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.

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 (LASV) is the causative agent of Lassa fever, an often fatal viral hemorrhagic fever (VHF). Cases are reported year round in Nigeria, Sierra Leone, Liberia, Guinea and other West African countries, with peak incidence in the dry season. Case-fatality rates (CFRs) among hospitalized Lassa fever patients vary from approximately 25% in Nigeria 1,2 to greater than 60% in Sierra Leone 3 . Subclinical infections appear to be common 4,5 . A variety of factors, including differential virulence of LASV strains and variations in human genetic susceptibility, immune responses or patient care, may account for the range of CFRs. The main reservoir of LASV is Mastomys natalensis, the natal multimammate rat (or mouse), an abundant peridomestic rodent [6][7][8] . Additional rodent reservoirs or intermediate hosts have been discovered 8,9 . While human-to-human transmission can occur, especially in hospital settings, most infections occur by exposure to rodent excreta or during preparation of rodents for food. Accurate estimates for the number of Lassa fever cases and deaths are not possible because of the limited availability of epidemiological data. Supportive care including management of fluid and electrolyte balance can improve survival 10,11 . The only available treatment is the off-label use of the nucleoside drug ribavirin 12 . There is currently no approved Lassa fever vaccine. Lassa fever has been recognized by the World Health Organization (WHO) as an important threat to global health that is in urgent need of countermeasure development 13 . The Coalition for Epidemic Preparedness Innovations (CEPI) has prioritized the accelerated development of a Lassa fever vaccine 14,15 . CEPI has also initiated an epidemiological study in five West African countries as part of their strategy to facilitate vaccine development efforts 16 .
LASV is a single-stranded RNA virus in the family Arenaviridae (Order: Bunyavirales) 17 . Its genome consists of two ambisense segments encoding four proteins: Z (zinc, matrix), L (polymerase), NP (nucleoprotein), and GPC (glycoprotein complex) 18 . GPC is post-translationally cleaved into glycoprotein 1 (GP1), glycoprotein 2 (GP2), and a stable signal peptide (SSP) that trimerize on the virion surface 19 . There are currently seven proposed genetically distinct LASV lineages (lineages I-VII) distributed throughout West Africa 20 . Phylogenetic analyses of the highly divergent LASV genomes suggest that the virus has been circulating in Nigeria for over a thousand years, followed by a more recent spread across West Africa 21 . LASV lineages and sublineages (clades) cluster geographically suggesting that once established in a region the virus remains stably separated in the rodent reservoirs [22][23][24] . The initial isolate of LASV representing lineage I has been detected in northern Nigeria 25 , but has rarely been observed in recent samplings 20,23,26 . lineages II and III are the most common lineages in Nigeria and are found in the southern and central regions, respectively 27,28 . Recently, lineages and phylogenetic clusters in Nigeria have been geographically mapped at a higher resolution 20 . Within lineage II five sublineages were distinguishable. Seven sublineages were distinguishable in lineage III. Lineage IV LASV, including the prototypical Josiah strain commonly used in laboratory studies, is present in Sierra Leone, Liberia and Guinea 21,29 . LASV lineage V is found in Mali and Ivory Coast 30,31 . LASV isolates from the Hylomyscus pamfi rodent trapped in Nigeria 8 and from a nosocomial outbreak in Togo 32 have been proposed to represent new lineage VI and lineage VII, respectively. Development of Lassa fever countermeasures is potentially challenged by the high genetic diversity of LASV 14,33 . The genetic variability of LASV could produce differences in the antigenicity of LASV proteins [34][35][36] . It is unclear whether a vaccine produced with antigens of LASV from a particular lineage will induce a human immune response that is protective against LASV of other lineages. Most Lassa vaccines in development have employed antigens of the Josiah strain (LIV) of LASV 37,38 . As a component of CEPI's efforts to facilitate and accelerate Lassa vaccine development, serum or plasma from Lassa fever survivors living in different geographical areas and thus representing different lineages has been collected through a contractual arrangement between CEPI and the Viral Hemorrhagic Fever Consortium (VHFC). Vaccine developers are being supplied with panels consisting of samples with high, medium and low titres from survivors of infection with different LASV lineages. These samples in addition to Lassa negative control samples are being used to establish and fine-tune immunoassays for vaccine development.

Results
igG and igM capture eLiSA based on recombinant antigens representing LASV of divergent lineages. Recombinant NP N-and C-terminal domains and Z proteins from lineages II-IV were expressed in codon-optimized E. coli strains and purified. The proteins were then visualized on SDS-PAGE and immunoblotting for identity and purity for use as coating antigens for ELISA (Fig. 1A,B, Fig. S1A,B). The NP N-and C-terminal domains from the three lineages are homogenous monomers (Fig. 1A). Minor levels of contaminants and breakdown products are present in the purified NP preparations. In contrast, Z proteins exhibit multimerization; lineage II Z protein forms higher order multimers than Z proteins of lineages III and IV (Fig. 1B).
Two forms of the LASV glycoprotein are utilized in the current studies. Linked LASV GP tethers the GP1 and GP2 subunits together via a flexible linker and is designed to present both pre-and post-fusion epitopes (Fig. 1C, Fig. S1C-F, Fig. S2A). Linked GP has been successfully used for the identification and characterization of anti-LASV GP antibodies from human survivors of Lassa virus infection 48 . Prefusion, stabilized GP is a disulphide linked LASV GP used in the determination of the only pre-fusion structures available for any arenavirus glycoprotein trimer 49,50 . Pre-fusion GP (Pf-GP) contains only the prefusion form (Fig. 1D, Fig. S1C-F, Fig. S2B) and is best suited for detection of serum/plasma IgG and IgM against the complex, neutralizing, domain-spanning epitopes that are a desired product of vaccination efforts. Linked GP and Pf-GP are expressed in Drosophila S2 cells and purified via streptactin-affinity chromatography. Following strepII tag removal with enterokinase, the GPs are further purified by size-exclusion chromatography (SEC). Both linked GP and Pf-GP show appropriate reactions with human monoclonal antibodies (huMAbs) that react with divergent epitopes (Fig. S2C,D) 48 .
A mixture of NP or Pf-GP GP from LASV lineages II, III, and IV immobilized in the ELISA microwell plates was used for the direct absorption of LASV-specific IgM or IgG antibodies from serum or plasma of Lassa fever survivors. Pan-Lassa NP or Pf-GP IgG-and IgM-capture ELISAs exhibited significant differences in reactivity by serum from Sierra Leone Lassa fever survivors compared to serum from United States controls (healthy blood donors) (Fig. 1E). IgG seroreactivity to NP and Pf-GP was higher than IgM seroreactivity in the Sierra Leonean sample cohort. Additional studies on validation of the Pan-Lassa antibody and antigen capture assays is presented elsewhere 51 . nigerian and Sierra Leone Lassa fever survivors reactivity to LASV recombinant antigens. During Jan. 14th-29th, 2019 laboratory staff of Redeemer's University (RUN), Ede State VHF Laboratory in Nigeria and the Kenema Government Hospital (KGH) VHF Laboratory in Sierra Leone, with training and assistance from Zalgen Labs personnel, conducted studies on seroreactivity to LASV recombinant proteins. Nigerian laboratory personnel from Irrua Specialist Teaching Hospital (ISTH) in Edo State, Federal Medical Center (FMC) in Owo, Ondo State, FMC Abakaliki in Ebonyi State and Ibadan U. were trained to use the ReLASV immunoassays. Lassa fever survivor, contacts, and suspected Lassa fever samples were obtained from ISTH, FMC Owo, and FMC Abakaliki for the study. At KGH post-acute, convalescent and Lassa fever survivor samples were selected from a biorepository of samples obtained from other on-going studies.
Samples were screened using the ReLASV Pan-Lassa NP IgG/IgM ELISA Kit and the prototype ReLASV Pan-Lassa Prefusion GP IgG/IgM ELISA Kit. In Nigeria 140 plasma or serum samples were screened at a 1:100 sample dilution using mixed NP or mixed Prefusion GP ELISAs for IgG/IgM reactivity (Fig. 2). In Sierra Leone 80 plasma and serum samples were selected and screened at a 1:100 sample dilution for IgG and/or IgM reactivity. A range of reactivities to LASV NP and GP was observed in the prescreening of samples from both Sierra Leonean and Nigerian subjects. With the exception of IgM in Sierra Leoneans the correlations between NP and GP reactivity were significant. While many samples had both strong IgG and/or IgM reactivity to both NP and GP other samples had low reactivity to one protein or the other. Linear correlations between NP and GP reactivity Binding to antigens encoded by LASV of lineages ii, iii, and iV. We next assessed a subset of plasma or serum samples from Nigerian (n = 40) and Sierra Leonean (n = 61) with a range of immune responses (from high to low or negative) using ELISAs coated with single antigens, NP, linked GP, Pf-GP, or Z representing LASV of lineages II-IV. Results of the binding studies using a 1:100 sample dilution are presented as scatterplots of the absorbance with linear correlations (Fig. 3). Overall substantial cross-reactivity for IgG binding exists between both NP and linked GP or Pf-GP of LASV of lineages II-IV ( Fig. 3A-F). The slopes for all cross-reactivity comparisons were near 1 (range from 0.80x to 1.15x) with the exception of lineage II Pf-GP versus lineage IIwI Pf-GP (slope = 0.49x, Fig. 3E). Binding of plasma or serum IgG to NP and both forms of GP across lineages gave significant R 2 values (> 0.80, P < 0.001) indicating that there were few samples that bound strongly to protein of one lineage, but weakly to the protein from another lineage. In contrast to the results with NP and GP we found that there was less IgG cross-reactivity between Z proteins of lineages II-IV (Fig. 3G,H). Slopes of 0.82x and 0.57x were observed for comparison of binding of lineage II Z versus lineage III Z for Nigerian and Sierra Leonean samples, respectively. However, for the comparison of lineage IV Z versus lineage II Z the slopes were 0.27x and 0.39x indicating a bias for binding of the lineage II protein. Similar results were obtained with the Z reactivity of Sierra Leonean plasma samples. The differences in the Z protein multimerization may contribute to these differences. Lineage II Z tended to form higher order multimers that may display epitopes more effectively (Fig. 1B). Z is a small protein with a limited number of epitopes that appear to be poorly cross-reactive. Similar correlations for IgG cross-reactivity to NP, linked GP, Pf-GP, or Z representing LASV of lineages II-IV were obtained using endpoint titres rather than quantifying absorbance at a 1:100 dilution (Fig. S3). Typical examples of the reactivities in serial dilution is shown for two Nigerian subjects and one Sierra Leonean subject (Fig. 4). Endpoint titres to LASV NP were typically higher than those to either linked GP, Pf-GP or Z. IgM cross-reactivity among lineages II-IV followed a similar pattern to that observed with IgG (Fig. 5). Class switching from IgM to IgG reactivity is delayed in Lassa fever survivors, which is reflected in the high levels of IgM detected 41 . cross-neutralization studies with LASV pseudoviruses expressing Gpc of LASV lineages ii-iV. Pseudoviruses expressing the GPC of LASV from lineages II, III or IV were employed to assess the ability of antibodies from Lassa fever survivors to cross-neutralize different lineages of LASV (Fig. 6). The average reciprocal 50% neutralization titres were higher in Nigerians than in Sierra Leoneans ( Fig. 6A-C). Neutralization by Nigerian samples was biased towards LASVpv lineage II. Reciprocal 50% neutralization titres against LASVpv lineage II were higher than titres against LASVpv expressing lineage III or IV in 23/30 (77%) Nigerian subjects (Fig. 6A). This does not appear to be due to LASVpv lineage II being easier to neutralize by human antibodies. 6/32 Sierra Leonean subjects (13%, p < 0.0001 Fisher's exact test) had reciprocal 50% neutralization titres against LASVpv lineage II that were higher than the titres for LASVpv lineage III or IV (Fig. 6B). Samples from  Neutralization curves for LASV expressing GPC of LII-IV had similar slopes even when there were differences in the reciprocal 50% neutralization titres for the distinct lineages ( Fig. 6C-E). Some samples failed to neutralize 100% of any lineage at any dilution tested (Fig. 6C). The failure to neutralize 100% of viruses even at high antibody levels has been noted in prior monoclonal antibody studies 50 . Certain dilutions of serum or plasma resulted in increased infectivity of the pseudovirus (asterisks in Fig. 6D,E). The possible presence of enhancing antibodies in serum from Lassa fever survivors has previously been reported 52 .
Cross-neutralization in neutralizing responses by Nigerian and Sierra Leonean plasma or serum samples was examined using linear correlations (Fig. 7). This analysis confirmed the bias for neutralization of LASVpv lineage II by Nigerian samples (Fig. 7A,C,E). In contrast, bias for neutralization of LASVpv of lineage IV was not demonstrated by Sierra Leonean samples (Fig. 7B,D,F). comparison of recombinant binding assays to pseudovirus neutralization assays and plaque reduction assays using BSL-4 LASV. A subset of samples (n = 20) was further evaluated in additional neutralization assays and these results were compared to the results of ELISA binding assays (Fig. 8). For this subanalysis plaque reduction neutralization tests (PRNT) at Biosafety Level-4 (BSL-4) were compared to 50% binding titres using pseudoviruses expressing GPC or binding to recombinant Pf-GP. Infectious LASV of lineage II and lineage IV showed a significant correlation (R 2 = 0.73, P < 0.001) when compared in PRNT (Fig. 8A). A comparison of neutralization titres using LASVpv expressing either the GPC of LASV lineage II or IV on an HIV-1 core also showed a significant correlation (R 2 = 0.30, P = 0.012). LASVpv neutralization assays were more sensitive than the PRNT, however, there were significant correlations between these neutralization assays. PRNT using LASV of LII showed a significant correlation (R 2 = 0.40, P = 0.003) to neutralization titres using LASVpv expressing lineage II GPC (Fig. 8C). Similarly, PRNT using LASV lineage IV showed a significant correlation www.nature.com/scientificreports/ (R 2 = 0.53, P < 0.001) to neutralization titres using LASV expressing lineage IV GPC. Although less sensitive, LASVpv using a vesicular stomatitis virus core showed significant correlations with neutralization of LASVpv using a HIV core (R 2 = 0.40, P = 0.001) and with PRNT (R 2 = 0.37, P = 0.004) (Fig. S4). PRNT titres were also lower than the binding titres to LASV Pf-GP. Significant correlations between PRNT titres and binding titres to LASV Pf-GP of lineage II (Fig. 8E) or lineages IV (Fig. 8F) were not observed. Likewise, significant correlations between pv neutralization titres and binding titres to LASV Pf-GP of lineage II (Fig. 8G) or lineages IV (Fig. 8H) were not observed. These results are expected since the majority of antibodies to LASV GP are nonneutralizing 48 .

Discussion
LASV infection induces igG or igM that cross-reacts with np or Gp of multiple lineages. As an initial step to evaluate the cross-reactivity potential of Lassa vaccine antigen(s) to cover different lineages, we assessed the ability of the human immune system to mount cross-reactive humoral immune responses during natural infections. Humoral responses to LASV proteins by Nigerian and Sierra Leonean Lassa fever survivors are heterogeneous. Lassa survivors may show IgG and/or IgM reactivity to NP and GP or reactivity to only NP or GP. NP, linked-GP, and Pf-GP antigens representing LASV lineages II-IV were shown to be reactive to LASVspecific antibodies produced by both Nigerian and Sierra Leonean survivors. These results suggest that infection with LASV induces IgG or IgM that is able to effectively cross-react with NP or GP of multiple lineages. Reactivity to Z was variable and there was minimal cross-reactivity between Z of lineages II-IV. These results differ from a previous study that reported that anti-LASV antibodies preferentially react with the antigens of virus strains present in the local areas 34 . Different techniques were used in this prior study including an indirect immunofluorescence assay (IFA) employing cells infected with different LASV strains and a reverse ELISA that utilized lysates from cells infected with different LASV strains. It is possible that differences in the sensitivities of these assays compared to the recombinant antigen ELISAs account for the more extensive crossreactivity observed in the current study.

Heterogeneous induction of LASV cross-neutralizing antibodies by Lassa fever survivors.
We also assessed the ability of natural LASV infection to induce cross-neutralizing antibodies. As in previous studies 48 , we observed that not all Lassa fever survivors produce LASV neutralizing antibodies. Neutralizing antibody titres when present are lower than binding titres, which is expected since most antibodies to the LASV GP do not neutralize 48 . Infection with LASV lineage II in Nigerians induces strong neutralizing titres to LASVpv lineage II, but neutralization titres to LASVpv lineages III and IV were lower. In contrast, infection with LASV lineage IV in Sierra Leoneans generally induces cross-reactive neutralizing immune responses to LASVpv of lineage II and III . These results suggest that GP of LASV lineage IV may be more effective than GP of LASV lineage II at presenting epitopes that induce broadly neutralizing antibodies.
Guidance for procedures to quantify humoral aspects of vaccine-induced immunity to LASV of distinct lineages. Recombinant LASV NP of lineages II-IV produced in E. coli appear to be appropriate to assess IgG and IgM responses. NP is not expressed on the surface of the LASV virion or infected cells. Therefore, it is unlikely that anti-NP humoral immune responses are involved in protection from LASV infection. However, anti-NP antibodies can serve as a marker for exposure to infectious LASV either prior to or during Lassa vaccine trials conducted in West Africa. NP could be involved in protective cellular immune response to LASV [53][54][55] . Most of the LASV vaccines in the CEPI portfolio, with the exception of the measles vectored vaccine do not express LASV NP 14,38 . Recombinant Pan-Lassa linked and Pf-GP IgG/IgM ELISA kits are capable of accurate detection of LASV GP-specific IgG and IgM antibody titres. The Pan-Lassa IgG/IgM ELISA has demonstrated sensitivity to Lassa fever in both Sierra Leone and Nigeria. This study confirms that the Pf-GP construct is a superior reagent for detecting serological responses to LASV GP, which are of prime importance for future vaccine studies.
A LASV pseudovirus platform was more sensitive for quantifying neutralization than PRNT, which is based on replication competent LASV in BSL-4. These findings agree with previous studies that compared several virus neutralization platforms in the evaluation of monoclonal antibodies against LASV 48 . Envelope glycoprotein levels, mechanisms of viral entry, transport, fusion, uncoating, pre-nuclear localization of replicative viral nucleic acids, tropism, and specificity versus passive incorporation of heterologous glycoproteins into particles may all contribute in some degree and affect the sensitivity of each neutralization assay platform. Given the imperfect correlation caution, should be employed when extrapolating the results from pseudovirus platforms to replication competent LASV. While a sensitive pv assay is useful for preclinical vaccine development, comparison of the results to BSL-4 assays would be prudent for vaccine trials. Lassa fever and for Lassa fever vaccines have yet to be defined. These CoP might also be different. Cellular immunity appears to be the primary effective arm of the adaptive immune response against LASV during natural infection 56 . High-titred anti-LASV IgG and virus can be simultaneously present in the blood of human Lassa fever patients 3 . Only a subset of Lassa fever survivors produce LASV neutralizing antibodies, and production of neutralizing antibodies is delayed months into convalescence 48,57 . In contrast to the situation during natural infection, passive serum transfer therapy from survivors protects against disease and death in animal models 58,59 . However, the ability of serum to neutralize LASV of particular strains determined the effectiveness of passive antibody transfer in treating LASV-infected nonhuman primates 59 . Furthermore, neutralizing huMAbs have shown high efficacy in passive immunotherapy of LASV-infected guinea pigs and NHPs [60][61][62] . In NHP models of lethal Lassa fever infection, treatment with cocktails of selected neutralizing huMAbs completely protected animals even when the first treatment was delayed until eight days post infection, a time when severe disease and dysregulation were clinically evident 61 . Moreover, this huMAb cocktail protected NHPs in late stage Lassa fever, highlighting the relevance of humoral immunity in protecting against lethal Lassa fever 60,61 . Even though neutralizing antibodies are not involved in clearing LASV during natural human infection, they could be important for an induced protective immune response. These studies educate future approaches toward evaluation of the quantity and quality of the humoral response generated by vaccination with LASV specific antigens, namely the viral glycoprotein. The conformation and presentation of the viral glycoprotein is critical to the level of protection 63 . Previous studies demonstrated that multiple doses of inactivated LASV failed to protect NHP from lethal challenge, despite induction of a substantial antibody response 64 . It remains to be determined if protective neutralizing antibodies could be elicited by vaccination, perhaps with engineered forms of LASV GP 63 .  48 . Coupled with the current observation that natural infection with lineage IV viruses (but not lineage II LASV) induces a broad cross-reactive and cross-neutralizing response suggests that it may be possible to induce an immune response with a LASV Josiah (lineage IV) GP-based vaccine that will be cross-protective for infections with LASV of other lineages. Further study on the differences in the induced immune responses to LASV in Nigerians and Sierra Leoneans is warranted and has important implications for vaccine development.

induction of cross-protective immune responses by Lassa vaccines.
Limitations to the current study. Representative strains from each of the three most prevalent lineages (II-IV) were selected, but a variety of sublineages (clades) exist within each lineages 20,23 . Due to the high heterogeneity among LASV lineages, continuous monitoring of its mutational spectrum and evolutionary change will be critical for maintaining effective vaccines. The large number of assays employed in this study required the use of a limited number of samples with sufficient volume to comprehensively evaluate binding and neutralizing activities.
implications of Lassa diversity for vaccine development. The genetic diversity of LASV presents potentially a major challenge to development of Lassa fever vaccines 14     igM and igG eLiSA. The ReLASV Pan-Lassa NP-specific IgM and IgG ELISA utilize microwell plates coated with a mixture of recombinant nucleoprotein NP from lineages II, III, and IV and performed according to the manufacturer's recommendations 66 . ELISAs utilizing recombinant LASV antigen (NP, linked GP, Pf-GP and Z from LII-IV were performed similarly. LASV antigens (either singular or combined lineages) were coated at 200 to 500 ng/well in 96-well microtiter plates (Nunc A/S, Denmark) using Carb-Bicarb buffer, pH 9.6. After antigens were immobilized, the coated microwell plates were stabilized using proprietary blocking solution, dried and packaged with desiccant. For the ELISA, lyophilized human monoclonal calibrator and negative control plasma were reconstituted with 0.25 mL laboratory-grade water. Calibrator was diluted 1:101 (0.01 mL/1.0 mL followed by four threefold serial dilutions to create a calibration curve for antibody concentration estimation. For Z protein ELISA screening a well characterized LF convalescent sample with sufficient anti-Z antibody titer was used as assay Reference and similarly diluted to create Reference curve dilutions. LF patient samples were diluted 1:101 in provided Sample Diluent prior to assay. Calibrator (or Reference) dilutions, diluted Negative Control and patient samples were transferred (0.1 mL/well) in duplicate wells. Microwell plates were incubated at ambient temperature (18-30 °C) for 30 min. Microwell plates were washed four times with PBS-Tween wash buffer. Anti-Hu IgG or IgM-horseradish peroxidase conjugate reagent (Jackson ImmunoResearch, West Grove, PA, USA) was added to microwell plate (0.1 mL/well) followed by a 30 min incubation at ambient temperature. After repeating the PBS-Tween wash, 3,3′,5,5′-Tetramethylbenzidine (TMB) Substrate (Moss Biotech Inc., Hanover, Maryland, USA) was added to each well (0.1 mL/well). The TMB substrate was incubated for 10 min followed by addition (0.1 mL/well) of Stopping Solution (0.36 N Sulfuric Acid). Developed ELISA plates were read at 450 nm (with 650 nm reference). IgG or IgM estimated concentration was calculated from Calibrator/Reference Curve plot using 4-paramenter logistic fit. Negative cut-off was determined as the 95 th percentile distribution of the study population.  www.nature.com/scientificreports/ pseudovirus assay. LASV pseudoviruses (LASVpv) were generated by co-transfection of HEK293T cells with LASV GPC plasmids and pSG3Denv encoding the envelope-deficient core of HIV-1 as previously described 48 . The pseudoviruses express LASV GPC of different lineages on a particle containing the core proteins of HIV and are capable of a single round of replication. LASV pseudoviruses (LASVpv) capable of a single round of replication were produced by co-transfection of HEK293T cells with LASV GPC plasmids (lineages II-IV) and pSG3Denv, encoding the envelope-deficient core of HIV-1. These LASVpv were assayed in TZMbl cells, a HeLa cell derivative that contains integrated luciferase and β-galactosidase genes under regulatory control of an HIV-1 long terminal repeat, which is activated by HIV-1 Tat after virion entry 67   . Note that multiple samples had the same 50% reciprocal titres producing overlapping data points.