Evidence brief: Development and qualification of an enzyme-linked immunosorbent assay to detect human serum immunoglobulin G reactive to multiple lineages of Lassa virus nucleoprotein

Abstract

Lassa fever is a severe, often fatal febrile illness endemic to West Africa caused by Lassa virus (LASV), with different virus lineages predominating across West African countries. The viral nucleoprotein (NP) is a target antigen for serological assays to identify previous exposure to LASV. To our knowledge, there is no commercially available assay that reliably quantifies anti-LASV-NP IgG antibodies in human serum. We report the development and qualification of an ELISA designed to detect and quantify anti-LASV-NP IgG in human serum samples. Following assay optimization, performance was assessed through assay qualification at clinical trial laboratories within Ghana. Assay positivity criteria, lower limit of detection, upper and lower limits of quantification, inter-assay precision, selectivity and dilutional linearity were determined. A new reference standard prepared from pooled sera from donors in endemic Lassa fever regions was established and calibrated to the first WHO international standard for LASV antibodies. One ELISA assay utilizing lineage IV LASV-NP was applicable for detection of anti-LASV-NP IgG antibodies in serum samples from different West African countries where either LASV lineages I, II, III and IV predominate. The ELISA remained selective in hemolysed serum samples with minimal loss of signal across repeated sample freeze-thaw cycles. Crucially, the developed ELISA was fully concordant with a now discontinued commercially available ELISA kit for quantification of anti-LASV-NP antibodies. Our anti-LASV-NP IgG ELISA was shown to reliably measure anti-LASV-NP IgG levels in human serum. Establishing and conducting this assay within West Africa represents an essential step towards strengthening LASV epidemiology research and supporting urgently needed development of a vaccine to prevent Lassa Fever.

Introduction

Lassa fever (LF) is a severe, often fatal febrile illness caused by one of at least seven phylogenetic lineages of Lassa virus (LASV), a member of the Arenaviridae family [1–3] endemic to West Africa [4]. LASV is a zoonotic infection, the predominant host being the multimammate rat Mastomys natalensis, with transmission to humans via the gastrointestinal, respiratory or skin abrasion routes following contact with rodent urine or feces. Human to human transmission, including nosocomial outbreaks, may also occur via contact with blood, bodily fluids or excreta with virus excreted in urine for up to nine weeks [5]. There is currently no approved LF vaccine and LF is defined by the World Health Organization (WHO) as a public health emergency of international concern [6]. IAVI has been developing a vaccine candidate targeting LASV surface glycoprotein complex (GPC) encoded in a replication competent recombinant Vesicular Stomatitis Virus platform (rVSVΔG-LASV-GPC) [7]. The rVSVΔG platform has been successfully applied in development of a prophylactic Zaire Ebola virus vaccine rVSVΔG-ZEBOV-GP (ERVEBO®, Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA) that has been approved by the FDA, EMA and African countries [8–12]. rVSVΔG-LASV-GPC vaccine candidate has been tested in a Phase 1 clinical trial (IAVI C102 study: NCT04794218) [13] and is under evaluation in a Phase 2a study (IAVI C105 study: NCT05868733), across West African clinical trial sites in Liberia, Ghana and Nigeria. Such studies aim to evaluate vaccine candidate safety and immunogenicity, thereby informing on vaccine design and further evaluation in clinical trials. Vaccine candidate immunogenicity is evaluated by assessments of humoral and cellular immune responses to LASV-GPC prior to and following vaccine candidate administration.