Development and evaluation of a recombinant antigen-based indirect immunofluorescence assay for the diagnosis of Lassa fever

Abstract

Lassa virus (LASV) forms part of the mammarenavirus genus from the Arenaviridae family and is the arenavirus with the most significant public health impact. It is endemic to West Africa as one of seven mammarenaviruses capable of causing viral haemorrhagic fever (VHF). Outbreaks of LASV in West Africa appear annually, infecting 300 000 people with a case fatality rate of <2%. Clinically, LASV causes diverse symptoms, reasoning it indistinguishable from other febrile diseases, complicating clinical diagnosis and reliability. LASV is classified as a biosafety level 4 (BSL-4) pathogen since it causes VHF. The live virus can be handled only in a high-containment laboratory, of which there are few facilities globally, encumbering diagnostic work. LASV diagnostics are hampered because of unaffordable rapid, validated diagnostic serological tests. A need exists to develop improved serological tests for use outside of containment facilities. This would allow accessible and quicker diagnosis, with the speedy implementation of preventive measures, such as the quarantine of infected patients, limiting the spread of the virus. An immunofluorescence assay (IFA) is a diagnostic device using fixed virus-infected mammalian or insect cells expressing a specific antigen to detect antibodies by microscopy. IFAs offer quick results, crucial for suspected VHF cases as the spread of the virus can be prevented. Recombinant technology can be employed in developing diagnostic assays for VHFs as a safer alternative for assays reliant on live virus culture. These recombinant diagnostic assays could be designed and implemented outside high-containment facilities once infectious samples were inactivated. During this study, indirect IFA tests were developed, using recombinant LASV nucleoprotein (NP) and LASV glycoprotein (GP) as individual antigens. We then evaluated their utility to approach the need for a quick, standardised serological test for LASV diagnosis. Expression cassettes for recombinant expression of the LASV NP and LASV GP in mammalian cells were designed and developed. The LASV NP and LASV GP were chosen for this project, forming the major structural proteins for LASV, as most antibodies target these proteins. The study data disclose the successful expression of these recombinant proteins. A LASV NP stable cell line was also generated. This stable cell line would allow the IFA to be standardised, removing the batch-to-batch variation with recombinant antigen IFA slides prepared by transiently transfected cells. With transiently expressed LASV GP mammalian cells, and the stable LASV NP mammalian cells we developed IFA slides. Indirect IFAs based on these recombinant antigens were examined for their ability to detect Lassa-specific antibodies in patient serum. The results were compared to a LASV IFA using virus-infected cells. The study results present a compelling correlation among the assays regarding Lassa-specific antibody detection in a panel of known Lassa antibody-negative samples, such as detection accuracy. When the LASV NP and LASV GP IFAs were used in combination on the panel of known Lassa negatives, 90% of the samples tested negative using recombinant LASV antigen-based IFAs. The virus-infected cell-based IFA accurately identified negative samples in 96.67% of the cases. The recombinant antigen-based assays were established as less sensitive (five-fold less) than the virus-infected cell-based IFA. This is anticipated since whole-virus antigen extracts allow antibody detection against any viral protein and are not restricted to specific antigens, accounting for better sensitivity. Conjointly, although limited, our initial evaluation of the recombinant LASV antigen-based indirect IFA displays it is a method that can detect LASV antibodies; it is comparable in its reliability and accuracy to the whole virus-based IFA. Based on this, the recombinant protein-based LASV IFA warrants further development and evaluation as a diagnostic device for Lassa fever diagnostics.