Lassa virus (LASV) is the causative agent of Lassa Fever (LF), an acute and occasionally fatal disease with hemorrhagic features. Current treatments for LF are limited to ribavirin and supportive care. The objective of this dissertation project was to characterize a human monoclonal antibody (mAb) with the potential to treat LASV infections. This antibody, mAb 8.9F, was isolated from the serum of a convalescent patient and has been shown to protect guinea pigs from lethal LASV challenge. Our approach focused on confirming that mAb 8.9F bound a quaternary neutralizing epitope (QNE), determining which residues comprised the epitope, and examining how mutations to critical residues would impact viral fitness. Our results showed that mAb 8.9F shared a number of features similar to previously characterized QNEs including broad neutralization of viral subtypes and high sensitivity to epitope disruption by detergents. We found that mAb 8.9F recognizes sites on both the GP1 and GP2 subunits of the LASV glycoprotein. Both subunits dissociate from the antibody at similar rates under chaotropic conditions, suggesting that these subunits have equal binding affinity with mAb 8.9F. To identify amino acids important to the epitope, we used a method of knockout site-directed mutagenesis that replaced sequences of LASV glycoprotein with homologous sequences from lymphocytic choriomeningitis virus. We created single mutations for those regions unreactive with mAb 8.9F and identified H124F, P145R, and F147N as mutations capable of abrogating mAb 8.9F reactivity. Viral fitness of these mutants was evaluated by measuring glycoprotein processing and viral infectivity. Our results showed that mutations abrogating mAb 8.9F binding did not significantly inhibit glycoprotein production, processing, or surface transport; neither was pseudovirus formation affected. However, these mutations did significantly reduce pseudovirus infectivity suggesting that mutants escaping mAb 8.9F neutralization may be less fit. The role of N-linked glycosylation in mAb 8.9F recognition was also examined but results proved inconclusive. In summary, this work provides a detailed analysis of the first documented QNE found on the Lassa virus glycoprotein. This work will help direct rational vaccine design and post-exposure antibody therapy.
|Advisor:||Robinson, James E.|
|Commitee:||Bunnell, Bruce A., Garry, Robert F., Morris, Cindy A., Schieffelin, John S.|
|School Location:||United States -- Louisiana|
|Source:||DAI-B 79/01(E), Dissertation Abstracts International|
|Keywords:||Immunology, Lassa virus, Quaternary neutralizing epitope|
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