Search

Lassa fever continues to be a major public health burden in West Africa, yet effective therapies or vaccines are lacking. The isolation of protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccine candidates have generally been unsuccessful at doing so, and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron microscopy-based epitope mapping workflow that enables high-resolution structural characterization of polyclonal antibodies to the GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization that involve epitopes of the GPC-A competition cluster. Furthermore, by identifying undescribed immunogenic off-target epitopes, we expose the challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests J.D.B. is on the scientific advisory boards of Apriori Bio, Invivyd, Aerium Therapeutics, and the Vaccine Company. K.G.A. is a consultant to and on the scientific advisory board of Invivyd. J.D.B. and K.H.D.C. receive royalty payments as inventors on Fred Hutch licensed patents related to viral deep mutational scanning. N.P.K. is a co-founder, shareholder, paid consultant, and chair of the scientific advisory board of Icosavax, Inc. The King lab has received unrelated sponsored research agreements from Pfizer and GSK. M.M. is currently an employee of Seagen, though his contributions to this manuscript were performed when he was an employee of the Institute for Protein Design., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: TL reports consulting fees from Vaccitech on an unrelated project, an honorarium from Seqirus, grant support from the DHSC for this work, and is named as an inventor on a patent application for a vaccine against SARS-CoV-2. SG is named as an inventor on patents covering use of ChAdOx1-vectored vaccines and a vaccine against SARS-CoV-2., (Copyright: © 2024 Flaxman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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/LASgpc 2m ) 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/LASgpc 2m . High levels of viremia and lethality were observed in CBA mice inoculated i.v. with rLCMV/LASgpc 2m , 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., Competing Interests: M.H. and M.I. are the inventors of a patent application related to the findings from this study. A.T. declares no competing interests.

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., (© 2023. The Author(s).)

Background: The objective of this study is to evaluate the immunogenicity of adjuvanted monovalent rabies virus (RABV)-based vaccine candidates against Ebola virus (FILORAB1), Sudan virus (FILORAB2), Marburg virus (FILORAB3), Lassa virus (LASSARAB1), and combined trivalent vaccine candidate (FILORAB1-3) and tetravalent vaccine candidate (FILORAB1-3 and LASSARAB) in nonhuman primates., Methods: Twenty-four Macaca fascicularis were randomly assigned into 6 groups of 4 animals. Each group was vaccinated with either a single adjuvanted vaccine, the trivalent vaccine, or the tetravalent vaccine at days 0 and 28. We followed the humoral immune responses for 1 year by antigen-specific enzyme-linked immunosorbent assays and RABV neutralization assays., Results: High titers of filovirus and/or Lassa virus glycoprotein-specific immunoglobulin G were induced in the vaccinated animals. There were no significant differences between immune responses in animals vaccinated with single vaccines vs trivalent or tetravalent vaccines. In addition, all vaccine groups elicited strong rabies neutralizing antibody titers. The antigen-specific immune responses were detectable for 1 year in all groups., Conclusions: In summary, this study shows the longevity of the immune responses up to 365 days for a pentavalent vaccine-against Ebola virus, Sudan virus, Marburg virus, Lassa virus, and RABV-using a safe and effective vaccine platform., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The Lassa virus one of the main etiological agent of hemorrhagic fevers in the world: according to WHO estimates, it affects 100,000 to 300,000 people annually, which results in up to 10,000 deaths [1]. Although expansion of Lassa fever caused by this pathogen is mostly limited to the West African countries: Sierra Leone, Liberia, Guinea and Nigeria, imported cases have been historically documented in Europe, the United States of America (USA), Canada, Japan, and Israel [2]. In 2017, WHO included the Lassa virus in the list of priority pathogens in need of accelerated research, development of vaccines, therapeutic agents and diagnostic tools regarding infections they cause [3]. This review describes main technological platforms used for the development of vaccines for the prevention of Lassa fever.

Background: The increasing trends of morbidity and mortality of Lassa fever is becoming more alarming in Nigeria. Information about immune response to the virus is limited. At exposure, the level of immunity plays a vital role in the vulnerability of individuals infected., Objective: Investigating the immune status of health workers, infected cases and contacts of infected cases of Lassa fever in Ondo State., Study Design: Blood samples were collected from 233 individuals comprising 102 health workers, 22 infected cases and 109 contacts of infected cases from Owo and Ose Local Government Areas and transported in triple level packaging. Plasma samples were analyzed for IgG and IgM markers using ReLASV® Pan-Lassa NP IgG/IgM ELISA Kit (Zalgen Labs, LLC, USA) while RNAs extracted from IgM positive samples were analyzed for LASV RNA according to manufacturers' instructions., Result: Among the health workers, 20/102 (19.6%) and 2/102 (2.0%) were IgG and IgM positive respectively. While 16/22 (72.7%) and 14/22 (63.6%) were IgG and IgM positive respectively among the infected cases. Of the contacts of infected cases screened, 64/109 (58.7%) were IgG positive while 4/109 (3.7%) were positive for IgM. There was no detectable LASV RNA in the samples analyzed., Conclusion: These findings suggest that majority of the health workers are naïve to the virus and hence may be prone to the viral infection. It could also be suggestive that a good personal protective procedure is been practiced by the health workers, hence the low exposure. However, most of the contacts of infected cases show exposure to the virus., (Copyright © 2021. Published by Elsevier GmbH.)

Background: Despite identification 50 years ago, the true burden of Lassa Fever (LF) across Africa remains undefined for reasons including research focus on hospitalised patients, lack of validated field-feasible tools which reliably identify past infection, and the fact that all assays require blood samples making large-scale surveys difficult. Designated a priority pathogen of epidemic potential requiring urgent research by the World Health Organisation, a better understanding of LF sero-epidemiology is essential to developing and evaluating new interventions including vaccines. We describe the first field testing of a novel species-neutral Double Antigen Binding Assay (DABA) designed to detect antibodies to LF in plasma and oral fluid., Methodology/principal Findings: Paired plasma and oral fluid were collected in Sierra Leone from survivors discharged from Kenema Government Hospital Lassa Fever Unit between 1980 and 2018, and from controls recruited in Freetown in 2019. Epidemiological sensitivity and specificity of the DABA measured against historical diagnosis in survivors and self-declared non-exposed controls was 81.7% (95% CI 70.7%- 89.9%) and 83.3% (72.7%- 91.1%) respectively in plasma, and 71.8% (60.0%- 81.9%) and 83.3% (72.7%- 91.1%) respectively in oral fluid. Antibodies were identified in people infected up to 15 years and, in one case, 40 years previously. Participants found oral fluid collection easy and painless with 80% happy to give an oral fluid sample regularly., Conclusions/significance: Given the difficulties of assay validation in a resource-limited setting, including unexpected exposures and diagnostics of varying accuracy, the new assay performed well in both plasma and oral fluid. Sensitivity and specificity are expected to be higher when case/control ascertainment is more definitive and further work is planned to investigate this. Even at the performance levels achieved, the species-neutral DABA has the potential to facilitate the large-scale seroprevalence surveys needed to underpin essential developments in LF control, as well as support zoonotic investigations., Competing Interests: The authors have declared that no competing interests exist.

As a consequence of the Ebola outbreak, human-animal contact has gained importance for zoonotic transmission surveillance. In Faranah (Upper Guinea), daily life is intertwined with rodents, such as the Natal multimammate mouse, Mastomys natalensis; a reservoir for Lassa virus (LASV). However, this contact is rarely perceived as a health risk by residents, although Lassa fever (LF) is known to be endemic to this region. Conversely, these observations remain a great concern for global health agendas. Drawing on ethnographic research involving interviews, focus group discussions, participant observations, and informal discussions over four months, we first identified factors that motivated children to hunt and consume rodents in Faranah villages, and thereafter, explored the knowledge of LF infection in children and their parents. Furthermore, we studied two dimensions of human-rodent encounters: 1) space-time of interaction and 2) factors that allowed the interaction to occur and their materiality. This approach allowed us to contextualize child-rodent contacts beyond domestic limits in the fallow fields, swamps, and at other times for this practice. A close look at these encounters provided information on rodent trapping, killing, and manipulation of cooking techniques and the risk these activities posed for the primary transmission of LASV. This research facilitated the understanding of children's exposure to M. natalensis during hunting sessions and the importance of rodent hunting, which is a part of their boyish identity in rural areas. Determination of when, where, why, and how children, rodents, and environments interacted allowed us to understand the exposures and risks important for human and animal surveillance programs in the Lassa-endemic region., Competing Interests: The authors have declared that no competing interests exist.

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-GPC LASV 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-GPC LASV represents a potential vaccine candidate against LF.

Ovarian cancer is the third most common female cancer, with poor survival in later stages of metastatic spread. We test a chimeric virus consisting of genes from Lassa and vesicular stomatitis viruses, LASV-VSV; the native VSV glycoprotein is replaced by the Lassa glycoprotein, greatly reducing neurotropism. Human ovarian cancer cells in immunocompromised nude mice were lethal in controls. Chemotherapeutic paclitaxel and cisplatin showed modest cancer inhibition and survival extension. In contrast, a single intraperitoneal injection of LASV-VSV selectively infected and killed ovarian cancer cells, generating long-term survival. Mice with human ovarian cancer cells in brain showed rapid deterioration; LASV-VSV microinjection into brain blocked cancer growth, and generated long-term survival. Treatment of immunocompetent mice with infected mouse ovarian cancer cells blocked growth of non-infected ovarian cancer cells peritoneally and in brain. These results suggest LASV-VSV is a viable candidate for further study and may be of use in the treatment of ovarian cancer., (Copyright © 2020. Published by Elsevier Inc.)

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).

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.

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., (Published by Elsevier B.V.)

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., (Copyright © 2020 Port et al.)

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.

Lassa virus is a significant burden on human health throughout its endemic region in West Africa, with most human infections the result of spillover from the primary rodent reservoir of the virus, the natal multimammate mouse, M. natalensis. Here we develop a Bayesian methodology for estimating epidemiological parameters of Lassa virus within its rodent reservoir and for generating probabilistic predictions for the efficacy of rodent vaccination programs. Our approach uses Approximate Bayesian Computation (ABC) to integrate mechanistic mathematical models, remotely-sensed precipitation data, and Lassa virus surveillance data from rodent populations. Using simulated data, we show that our method accurately estimates key model parameters, even when surveillance data are available from only a relatively small number of points in space and time. Applying our method to previously published data from two villages in Guinea estimates the time-averaged R0 of Lassa virus to be 1.74 and 1.54 for rodent populations in the villages of Bantou and Tanganya, respectively. Using the posterior distribution for model parameters derived from these Guinean populations, we evaluate the likely efficacy of vaccination programs relying on distribution of vaccine-laced baits. Our results demonstrate that effective and durable reductions in the risk of Lassa virus spillover into the human population will require repeated distribution of large quantities of vaccine., Competing Interests: The authors have declared that no competing interests exist.

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-β 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.

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., (Copyright © 2020 Sakabe et al.)

Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development focuses on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein. Coronavirus S proteins are extensively glycosylated, encoding around 66-87 N-linked glycosylation sites per trimeric spike. Here, we reveal a specific area of high glycan density on MERS S that results in the formation of oligomannose-type glycan clusters, which were absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Overall, our data reveal how organisation of glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the protein surface.

Lassa virus (LASV) is the causative agent of Lassa fever (LF), an often-fatal hemorrhagic disease. LF is endemic in Nigeria, Sierra Leone and other West African countries. Diagnosis of LASV infection is challenged by the genetic diversity of the virus, which is greatest in Nigeria. The ReLASV Pan-Lassa Antigen Rapid Test (Pan-Lassa RDT) is a point-of-care, in vitro diagnostic test that utilizes a mixture of polyclonal antibodies raised against recombinant nucleoproteins of representative strains from the three most prevalent LASV lineages (II, III and IV). We compared the performance of the Pan-LASV RDT to available quantitative PCR (qPCR) assays during the 2018 LF outbreak in Nigeria. For patients with acute LF (RDT positive, IgG/IgM negative) during initial screening, RDT performance was 83.3% sensitivity and 92.8% specificity when compared to composite results of two qPCR assays. 100% of samples that gave Ct values below 22 on both qPCR assays were positive on the Pan-Lassa RDT. There were significantly elevated case fatality rates and elevated liver transaminase levels in subjects whose samples were RDT positive compared to RDT negative.

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.

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., (Copyright © 2020 American Society for Microbiology.)

Lassa virus (LASV), which causes considerable morbidity and mortality annually, has a high genetic diversity across West Africa. LASV glycoprotein (GP) expresses this diversity, but most LASV vaccine candidates utilize only the Lineage IV LASV Josiah strain GP antigen as an immunogen and homologous challenge with Lineage IV LASV. In addition to the sequence variation amongst the LASV lineages, these lineages are also distinguished in their presentations. Inter-lineage variations within previously mapped B-cell and T-cell LASV GP epitopes and the breadth of protection in LASV vaccine/challenge studies were examined critically. Multiple alignments of the GP primary sequence of strains from each LASV lineage showed that LASV GP has diverging degrees of amino acid conservation within known epitopes among LASV lineages. Conformational B-cell epitopes spanning different sites in GP subunits were less impacted by LASV diversity. LASV GP diversity should influence the approach used for LASV vaccine design. Expression of LASV GP on viral vectors, especially in its prefusion configuration, has shown potential for protective LASV vaccines that can overcome LASV diversity. Advanced vaccine candidates should demonstrate efficacy against all LASV lineages for evidence of a pan-LASV vaccine.

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.

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., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: RFG and LMB are co-founders of Zalgen Labs, LLC. The Viral Hemorrhagic Fever Consortium (vhfc.org) is a partnership of academic and industry scientists who are developing diagnostics, therapeutics and vaccines for LF and other severe diseases. Tulane University and various industry partners have filed United States and foreign patent applications on behalf of the consortium for several of these technologies. If commercial products are developed, consortium members may receive royalties or profits.

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., (Copyright © 2020 Cai et al.)

Recent occurrences of filoviruses and the arenavirus Lassa virus (LASV) in overlapping endemic areas of Africa highlight the need for a prophylactic vaccine that would confer protection against all of these viruses that cause lethal hemorrhagic fever (HF). We developed a quadrivalent formulation of VesiculoVax that contains recombinant vesicular stomatitis virus (rVSV) vectors expressing filovirus glycoproteins and that also contains a rVSV vector expressing the glycoprotein of a lineage IV strain of LASV. Cynomolgus macaques were vaccinated twice with the quadrivalent formulation, followed by challenge 28 days after the boost vaccination with each of the 3 corresponding filoviruses (Ebola, Sudan, Marburg) or a heterologous contemporary lineage II strain of LASV. Serum IgG and neutralizing antibody responses specific for all 4 glycoproteins were detected in all vaccinated animals. A modest and balanced cell-mediated immune response specific for the glycoproteins was also detected in most of the vaccinated macaques. Regardless of the level of total glycoprotein-specific immune response detected after vaccination, all immunized animals were protected from disease and death following lethal challenges. These findings indicate that vaccination with attenuated rVSV vectors each expressing a single HF virus glycoprotein may provide protection against those filoviruses and LASV most commonly responsible for outbreaks of severe HF in Africa.

Lassa fever is an acute viral haemorrhagic illness caused by Lassa virus (LASV), which is endemic throughout much of West Africa. The virus primarily circulates in the Mastomys natalensis reservoir and is transmitted to humans through contact with infectious rodents or their secretions; human-to-human transmission is documented as well. With the exception of Dengue fever, LASV has the highest human impact of any haemorrhagic fever virus. On-going outbreaks in Nigeria have resulted in unprecedented mortality. Consequently, the World Health Organization (WHO) has listed LASV as a high priority pathogen for the development of treatments and prophylactics. Currently, there are no licensed vaccines to protect against LASV infection. Although numerous candidates have demonstrated efficacy in animal models, to date, only a single candidate has advanced to clinical trials. Lassa fever vaccine development efforts have been hindered by the high cost of biocontainment requirements, the absence of established correlates of protection, and uncertainty regarding the extent to which animal models are predictive of vaccine efficacy in humans. This review briefly discusses the epidemiology and biology of LASV infection and highlights recent progress in vaccine development., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Lassa virus (LASV), the causative agent of Lassa fever (LF), was first identified in 1969. Since then, outbreaks in the endemic countries of Nigeria, Liberia, and Sierra Leone occur on an annual basis resulting in a case-fatality rate of 15-70% in hospitalized patients. There is currently no licensed vaccine and there are limited animal models to test vaccine efficacy. An estimated 37.7 million people are at risk of contracting LASV; therefore, there is an urgent need for the development of a safe, effective vaccine against LASV infection. The LF endemic countries are also inflicted with HIV, Ebola, and malaria infections. The safety in immunocompromised populations must be considered in LASV vaccine development. The novel adenovirus vector-based platform, Ad5 (E1-,E2b-) has been used in clinical trial protocols for treatment of immunocompromised individuals, has been shown to exhibit high stability, low safety risk in humans, and induces a strong cell-mediated and pro-inflammatory immune response even in the presence of pre-existing adenovirus immunity. To this nature, our lab has developed an Ad5 (E1-,E2b-) vector-based vaccine expressing the LASV-NP or LASV-GPC. We found that guinea pigs vaccinated with two doses of Ad5 (E1-,E2b-) LASV-NP and Ad5 (E1-,E2b-) LASV-GPC were protected against lethal LASV challenge. The Ad5 (E1-,E2b-) LASV-NP and LASV-GPC vaccine represents a potential vaccine candidate against LF., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Lassa fever is a frequently severe human disease that is endemic to several countries in West Africa. To date, no licensed vaccines are available to prevent Lassa virus (LASV) infection, even though Lassa fever is thought to be an important disease contributing to mortality and both acute and chronic morbidity. We have previously described a vaccine candidate composed of single-cycle LASV replicon particles (VRPs) and a stable cell line for their production. Here, we refine the genetic composition of the VRPs and demonstrate the ability to reproducibly purify them with high yields. Studies in the guinea pig model confirm efficacy of the vaccine candidate, demonstrate that single-cycle replication is necessary for complete protection by the VRP vaccine, and show that postexposure vaccination can confer protection from lethal outcome., (Published by Oxford University Press for the Infectious Diseases Society of America 2019.)

Following the Ebola outbreak in Western Africa in 2013-16, a global effort has taken place for preparedness for future outbreaks. As part of this response, the development of vaccines, treatments and diagnostic tools has been accelerated, especially towards pathogens listed as likely to cause an epidemic and for which there are no current treatments. Several of the priority pathogens identified by the World Health Organisation are haemorrhagic fever viruses. This review provides information on the role of reference materials as an enabling tool for the development and evaluation of assays, and ultimately vaccines and treatments. The types of standards available are described, along with how they can be applied for assay harmonisation through calibration as a relative potency to a common arbitrary unitage system (WHO International Unit). This assures that assay metrology is accurate and robust. We describe reference materials that have been or are being developed for haemorrhagic fever viruses and consider the issues surrounding their production, particularly that of biosafety where the viruses require specialised containment facilities. Finally, we advocate the use of reference materials at early stages, including research and development, as this helps produce reliable assays and can smooth the path to regulatory approval.

Lassa fever remains the most imported viral haemorrhagic fever in Europe and is responsible for 5000 deaths per year throughout Western Africa. There is no vaccine and treatment is often ineffective. We have developed a vaccine based on modified Vaccinia Ankara expressing the nucleoprotein from Lassa virus (MVALassaNP). This study investigated the immunogenicity (in mice) and efficacy (in guinea pigs) of the MVALassaNP vaccine as a prime/boost or single vaccination regime. ELISA and ELISpot assays confirmed humoral and T-cell immunity following both a prime and prime/boost vaccination, with the prime/boost regime producing a statistically increased response compared to a prime only vaccine (P < 0.0001). The vaccine offered protection in guinea pigs against disease manifestations after challenge with virulent Lassa virus. Clinical signs, weight loss and temperature increases were observed in all animals receiving a control MVA vaccine, after challenge with Lassa virus. In contrast, no clinical signs, fever or weight loss were observed in any of the MVALassaNP vaccinated animals demonstrating that both a single immunisation, and prime/boost regime confer protection against disease progression. In conclusion, the MVALassaNP vaccine candidate elicits an immune response, demonstrates efficacy against Lassa virus disease and is suitable for further preclinical and clinical development., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Lassa virus (LASV) causes hemorrhagic fever and is endemic in West Africa. Protective antibody responses primarily target the LASV surface glycoprotein (GPC), and GPC-B competition group antibodies often show potent neutralizing activity in humans. However, which features confer potent and broadly neutralizing antibody responses is unclear. Here, we compared three crystal structures of LASV GPC complexed with GPC-B antibodies of varying neutralization potency. Each GPC-B antibody recognized an overlapping epitope involved in binding of two adjacent GPC monomers and preserved the prefusion trimeric conformation. Differences among GPC-antibody interactions highlighted specific residues that enhance neutralization. Using structure-guided amino acid substitutions, we increased the neutralization potency and breadth of these antibodies to include all major LASV lineages. The ability to define antibody residues that allow potent and broad neutralizing activity, together with findings from analyses of inferred germline precursors, is critical to develop potent therapeutics and for vaccine design and assessment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lassa fever is a zoonotic disease caused by the Lassa virus, a rodent-borne arenavirus endemic to West Africa. Recent steady increase in reported cases of the disease in Nigeria, where 123 deaths occurred in 546 confirmed cases in 2019 has further underlined the need to accelerate the development of vaccines for preventing the disease. Intensified research and development of Lassa fever medical countermeasures have yielded some vaccine candidates with preclinical scientific plausibility using predominantly novel technology. The more advanced candidates are based on recombinant measles, Vesicular Stomatitis Virus or Mopiea and Lassa virus reassortants expressing Lassa virus antigens, and the deoxyribonucleic acid platform. However, the Lassa fever portfolio still lags behind other neglected tropical diseases', and further investments are needed for continued development and additional research, such as the safety and efficacy of these vaccine candidates in special populations., (Copyright © 2019. Published by Elsevier B.V.)

Lassa fever is a unique viral hemorrhagic fever that is endemic in parts of West Africa, primarily Sierra Leone, Guinea, Liberia, and Nigeria. The disease is caused by the Lassa virus, an Old World arenavirus that has as primary reservoir host the multimammate rodent Mastomys nataliensis, which lives in association with humans. Recent estimates suggest LF causes two million cases and 5000-10000 deaths annually, mainly in West Africa. Clinical diagnosis and laboratory confirmation have always been major challenges for effective management and control of the disease in afflicted areas of West Africa. Recent advancements in molecular biology, recombinant DNA technology, and genomics sequencing has facilitated major advancement in development of better diagnostic and surveillance tools for Lassa fever virus. These include, the multiplex, magnetic bead-based immunodiagnostics for both Lassa virus antigens and antibodies; molecular probe-based quantitative real-time PCR for genomic signatures; rapid diagnostics tests that detects the most prevalent West African lineages; and the successful utilization of next-generation sequencing technology to diagnose and characterize Lassa virus in West Africa. These advances will continue to improve disease treatment, control, and prevention. In this review we will discuss progression of Lassa virus diagnostics from the past and into the future., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Lassa fever is an acute viral illness caused by Lassa virus (LASV), a rodent-borne pathogen. LASV is endemic to much of Sub-Saharan West Africa, where seasonal outbreaks cause significant morbidity and mortality. Increased global awareness of LASV has led to development of improved diagnostic tests, treatments and vaccines. As vaccine candidates are trialled, it is essential to assess the potential outcomes of introducing a LASV vaccination program in endemic regions. This study investigates the potential outcomes of routine and pulse vaccination strategies using a deterministic mathematical model that captures seasonal LASV transmission between rodents and humans. For plausible parameter values, we find that immunization of 40% of infants at 70% vaccine effectiveness achieves a population-level reduction in infectious case numbers of 30%, while coverage of 60% at 90% vaccine effectiveness achieves a 56% reduction. Similar reductions can be achieved more rapidly via population-wide pulse vaccination at 11% coverage (30% reduction at 70% effectiveness) or 23% coverage (56% reduction at 90% effectiveness) repeated every 10 years. Similar pulse vaccine doses delivered at reduced frequency, but increased coverage achieves a greater reduction in infectious cases. Findings around infant vaccination are sensitive to our assumption that immunity is life-long, while pulse-vaccination has only slightly reduced effect if immunity lasts 10-30 years. An effective LASV vaccination program would incorporate pulse vaccination in addition to routine childhood immunization to limit disease. Estimates of feasible vaccine coverage and effectiveness are needed to fully quantify the likely benefits of a vaccination program in LASV endemic regions., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Lassa fever (LF) survivors develop various clinical manifestations including polyserositis, myalgia, epididymitis, and hearing loss weeks to months after recovery from acute infection. We demonstrate a systemic lymphoplasmacytic and histiocytic arteritis and periarteritis in guinea pigs more than 2 months after recovery from acute Lassa virus (LASV) infection. LASV was detected in the arterial tunica media smooth muscle cells by immunohistochemistry, in situ hybridization, and transmission electron microscopy. Our results suggest that the sequelae of LASV infection may be due to virus persistence resulting in systemic vascular damage. These findings shed light on the pathogenesis of LASV sequelae in convalescent human survivors., (Published by Oxford University Press for the Infectious Diseases Society of America 2018.)

Rodents serve as reservoirs and/or vectors for several human infections of high morbidity and mortality in the tropics. Population growth and demographic shifts over the years have increased contact with these mammals, thereby increasing opportunities for disease transmission. In Africa, the burden of rodent-borne diseases is not well described. To investigate human seroprevalence of selected rodent-borne pathogens, sera from 657 healthy adults in ten rural communities in Ghana were analyzed. An in-house enzyme-linked immunosorbent assay (ELISA), for immunoglobulin G (IgG) antibodies to Lassa virus was positive in 34 (5%) of the human samples. Using commercial kits, antibodies to hantavirus serotypes, Puumala and Dobrava, and Leptospira bacteria were detected in 11%, 12% and 21% of the human samples, respectively. Forty percent of residents in rural farming communities in Ghana have measurable antibodies to at least one of the rodent-borne pathogens tested, including antibodies to viral hemorrhagic fever viruses. The high seroprevalence found in rural Ghana to rodent-borne pathogens associated with both sporadic cases and larger disease outbreaks will help define disease threats and inform public health policy to reduce disease burden in underserved populations and deter larger outbreaks., Competing Interests: Dr. Nimo-Paintsil, Dr. Mohareb, Dr. Schoepp, CDR Letizia, and CAPT Kronmann are military service members or employees of the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

There are two predominant subgroups in the Arenaviridae family of viruses, the Old World and the New World viruses, that use distinct cellular receptors for entry. While New World viruses typically elicit good neutralizing antibody responses, the Old World viruses generally evade such responses. Antibody-based immune responses are directed against the glycoprotein spike complexes that decorate the viruses. A thick coat of glycans reduces the accessibility of antibodies to the surface of spike complexes from Old World viruses, but other mechanisms may further hamper the development of efficient humoral responses. Specifically, it was suggested that the GP1 receptor-binding module of the Old World Lassa virus might help with evasion of the humoral response. Here we investigated the immunogenicity of the GP1 domain from Lassa virus and compared it to that of the GP1 domain from the New World Junín virus. We found striking differences in the ability of antibodies that were developed against these immunogens to target the same GP1 receptor-binding domains in the context of the native spike complexes. Whereas GP1 from Junín virus elicited productive neutralizing responses, GP1 from Lassa virus elicited only nonproductive responses. These differences can be rationalized by the conformational changes that GP1 from Lassa virus but not GP1 from Junín virus undergoes after dissociating from the trimeric spike complex. Hence, shedding of GP1 in the case of Lassa virus can indeed serve as a mechanism to subvert the humoral immune response. Moreover, the realization that a recombinant protein may be used to elicit a productive response against the New World Junín virus may suggest a novel and safe way to design future vaccines. IMPORTANCE Some viruses that belong to the Arenaviridae family, like Lassa and Junín viruses, are notorious human pathogens, which may lead to fatal outcomes when they infect people. It is thus important to develop means to combat these viruses. For developing effective vaccines, it is vital to understand the basic mechanisms that these viruses utilize in order to evade or overcome host immune responses. It was previously noted that the GP1 receptor-binding domain from Lassa virus is shed and accumulates in the serum of infected individuals. This raised the possibility that Lassa virus GP1 may function as an immunological decoy. Here we demonstrate that mice develop nonproductive immune responses against GP1 from Lassa virus, which is in contrast to the effective neutralizing responses that GP1 from Junín virus elicits. Thus, GP1 from Lassa virus is indeed an immunological decoy and GP1 from Junín virus may serve as a constituent of a future vaccine., (Copyright © 2019 American Society for Microbiology.)

Situated in southeastern Sierra Leone, Kenema Government Hospital (KGH) maintains one of the world's only Lassa fever isolation wards and was a strategic Ebola virus disease (EVD) treatment facility during the 2014 EVD outbreak. Since 2006, the Viral Hemorrhagic Fever Consortium (VHFC) has carried out research activities at KGH, capturing clinical and laboratory data for suspected cases of Lassa fever. Here we describe the approach, progress, and challenges in designing and maintaining a data capture and management system (DCMS) at KGH to assist infectious disease researchers in building and sustaining DCMS in low-resource environments. Results on screening patterns and case-fatality rates are provided to illustrate the context and scope of the DCMS covered in this study. A medical records system and DCMS was designed and implemented between 2010 and 2016 linking historical and prospective Lassa fever data sources across KGH Lassa fever units and its peripheral health units. Data were captured using a case report form (CRF) system, enzyme-linked immunosorbent assay (ELISA) plate readers, polymerase chain reaction (PCR) machines, blood chemistry analyzers, and data auditing procedures. Between 2008 and 2016, blood samples for 4,229 suspected Lassa fever cases were screened at KGH, ranging from 219 samples in 2008 to a peak of 760 samples in 2011. Lassa fever case-fatality rates before and following the Ebola outbreak were 65.5% (148/226) and 89.5% (17/19), respectively, suggesting that fewer, but more seriously ill subjects with Lassa fever presented to KGH following the 2014 EVD outbreak (p = .040). DCMS challenges included weak specificity of the Lassa fever suspected case definition, limited capture of patient survival outcome data, internet costs, lapses in internet connectivity, low bandwidth, equipment and software maintenance, lack of computer teaching laboratories, and workload fluctuations due to variable screening activity. DCMS are the backbone of international research efforts and additional literature is needed on the topic for establishing benchmarks and driving goal-based approaches for its advancement in developing countries., Competing Interests: The Viral Hemorrhagic Fever Consortium (VHFC; vhfc.org) is a partnership of academic and industry scientists developing diagnostics, therapeutics, and vaccines for Lassa fever and other severe diseases. Tulane University and various industry partners have filed United States and foreign patent applications on behalf of the VHFC for several of these technologies. If commercial products are developed, VHFC members may receive royalties or profits. The authors MLB, LMB, and RFG are current employees or affiliates of Zalgen Labs, LLC. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Lassa virus (LASV) causes a viral haemorrhagic fever in humans and is a major public health concern in West Africa. An efficient immune response to LASV appears to rely on type I interferon (IFN-I) production and T-cell activation. We evaluated the response of plasmacytoid dendritic cells (pDC) to LASV, as they are an important and early source of IFN-I. We compared the response of primary human pDCs to LASV and Mopeia virus (MOPV), which is very closely related to LASV, but non-pathogenic. We showed that pDCs are not productively infected by either MOPV or LASV, but produce IFN-I. However, the activation of pDCs was more robust in response to MOPV than LASV. In vivo, pDC activation may support the control of viral replication through IFN-I production, but also improve the induction of a global immune response. Therefore, pDC activation could play a role in the control of LASV infection.

Background: Lassa virus (LASV) is the etiologic agent of an acute hemorrhagic fever endemic in West Africa. Natural killer (NK) cells control viral infections in part through the interaction between killer cell immunoglobulin-like receptors (KIRs) and their ligands. LASV infection is associated with defective immune responses, including inhibition of NK cell activity in the presence of MHC-class 1 + -infected target cells., Methods: We compared individual KIR and HLA-class 1 genotypes of 68 healthy volunteers to 51 patients infected with LASV in Sierra Leone, including 37 survivors and 14 fatalities. Next, potential HLA-C1, HLA-C2, and HLA-Bw4 binding epitopes were in silico screened among LASV nucleoprotein (NP) and envelope glycoprotein (GP). Selected 10-mer peptides were then tested in peptide-HLA stabilization, KIR binding and polyfunction assays., Findings: LASV-infected patients were similar to healthy controls, except for the inhibitory KIR2DL2 gene. We found a specific increase in the HLA-C1:KIR2DL2 interaction in fatalities (10/11) as compared to survivors (12/19) and controls (19/29). We also identified that strong of NP and GP viral epitopes was only observed with HLA-C molecules, and associated with strong inhibition of degranulation in the presence of KIR2DL + NK cells. This inhibitory effect significantly increased in the presence of the vGP 420 variant, detected in 28.1% of LASV sequences., Interpretation: Our finding suggests that presentation of specific LASV epitopes by HLA-C alleles to the inhibitory KIR2DL2 receptor on NK cells could potentially prevent the killing of infected cells and provides insights into the mechanisms by which LASV can escape NK-cell-mediated immune pressure., (Copyright © 2019. Published by Elsevier B.V.)

The Natal multimammate mouse (Mastomys natalensis) is the reservoir host of Lassa virus, an arenavirus that causes Lassa haemorrhagic fever in humans in West Africa. Because no vaccine exists and therapeutic options are limited, preventing infection through rodent control and human behavioural measures is currently considered to be the only option. In order to assess the efficacy of rodent control, we performed a 4-year field experiment in rural Upper Guinea and developed a mathematical model to simulate different control strategies (annual density control, continuous density control, and rodent vaccination). For the field study, rodenticide baits were placed each year in three rural villages, while three other villages were used as controls. Rodents were trapped before and after every treatment and their antibody status and age were determined. Data from the field study were used to parameterize the mathematical model. In the field study, we found a significant negative effect of rodent control on seroprevalence, but this effect was small especially given the effort. Furthermore, the rodent populations recovered rapidly after rodenticide application, leading us to conclude that an annual control strategy is unlikely to significantly reduce Lassa virus spillover to humans. In agreement with this finding, the mathematical model suggests that the use of continuous control or rodent vaccination is the only strategy that could lead to Lassa virus elimination. These field and model results can serve as a guide for determining how long and frequent rodent control should be done in order to eliminate Lassa virus in rural villages.

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.

Lassa virus (LASV) is responsible for a viral hemorrhagic fever in humans and the death of 3,000 to 5,000 people every year. The immune response to LASV is poorly understood, but type I interferon (IFN-I) and T-cell responses appear to be critical for the host. We studied the response of myeloid dendritic cells (mDC) to LASV, as mDCs are involved in both IFN-I production and T-cell activation. We compared the response of primary human mDCs to LASV and Mopeia virus (MOPV), which is similar to LASV, but non-pathogenic. We showed that mDCs produced substantial amounts of IFN-I in response to both LASV and MOPV. However, only MOPV-infected mDCs were able to activate T cells. More surprisingly, coculture with T cells completely inhibited the activation of LASV-infected mDCs. These differences between LASV and MOPV were mostly due to the LASV nucleoprotein, which has major immunosuppressive properties, but the glycoprotein was also involved. Overall, these results suggest that mDCs may be important for the global response to LASV and play a role in the outcome of Lassa fever., Competing Interests: The authors have declared that no competing interests exist.