Yellow fever virus (YFV) is a mosquito-transmitted, enveloped, positive stranded RNA virus belonging to the genus flavivirus, which causes hemorrhagic fever in humans in Africa and South America. The YFV is responsible for 200 000 clinical infections per year including 40 000 deaths. Despite the presence of a highly effective YF vaccine called 17D vaccine, this disease is now strongly re-emerging and has to be considered as a public health problem. The present live attenuated 17D vaccine has two major drawbacks: 1) the ancient production method by inoculating viable embryonated eggs which limits the vaccine production capacity and, therefore, impairs attempts to control the disease and may contribute to vaccine supply shortage. 2) this vaccine is a non clonal vaccine which is constituted of heterogenous virion sub-populations. Furthermore, recent reports of several cases of viscerotropic and neurotropic disease associated with 17D vaccination have raised the obvious question of vaccine safety. Taken together, these data show that it appears essential to design a new clonal vaccine which could be based on infectious cDNA clone and produced in animal cell culture. For this purpose, the knowledge of YFV neutralizing epitopes is essential. Because YFV immunity is mainly antibody-mediated, we wanted to isolate human neutralizing antibodies specific for YFV and use them as a tool to characterize the neutralizing epitopes of YFV. The phage display technology provides one of the most convenient systems to isolate such neutralizing recombinant antibody fragments. We generated YF patient-derived antibody phage libraries which were screened against purified virions of the YFV-204-WHO vaccine strain. This step led to the isolation of several single-chain antibody fragments (scFv) which recognized conformational and pH sensitive epitopes in the envelope E protein. Three genetically closely-related and competing scFvs were found to be able to neutralize in vitro the 17D vaccine strain and five wild-type African strains of YFV. To map their epitopes, neutralization escape variants of the YFV-17D-204-WHO were generated using one high-affinity scFv (scFv-7A). Amino acids (aa) E-153, E-154 and E-155 in domain I and aa E-71 in domain II of the E protein were shown to be the critical components of one complex neutralizing epitope. These aa do not form a contiguous epitope on the monomeric E protein, but are in close vicinity in the dimeric form the E protein is predicted to adopt, based on the crystal structures of related flaviviruses. The neutralizing epitope is thus predicted to be formed by contribution of aa from domain I and II of opposing E monomers. The nature of this epitope was supported by the analysis of one wild-type YFV strain (Senegal 90) which is naturally resistant to neutralization by scFv-7A. Microneutralization assays using sera from YFV-infected patients and 17D-immunized travelers confirm the importance of E-71 in YFV neutralization but also showed that those escape variants, originally present in the vaccine lot, do not carry a risk of neutralization escape in persons who are immunized with the 17D vaccine. The potential neutralization mechanism by which these scFvs act, particularly by preventing the fusion process, and their potential use as a therapeutical tool are discussed. The structural complexity of the epitope identified in this work has implications for understanding the mechanism of antibody-mediated neutralization of YFV and these data may be useful for the design of a new recombinant yellow fever vaccine based on a cDNA-derived infectious clone
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