RESUMO
BACKGROUND: There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). RESULTS: Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA). CONCLUSION: These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.
Assuntos
Antígenos Virais/biossíntese , Antígenos Virais/isolamento & purificação , Vírus Lassa/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Virais/biossíntese , Proteínas Virais/isolamento & purificação , Animais , Anticorpos Antibacterianos/sangue , Antígenos Virais/genética , Arenavirus/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Cromatografia de Afinidade , Reações Cruzadas , Escherichia coli/genética , Humanos , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Proteínas Virais/genéticaRESUMO
Unlike many viral hemorrhagic fevers (VHFs), Lassa fever (LF) is not a rare disease that emerges only as sporadic cases or in outbreak form. Although surveillance is inadequate to determine the true incidence, up to 300,000 infections and 5000 deaths from LF are estimated to occur yearly. The highest incidence is in the "Mano River Union (MRU) countries" of Sierra Leone, Liberia, and Guinea. Although civil unrest in this region over the past two decades has impeded capacity building and research, new-found peace in recent years presents new opportunities. In 2004, the Mano River Union Lassa Fever Network (MRU LFN) was established to assist MRU countries in the development of national and regional surveillance, diagnosis, treatment, control, and prevention of LF. Here, we review the present literature on treatment and pathogenesis of LF and outline priorities for future research in the field made possible by the improved research capacity of the MRU LFN.
