Characterization of a major neutralizing epitope on the yellow fever virus envelope protein using human recombinant monoclonal antibody fragments generated by phage display

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

The role of myristoylation in the membrane association of the Lassa virus matrix protein Z

The Z protein is the matrix protein of arenaviruses and has been identified as the main driving force for budding. Both LCMV and Lassa virus Z proteins bud from cells in the absence of other viral proteins as enveloped virus-like particles. Z accumulates near the inner surface of the plasma membrane where budding takes place. Furthermore, biochemical data have shown that Z is strongly membrane associated. The primary sequence of Z lacks a typical transmembrane domain and until now it is not understood by which mechanism Z is able to interact with cellular membranes. In this report, we analyzed the role of N-terminal myristoylation for the membrane binding of Lassa virus Z. We show that disruption of the N-terminal myristoylation signal by substituting the N-terminal glycine with alanine (Z-G2A mutant) resulted in a significant reduction of Z protein association with cellular membranes. Furthermore, removal of the myristoylation site resulted in a relocalization of Z from a punctuate distribution to a more diffuse cellular distribution pattern. Finally, treatment of Lassa virus-infected cells with various myristoylation inhibitors drastically reduced efficient Lassa virus replication. Our data indicate that myristoylation of Z is critical for its binding ability to lipid membranes and thus, for effective virus budding

Cell-Specific IRF-3 Responses Protect against West Nile Virus Infection by Interferon-Dependent and -Independent Mechanisms

Interferon regulatory factor (IRF)-3 is a master transcription factor that activates host antiviral defense programs. Although cell culture studies suggest that IRF-3 promotes antiviral control by inducing interferon (IFN)-β, near normal levels of IFN-α and IFN-β were observed in IRF-3−/− mice after infection by several RNA and DNA viruses. Thus, the specific mechanisms by which IRF-3 modulates viral infection remain controversial. Some of this disparity could reflect direct IRF-3-dependent antiviral responses in specific cell types to control infection. To address this and determine how IRF-3 coordinates an antiviral response, we infected IRF-3−/− mice and two primary cells relevant for West Nile virus (WNV) pathogenesis, macrophages and cortical neurons. IRF-3−/− mice were uniformly vulnerable to infection and developed elevated WNV burdens in peripheral and central nervous system tissues, though peripheral IFN responses were largely normal. Whereas wild-type macrophages basally expressed key host defense molecules, including RIG-I, MDA5, ISG54, and ISG56, and restricted WNV infection, IRF-3−/− macrophages lacked basal expression of these host defense genes and supported increased WNV infection and IFN-α and IFN-β production. In contrast, wild-type cortical neurons were highly permissive to WNV and did not basally express RIG-I, MDA5, ISG54, and ISG56. IRF-3−/− neurons lacked induction of host defense genes and had blunted IFN-α and IFN-β production, yet exhibited only modestly increased viral titers. Collectively, our data suggest that cell-specific IRF-3 responses protect against WNV infection through both IFN-dependent and -independent programs

IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity

The innate immune response is essential for controlling West Nile virus (WNV) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. Herein, we show that IPS-1, the central adaptor protein to RIG-I-like receptor (RLR) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic WNV. IPS-1−/− mice exhibited increased susceptibility to WNV infection marked by enhanced viral replication and dissemination with early viral entry into the CNS. Infection of cultured bone-marrow (BM) derived dendritic cells (DCs), macrophages (Macs), and primary cortical neurons showed that the IPS-1-dependent RLR signaling was essential for triggering IFN defenses and controlling virus replication in these key target cells of infection. Intriguingly, infected IPS-1−/− mice displayed uncontrolled inflammation that included elevated systemic type I IFN, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory DCs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific CD8+ T cells and non-specific immune cell proliferation in the periphery and in the CNS. This uncontrolled inflammatory response was associated with a lack of regulatory T cell expansion that normally occurs during acute WNV infection. Thus, the enhanced inflammatory response in the absence of IPS-1 was coupled with a failure to protect against WNV infection. Our data define an innate/adaptive immune interface mediated through IPS-1-dependent RLR signaling that regulates the quantity, quality, and balance of the immune response to WNV infection

Interferon Regulatory Factor-1 (IRF-1) Shapes Both Innate and CD8+ T Cell Immune Responses against West Nile Virus Infection

Interferon regulatory factor (IRF)-1 is an immunomodulatory transcription factor that functions downstream of pathogen recognition receptor signaling and has been implicated as a regulator of type I interferon (IFN)-αβ expression and the immune response to virus infections. However, this role for IRF-1 remains controversial because altered type I IFN responses have not been systemically observed in IRF-1-/- mice. To evaluate the relationship of IRF-1 and immune regulation, we assessed West Nile virus (WNV) infectivity and the host response in IRF-1-/- cells and mice. IRF-1-/- mice were highly vulnerable to WNV infection with enhanced viral replication in peripheral tissues and rapid dissemination into the central nervous system. Ex vivo analysis revealed a cell-type specific antiviral role as IRF-1-/- macrophages supported enhanced WNV replication but infection was unaltered in IRF-1-/- fibroblasts. IRF-1 also had an independent and paradoxical effect on CD8+ T cell expansion. Although markedly fewer CD8+ T cells were observed in naïve animals as described previously, remarkably, IRF-1-/- mice rapidly expanded their pool of WNV-specific cytolytic CD8+ T cells. Adoptive transfer and in vitro proliferation experiments established both cell-intrinsic and cell-extrinsic effects of IRF-1 on the expansion of CD8+ T cells. Thus, IRF-1 restricts WNV infection by modulating the expression of innate antiviral effector molecules while shaping the antigen-specific CD8+ T cell response

Induction of IFN-β and the Innate Antiviral Response in Myeloid Cells Occurs through an IPS-1-Dependent Signal That Does Not Require IRF-3 and IRF-7

Interferon regulatory factors (IRF)-3 and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-α/β) induction and innate immune defenses after virus infection. Prior studies in mice with single deletions of the IRF-3 or IRF-7 genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed reduced IFN-α levels after WNV infection, those lacking IRF-3 or IRF-7 had relatively normal IFN-b production. Here, we generated IRF-3−/−× IRF-7−/− double knockout (DKO) mice, analyzed WNV pathogenesis, IFN responses, and signaling of innate defenses. Compared to wild type mice, the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality. Ex vivo analysis showed complete ablation of the IFN-α response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-β response in DKO fibroblasts and cortical neurons. In contrast, the IFN-β response was minimally diminished in DKO macrophages and dendritic cells. However, pharmacological inhibition of NF-κB and ATF-2/c-Jun, the two other known components of the IFN-β enhanceosome, strongly reduced IFN-β gene transcription in the DKO dendritic cells. Finally, a genetic deficiency of IPS-1, an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling, completely abolished the IFN-β response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-β gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-β enhanceosome by canonical constituent transcriptional factors

A Temporal Role Of Type I Interferon Signaling in CD8+ T Cell Maturation during Acute West Nile Virus Infection

A genetic absence of the common IFN- α/β signaling receptor (IFNAR) in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR-/- mice is limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8+ T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8+ T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8+ T cell development requires type I IFN signaling. WNV infection experiments in BATF3-/- mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8+ T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8+ T cell response at a stage distinct from the initial priming event

Characterization of a major neutralizing epitope on the yellow fever virus envelope protein using human recombinant monoclonal antibody fragments generated by phage display

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