Immunization with recombinant, plant-produced yellow fever virus envelope (E) protein vaccine candidates in rhesus macaques

ABSTRACT Application of genetically modified, deficient-in-replication flaviviruses that are incapable of developing productive, spreading infection is a promising means of designing safe and effective vaccines. Here we describe a two-component genome yellow fever virus (YFV) replication system in which each of the genomes encodes complete sets of nonstructural proteins that form the replication complex but expresses either only capsid or prM/E instead of the entire structural polyprotein. Upon delivery to the same cell, these genomes produce together all of the viral structural proteins, and cells release a combination of virions with both types of genomes packaged into separate particles. In tissue culture, this modified YFV can be further passaged at an escalating scale by using a high multiplicity of infection (MOI). However, at a low MOI, only one of the genomes is delivered into the cells, and infection cannot spread. The replicating prM/E-encoding genome produces extracellular E protein in the form of secreted subviral particles that are known to be an effective immunogen. The presented strategy of developing viruses defective in replication might be applied to other flaviviruses, and these two-component genome viruses can be useful for diagnostic or vaccine applications, including the delivery and expression of heterologous genes. In addition, the achieved separation of the capsid-coding sequence and the cyclization signal in the YFV genome provides a new means for studying the mechanism of the flavivirus packaging process.

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Identification of Novel Vaccine Candidates against Yellow Fever Virus from the Envelope Protein: An Insilico Approach

Journal of Microbiology and Infectious Diseases ◽

10.5799/jmid.700510 ◽

2020 ◽

pp. 31-46

Author(s):

Hind Abdelrahman HASSAN ◽

Khoubieb Ali ABDELRAHMAN ◽

Nasr Mohammed NASR ◽

Yassir A. ALMOFTİ

Keyword(s):

Yellow Fever ◽

Envelope Protein ◽

Yellow Fever Virus ◽

Fever Virus ◽

Vaccine Candidates

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Vaccine and Wild-Type Strains of Yellow Fever Virus Engage Distinct Entry Mechanisms and Differentially Stimulate Antiviral Immune Responses

mBio ◽

10.1128/mbio.01956-15 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 32

Author(s):

Maria Dolores Fernandez-Garcia ◽

Laurent Meertens ◽

Maxime Chazal ◽

Mohamed Lamine Hafirassou ◽

Ophélie Dejarnac ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Virus Entry ◽

Fever Virus ◽

Host Cells ◽

Target Cells ◽

Wild Type ◽

E Protein ◽

Virulence Attenuation ◽

Molecular Determinants

ABSTRACTThe live attenuated yellow fever virus (YFV) vaccine 17D stands as a “gold standard” for a successful vaccine. 17D was developed empirically by passaging the wild-type Asibi strain in mouse and chicken embryo tissues. Despite its immense success, the molecular determinants for virulence attenuation and immunogenicity of the 17D vaccine are poorly understood. 17D evolved several mutations in its genome, most of which lie within the envelope (E) protein. Given the major role played by the YFV E protein during virus entry, it has been hypothesized that the residues that diverge between the Asibi and 17D E proteins may be key determinants of attenuation. In this study, we define the process of YFV entry into target cells and investigate its implication in the activation of the antiviral cytokine response. We found that Asibi infects host cells exclusively via the classical clathrin-mediated endocytosis, while 17D exploits a clathrin-independent pathway for infectious entry. We demonstrate that the mutations in the 17D E protein acquired during the attenuation process are sufficient to explain the differential entry of Asibi versus 17D. Interestingly, we show that 17D binds to and infects host cells more efficiently than Asibi, which culminates in increased delivery of viral RNA into the cytosol and robust activation of the cytokine-mediated antiviral response. Overall, our study reveals that 17D vaccine and Asibi enter target cells through distinct mechanisms and highlights a link between 17D attenuation, virus entry, and immune activation.IMPORTANCEThe yellow fever virus (YFV) vaccine 17D is one of the safest and most effective live virus vaccines ever developed. The molecular determinants for virulence attenuation and immunogenicity of 17D are poorly understood. 17D was generated by serially passaging the virulent Asibi strain in vertebrate tissues. Here we examined the entry mechanisms engaged by YFV Asibi and the 17D vaccine. We found the two viruses use different entry pathways. We show that the mutations differentiating the Asibi envelope (E) protein from the 17D E protein, which arose during attenuation, are key determinants for the use of these distinct entry routes. Finally, we demonstrate that 17D binds and enters host cells more efficiently than Asibi. This results in a higher uptake of viral RNA into the cytoplasm and consequently a greater cytokine-mediated antiviral response. Overall, our data provide new insights into the biology of YFV infection and the mechanisms of viral attenuation.

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Structure–Function of the Yellow Fever Virus Envelope Protein: Analysis of Antibody Epitopes

Viral Immunology ◽

10.1089/vim.2019.0107 ◽

2020 ◽

Vol 33 (1) ◽

pp. 12-21 ◽

Cited By ~ 1

Author(s):

Emily H. Davis ◽

Alan D.T. Barrett

Keyword(s):

Structure Function ◽

Yellow Fever ◽

Envelope Protein ◽

Yellow Fever Virus ◽

Protein Analysis ◽

Fever Virus ◽

Virus Envelope ◽

Virus Envelope Protein ◽

Antibody Epitopes

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E Protein Domain III Determinants of Yellow Fever Virus 17D Vaccine Strain Enhance Binding to Glycosaminoglycans, Impede Virus Spread, and Attenuate Virulence

Journal of Virology ◽

10.1128/jvi.02509-07 ◽

2008 ◽

Vol 82 (12) ◽

pp. 6024-6033 ◽

Cited By ~ 62

Author(s):

Eva Lee ◽

Mario Lobigs

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Fever Virus ◽

Protein Domain ◽

Type I ◽

Wild Type ◽

E Protein ◽

Virulence Attenuation ◽

Domain Iii

ABSTRACT The yellow fever virus (YFV) 17D strain is one of the most effective live vaccines for human use, but the in vivo mechanisms for virulence attenuation of the vaccine and the corresponding molecular determinants remain elusive. The vaccine differs phenotypically from wild-type YFV by the loss of viscerotropism, despite replicative fitness in cell culture, and genetically by 20 amino acid changes predominantly located in the envelope (E) protein. We show that three residues in E protein domain III inhibit spread of 17D in extraneural tissues and attenuate virulence in type I/II interferon-deficient mice. One of these residues (Arg380) is a dominant glycosaminoglycan-binding determinant, which mainly accounts for more rapid in vivo clearance of 17D from the bloodstream in comparison to 17D-derived variants with wild-type-like E protein. While other mutations will account for loss of neurotropism and phenotypic stability, the described impact of E protein domain III changes on virus dissemination and virulence is the first rational explanation for the safety of the 17D vaccine in humans.

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Exchanging the Yellow Fever Virus Envelope Proteins with Modoc Virus prM and E Proteins Results in a Chimeric Virus That Is Neuroinvasive in SCID Mice

Journal of Virology ◽

10.1128/jvi.78.14.7418-7426.2004 ◽

2004 ◽

Vol 78 (14) ◽

pp. 7418-7426 ◽

Cited By ~ 16

Author(s):

Nathalie Charlier ◽

Richard Molenkamp ◽

Pieter Leyssen ◽

Jan Paeshuyse ◽

Christian Drosten ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Vero Cells ◽

Envelope Proteins ◽

Scid Mice ◽

Fever Virus ◽

Chimeric Virus ◽

E Proteins ◽

Virus Envelope

ABSTRACT A chimeric flavivirus infectious cDNA was constructed by exchanging the premembrane (prM) and envelope (E) genes of the yellow fever virus vaccine strain 17D (YF17D) with the corresponding genes of Modoc virus (MOD). This latter virus belongs to the cluster of the “not-known vector” flaviviruses and is, unlike YF17D, neuroinvasive in SCID mice. Replication of in vitro-transcribed RNA from this chimeric flavivirus was shown by [3H]uridine labeling and RNA analysis. Expression of the MOD prM and E proteins was monitored by radioimmunoprecipitation and revealed that the MOD proteins were correctly and efficiently produced from the chimeric precursor protein. The MOD E protein was shown to be N-linked glycosylated, whereas prM, as predicted from the genome sequence, did not contain N-linked carbohydrates. In Vero cells, the chimeric virus replicated with a similar efficiency as the parental viruses, although it formed smaller plaques than YF17D and MOD. In SCID mice that had been infected intraperitoneally with the chimeric virus, the viral load increased steadily until death. The MOD/YF virus, like MOD from which it had acquired the prM and E structural proteins, but unlike YF, proved neuroinvasive in SCID mice. Animals developed neurological symptoms about 15 days after inoculation and died shortly thereafter. The distribution of MOD/YF RNA in the brain of infected mice was similar to that observed in MOD-infected mice. The observations provide compelling evidence that the determinants of neuroinvasiveness of flaviviruses are entirely located in the envelope proteins prM and E.

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