantation, the endometrial stroma, glands, arteries and immune cells undergo anatomical and functional transformation to create the decidua, the specialized secretory endometrium of pregnancy. The maternal decidua and the invading fetal trophoblast constitute a dynamic junction that facilitates a complex immunological dialogue between the two. The decidual and peripheral immune systems together assume a pivotal role in regulating the critical balance between tolerance and defense against infection. Throughout pregnancy, this equilibrium is repeatedly subjected to microbial challenge. Acute viral infection in pregnancy is associated with a wide spectrum of adverse consequences for both mother and fetus. Vertical transmission from mother to fetus can cause developmental anomalies, growth restriction, preterm birth and stillbirth, while the mother is predisposed to heightened morbidity and maternal death. A rapid, effective response to invasive pathogens is therefore essential in order to avoid overwhelming maternal infection and consequent fetal compromise. This sentinel response is mediated by the innate immune system: a heritable, highly evolutionarily conserved system comprising physical barriers, antimicrobial peptides (AMP) and a variety of immune cells-principally neutrophils, macrophages, dendritic cells, and natural killer cells-which express pattern-receptors that detect invariant molecular signatures unique to pathogenic micro-organisms. Recognition of these signatures during acute infection triggers signaling cascades that enhance antimicrobial properties such as phagocytosis, secretion of pro-inflammatory cytokines and activation of the complement system. As well as coordinating the initial immune response, macrophages and dendritic cells present microbial antigens to lymphocytes, initiating and influencing the development of specific, long-lasting adaptive immunity. Despite extensive progress in unraveling the immunological adaptations of pregnancy, pregnant women remain particularly susceptible to certain acute viral infections and continue to experience mortality rates equivalent to those observed in pandemics several decades ago. Here, we focus specifically on the pregnancy-induced vulnerabilities in innate immunity that contribute to the disproportionately high maternal mortality observed in the following acute viral infections: Lassa fever, Ebola virus disease (EVD), dengue fever, hepatitis E, influenza, and novel coronavirus infections.

Lassa virus antigen distribution and inflammation in the ear of infected strain 13/N Guinea pigs.

Sudden-onset sensorineuronal hearing loss (SNHL) is reported in approximately one-third of survivors of Lassa fever (LF) and remains the most prominent cause of Lassa virus (LASV)-associated morbidity in convalescence. Using a guinea pig model of LF, and incorporating animals from LASV vaccine trials, we investigated viral antigen distribution and histopathology in the ear of infected animals to elucidate the pathogenesis of hearing loss associated with LASV infection. Antigen was detected only in animals that succumbed to disease and was found within structures of the inner ear that are intimately associated with neural detection and/or translation of auditory stimuli and in adjacent vasculature. No inflammation or viral cytopathic changes were observed in the inner ear or surrounding structures in these animals. In contrast, no viral antigen was detected in the ear of surviving animals. However, all survivors that exhibited clinical signs of disease during the course of infection developed perivascular mononuclear inflammation within and adjacent to the ear, indicating an ongoing inflammatory response in these animals that may contribute to hearing loss. These data contribute to the knowledge of LASV pathogenesis in the auditory system, support an immune-mediated process resulting in LASV-associated hearing loss, and demonstrate that vaccination protecting animals from clinical disease can also prevent infection-associated auditory pathology.

Antibodies from Sierra Leonean and Nigerian Lassa fever survivors cross-react with recombinant proteins representing Lassa viruses of divergent lineages.

Lassa virus (LASV) is the causative agent of Lassa fever, an often-fatal hemorrhagic disease that is endemic in West Africa. Seven genetically distinct LASV lineages have been identified. As part of CEPI's (Coalition for Epidemic Preparedness Innovations) Lassa vaccine development program, we assessed the potential of the human immune system to mount cross-reactive and cross-protective humoral immune responses to antigens from the most prevalent LASV lineages, which are lineages II and III in Nigeria and lineage IV in Sierra Leone. IgG and IgM present in the blood of Lassa fever survivors from Nigeria or Sierra Leone exhibited substantial cross-reactivity for binding to LASV nucleoprotein and two engineered (linked and prefusion) versions of the glycoproteins (GP) of lineages II-IV. There was less cross-reactivity for the Zinc protein. Serum or plasma from Nigerian Lassa fever survivors neutralized LASV pseudoviruses expressing lineage II GP better than they neutralized lineage III or IV GP expressing pseudoviruses. Sierra Leonean survivors did not exhibit a lineage bias. Neutralization titres determined using LASV pseudovirus assays showed significant correlation with titres determined by plaque reduction with infectious LASV. These studies provide guidance for comparison of humoral immunity to LASV of distinct lineages following natural infection or immunization.

Effect of Strain Variations on Lassa Virus Z Protein-Mediated Human RIG-I Inhibition.

Mammarenaviruses include several known human pathogens, such as the prototypic lymphocytic choriomeningitis virus (LCMV) that can cause neurological diseases and Lassa virus (LASV) that causes endemic hemorrhagic fever infection. LASV-infected patients show diverse clinical manifestations ranging from asymptomatic infection to hemorrhage, multi-organ failures and death, the mechanisms of which have not been well characterized. We have previously shown that the matrix protein Z of pathogenic arenaviruses, including LASV and LCMV, can strongly inhibit the ability of the innate immune protein RIG-I to suppress type I interferon (IFN-I) expression, which serves as a mechanism of viral immune evasion and virulence. Here, we show that Z proteins of diverse LASV isolates derived from rodents and humans have a high degree of sequence variations at their N- and C-terminal regions and produce variable degrees of inhibition of human RIG-I (hRIG-I) function in an established IFN-ß promoter-driven luciferase (LUC) reporter assay. Additionally, we show that Z proteins of four known LCMV strains can also inhibit hRIG-I at variable degrees of efficiency. Collectively, our results confirm that Z proteins of pathogenic LASV and LCMV can inhibit hRIG-I and suggest that strain variations of the Z proteins can influence their efficiency to suppress host innate immunity that might contribute to viral virulence and disease heterogeneity.

Severe Human Lassa Fever Is Characterized by Nonspecific T-Cell Activation and Lymphocyte Homing to Inflamed Tissues.

Lassa fever (LF) is a zoonotic viral hemorrhagic fever caused by Lassa virus (LASV), which is endemic to West African countries. Previous studies have suggested an important role for T-cell-mediated immunopathology in LF pathogenesis, but the mechanisms by which T cells influence disease severity and outcome are not well understood. Here, we present a multiparametric analysis of clinical immunology data collected during the 2017-2018 Lassa fever outbreak in Nigeria. During the acute phase of LF, we observed robust activation of the polyclonal T-cell repertoire, which included LASV-specific and antigenically unrelated T cells. However, severe and fatal LF cases were characterized by poor LASV-specific effector T-cell responses. Severe LF was also characterized by the presence of circulating T cells with homing capacity to inflamed tissues, including the gut mucosa. These findings in LF patients were recapitulated in a mouse model of LASV infection, in which mucosal exposure resulted in remarkably high lethality compared to skin exposure. Taken together, our findings indicate that poor LASV-specific T-cell responses and activation of nonspecific T cells with homing capacity to inflamed tissues are associated with severe LF.IMPORTANCE Lassa fever may cause severe disease in humans, in particular in areas of endemicity like Sierra Leone and Nigeria. Despite its public health importance, the pathophysiology of Lassa fever in humans is poorly understood. Here, we present clinical immunology data obtained in the field during the 2018 Lassa fever outbreak in Nigeria indicating that severe Lassa fever is associated with activation of T cells antigenically unrelated to Lassa virus and poor Lassa virus-specific effector T-cell responses. Mechanistically, we show that these bystander T cells express defined tissue homing signatures that suggest their recruitment to inflamed tissues and a putative role of these T cells in immunopathology. These findings open a window of opportunity to consider T-cell targeting as a potential postexposure therapeutic strategy against severe Lassa fever, a hypothesis that could be tested in relevant animal models, such as nonhuman primates.

In-silico identification of the vaccine candidate epitopes against the Lassa virus hemorrhagic fever.

Lassa virus (LASV), a member of the Arenaviridae, is an ambisense RNA virus that causes severe hemorrhagic fever with a high fatality rate in humans in West and Central Africa. Currently, no FDA approved drugs or vaccines are available for the treatment of LASV fever. The LASV glycoprotein complex (GP) is a promising target for vaccine or drug development. It is situated on the virion envelope and plays key roles in LASV growth, cell tropism, host range, and pathogenicity. In an effort to discover new LASV vaccines, we employ several sequence-based computational prediction tools to identify LASV GP major histocompatibility complex (MHC) class I and II T-cell epitopes. In addition, many sequence- and structure-based computational prediction tools were used to identify LASV GP B-cell epitopes. The predicted T- and B-cell epitopes were further filtered based on the consensus approach that resulted in the identification of thirty new epitopes that have not been previously tested experimentally. Epitope-allele complexes were obtained for selected strongly binding alleles to the MHC-I T-cell epitopes using molecular docking and the complexes were relaxed with molecular dynamics simulations to investigate the interaction and dynamics of the epitope-allele complexes. These predictions provide guidance to the experimental investigations and validation of the epitopes with the potential for stimulating T-cell responses and B-cell antibodies against LASV and allow the design and development of LASV vaccines.

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.

A Lassa Virus Live-Attenuated Vaccine Candidate Based on Rearrangement of the Intergenic Region.

Lassa virus (LASV) poses a significant public health problem within the regions of Lassa fever endemicity in Western Africa. LASV infects several hundred thousand individuals yearly, and a considerable number of Lassa fever cases are associated with high morbidity and lethality. No approved LASV vaccine is available, and current therapy is limited to an off-label usage of ribavirin that is only partially effective and associated with significant side effects. The impact of Lassa fever on human health, together with the limited existing countermeasures, highlights the importance of developing effective vaccines against LASV. Here, we present the development and characterization of a recombinant LASV (rLASV) vaccine candidate [rLASV(IGR/S-S)], which is based on the presence of the noncoding intergenic region (IGR) of the small (S) genome segment (S-IGR) in both large (L) and S LASV segments. In cultured cells, rLASV(IGR/S-S) was modestly less fit than wild-type rLASV (rLASV-WT). rLASV(IGR/S-S) was highly attenuated in guinea pigs, and a single subcutaneous low dose of the virus completely protected against otherwise lethal infection with LASV-WT. Moreover, rLASV(IGR/S-S) was genetically stable during serial passages in cultured cells. These findings indicate that rLASV(IGR/S-S) can be developed into a LASV live-attenuated vaccine (LAV) that has the same antigenic composition as LASV-WT and a well-defined mechanism of attenuation that overcomes concerns about increased virulence that could be caused by genetic changes in the LAV during multiple rounds of multiplication.IMPORTANCE Lassa virus (LASV), the causative agent of Lassa fever, infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever cases. No U.S. Food and Drug Administration-licensed countermeasures are available to prevent or treat LASV infection. We describe the generation of a novel LASV live-attenuated vaccine candidate rLASV(IGR/S-S), which is based on the replacement of the large genomic segment noncoding intergenic region (IGR) with that of the small genome segment. rLASV(IGR/S-S) is less fit in cell culture than wild-type virus and does not cause clinical signs in inoculated guinea pigs. Importantly, rLASV(IGR/S-S) protects immunized guinea pigs against an otherwise lethal exposure to LASV.

A Lassa Fever Live-Attenuated Vaccine Based on Codon Deoptimization of the Viral Glycoprotein Gene.

Lassa virus (LASV) is endemic in Western Africa and is estimated to infect hundreds of thousands of individuals annually. A considerable number of these infections result in Lassa fever (LF), which is associated with significant morbidity and a case-fatality rate as high as 69% among hospitalized confirmed patients. U.S. Food and Drug Administration-approved LF vaccines are not available. Current antiviral treatment is limited to off-label use of a nucleoside analogue, ribavirin, that is only partially effective and associated with significant side effects. We generated and characterized a recombinant LASV expressing a codon-deoptimized (CD) glycoprotein precursor gene (GPC), rLASV-GPC/CD. Comparison of growth kinetics and peak titers showed that rLASV-GPC/CD is slightly attenuated in cell culture compared to wild-type (WT) recombinant LASV (rLASV-WT). However, rLASV-GPC/CD is highly attenuated in strain 13 and Hartley guinea pigs, as reflected by the absence of detectable clinical signs in animals inoculated with rLASV-GPC/CD. Importantly, a single subcutaneous dose of rLASV-GPC/CD provides complete protection against an otherwise lethal exposure to LASV. Our results demonstrate the feasibility of implementing a CD approach for developing a safe and effective LASV live-attenuated vaccine candidate. Moreover, rLASV-GPC/CD might provide investigators with a tool to safely study LASV outside maximum (biosafety level 4) containment, which could accelerate the elucidation of basic aspects of the molecular and cell biology of LASV and the development of novel LASV medical countermeasures.IMPORTANCE Lassa virus (LASV) infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever (LF) cases. Licensed LF vaccines are not available, and anti-LF therapy is limited to off-label use of the nucleoside analog ribavirin with uncertain efficacy. We describe the generation of a novel live-attenuated LASV vaccine candidate. This vaccine candidate is based on mutating wild-type (WT) LASV in a key region of the viral genome, the glycoprotein precursor (GPC) gene. These mutations do not change the encoded GPC but interfere with its production in host cells. This mutated LASV (rLASV-GPC/CD) behaves like WT LASV (rLASV-WT) in cell culture, but in contrast to rLASV-WT, does not cause disease in inoculated guinea pigs. Guinea pigs immunized with rLASV-GPC/CD were protected against an otherwise lethal exposure to WT LASV. Our results support the testing of this candidate vaccine in nonhuman primate models ofLF.

Lassa Virus, but Not Highly Pathogenic New World Arenaviruses, Restricts Immunostimulatory Double-Stranded RNA Accumulation during Infection.

The arenaviruses Lassa virus (LASV), Junín virus (JUNV), and Machupo virus (MACV) can cause severe and fatal diseases in humans. Although these pathogens are closely related, the host immune responses to these virus infections differ remarkably, with direct implications for viral pathogenesis. LASV infection is immunosuppressive, with a very low-level interferon response. In contrast, JUNV and MACV infections stimulate a robust interferon (IFN) response in a retinoic acid-inducible gene I (RIG-I)-dependent manner and readily activate protein kinase R (PKR), a known host double-stranded RNA (dsRNA) sensor. In response to infection with RNA viruses, host nonself RNA sensors recognize virus-derived dsRNA as danger signals and initiate innate immune responses. Arenavirus nucleoproteins (NPs) contain a highly conserved exoribonuclease (ExoN) motif, through which LASV NP has been shown to degrade virus-derived immunostimulatory dsRNA in biochemical assays. In this study, we for the first time present evidence that LASV restricts dsRNA accumulation during infection. Although JUNV and MACV NPs also have the ExoN motif, dsRNA readily accumulated in infected cells and often colocalized with dsRNA sensors. Moreover, LASV coinfection diminished the accumulation of dsRNA and the IFN response in JUNV-infected cells. The disruption of LASV NP ExoN with a mutation led to dsRNA accumulation and impaired LASV replication in minigenome systems. Importantly, both LASV NP and RNA polymerase L protein were required to diminish the accumulation of dsRNA and the IFN response in JUNV infection. For the first time, we discovered a collaboration between LASV NP ExoN and L protein in limiting dsRNA accumulation. Our new findings provide mechanistic insights into the differential host innate immune responses to highly pathogenic arenavirus infections.IMPORTANCE Arenavirus NPs contain a highly conserved DEDDh ExoN motif, through which LASV NP degrades virus-derived, immunostimulatory dsRNA in biochemical assays to eliminate the danger signal and inhibit the innate immune response. Nevertheless, the function of NP ExoN in arenavirus infection remains to be defined. In this study, we discovered that LASV potently restricts dsRNA accumulation during infection and minigenome replication. In contrast, although the NPs of JUNV and MACV also harbor the ExoN motif, dsRNA readily formed during JUNV and MACV infections, accompanied by IFN and PKR responses. Interestingly, LASV NP alone was not sufficient to limit dsRNA accumulation. Instead, both LASV NP and L protein were required to restrict immunostimulatory dsRNA accumulation. Our findings provide novel and important insights into the mechanism for the distinct innate immune response to these highly pathogenic arenaviruses and open new directions for future studies.