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.

[Review of candidate vaccines for the prevention of Lassa fever].

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.

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.

Boosting understanding of Lassa Fever virus epidemiology: Field testing a novel assay to identify past Lassa Fever virus infection in blood and oral fluids of survivors and unexposed controls in Sierra Leone.

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.

Immunological screening of Lassa Virus among Health workers and Contacts of patients of Lassa fever in Ondo State.

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.

Hunting and consumption of rodents by children in the Lassa fever endemic area of Faranah, Guinea.

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.

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).

Lassa-VSV chimeric virus targets and destroys human and mouse ovarian cancer by direct oncolytic action and by initiating an anti-tumor response.

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.

Tetravalent Rabies-Vectored Filovirus and Lassa Fever Vaccine Induces Long-term Immunity in Nonhuman Primates.

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.

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.

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.

Bayesian estimation of Lassa virus epidemiological parameters: Implications for spillover prevention using wildlife vaccination.

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.

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.

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.

Vulnerabilities in coronavirus glycan shields despite extensive glycosylation.

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.

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.

Field evaluation of a Pan-Lassa rapid diagnostic test during the 2018 Nigerian Lassa fever outbreak.

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.

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.