Modeling mRNA-based vaccine YFV.E1988 against yellow fever virus E-protein using immuno-informatics and reverse vaccinology approach

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Neuroadapted Yellow Fever Virus 17D: Genetic and Biological Characterization of a Highly Mouse-Neurovirulent Virus and Its Infectious Molecular Clone

Journal of Virology ◽

10.1128/jvi.75.22.10912-10922.2001 ◽

2001 ◽

Vol 75 (22) ◽

pp. 10912-10922 ◽

Cited By ~ 32

Author(s):

Thomas J. Chambers ◽

Michael Nickells

Keyword(s):

Cell Culture ◽

Survival Time ◽

Yellow Fever ◽

Yellow Fever Virus ◽

Scid Mice ◽

Fever Virus ◽

Nucleotide Substitutions ◽

Average Survival Time ◽

E Protein ◽

Average Survival

ABSTRACT A neuroadapted strain of yellow fever virus (YFV) 17D derived from a multiply mouse brain-passaged virus (Porterfield YF17D) was additionally passaged in SCID and normal mice. The virulence properties of this virus (SPYF) could be distinguished from nonneuroadapted virus (YF5.2iv, 17D infectious clone) by decreased average survival time in SCID mice after peripheral inoculation, decreased average survival time in normal adult mice after intracerebral inoculation, and occurrence of neuroinvasiveness in normal mice. SPYF exhibited more efficient growth in peripheral tissues of SCID mice than YF5.2iv, resulting in a more rapid accumulation of virus burden, but with low-titer viremia, at the time of fatal encephalitis. In cell culture, SPYF was less efficient in replication than YF5.2iv in all cell lines tested. The complete nucleotide sequence of SPYF revealed 29 nucleotide substitutions relative to YF5.2iv, and these were distributed throughout the genome. There were a total of 13 predicted amino acid substitutions, some of which correspond to known differences among the Asibi, French viscerotropic virus, French neurotropic vaccine, and YF17D vaccine strains. The envelope (E) protein contained five substitutions, within all three functional domains. Substitutions were also present in regions encoding the NS1, NS2A, NS4A, and NS5 proteins and in the 3′ untranslated region (UTR). Construction of YFV harboring all of the identified coding nucleotide substitutions and those in the 3′ UTR yielded a virus whose cell culture and pathogenic properties, particularly neurovirulence and neuroinvasiveness for SCID mice, generally resembled those of the original SPYF isolate. These findings implicate the E protein and possibly other regions of the genome as virulence determinants during pathogenesis of neuroadapted YF17D virus in mice. The determinants affect replication efficiency in both neural and extraneural tissues of the mouse and confer some limited host-range differences in cultured cells of nonmurine origin.

Download Full-text

Interaction of Yellow Fever Virus French Neurotropic Vaccine Strain with Monkey Brain: Characterization of Monkey Brain Membrane Receptor Escape Variants

Journal of Virology ◽

10.1128/jvi.74.6.2903-2906.2000 ◽

2000 ◽

Vol 74 (6) ◽

pp. 2903-2906 ◽

Cited By ~ 24

Author(s):

Haolin Ni ◽

Kate D. Ryman ◽

Heiman Wang ◽

Mohammad F. Saeed ◽

Robin Hull ◽

...

Keyword(s):

Amino Acid ◽

Yellow Fever ◽

Mouse Brain ◽

Amino Acid Substitution ◽

Yellow Fever Virus ◽

Fever Virus ◽

Monkey Brain ◽

Intracerebral Inoculation ◽

E Protein ◽

Monkey Liver

ABSTRACT Binding of yellow fever virus wild-type strains Asibi and French viscerotropic virus and vaccine strains 17D and FNV to monkey brain and monkey liver cell membrane receptor preparations (MRPs) was investigated. Only FNV bound to monkey brain MRPs, while French viscerotropic virus, Asibi, and FNV all bound to monkey liver MRPs. Four monkey brain and two mouse brain MRP escape (MRPR) variants of FNV were selected at pH 7.6 and 6.0. Three monkey brain MRPR variants selected at pH 7.6 each had only one amino acid substitution in the envelope (E) protein in domain II (E-237, E-260, or E274) and were significantly attenuated in mice following intracerebral inoculation. Two of the variants were tested in monkeys and retained parental neurotropism following intracerebral inoculation at the dose tested. We speculate that this region of domain II is involved in binding of FNV E protein to monkey brain and is, in part, responsible for the enhanced neurotropism of FNV for monkeys. A monkey brain MRPR variant selected at pH 6.0 and two mouse brain MRPR variants selected at pH 7.6 were less attenuated in mice, and each had an amino acid substitution in the transmembrane region of the E protein (E-457 or E-458).

Download Full-text

In Silico Approach To Design a B-cell Epitope Based Vaccine Target Against Yellow Fever Virus

Bangladesh Journal of Microbiology ◽

10.3329/bjm.v35i1.39801 ◽

2019 ◽

Vol 35 (1) ◽

pp. 27-35

Author(s):

Shamira Tabrejee ◽

M Mahboob Hossain

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Peptide Vaccine ◽

Cell Epitope ◽

Epitope Prediction ◽

Fever Virus ◽

Young Infants ◽

E Protein

Yellow fever virus is a prototype member of the Flaviviridae family causing high fever and jaundice. Though YF 17D vaccine is administered to yellow fever patients, however it can produce adverse effects in immunocompromised, older people and young infants. The aim of this study is to design an epitope-based peptide vaccine by targeting envelope (E) protein of Yellow Fever Virus. Thirty sequences of E protein of Yellow Fever Virus strains were retrieved from NCBI database. E protein was found to be mostly conserved among all the sequences with little variability and also was identified as a probable antigen. Different epitope prediction tools predicted 4 common epitopes, 3 of which were found to be antigenic. A peptide VKNPTDTGin E protein was predicted to have surface accessibility which overlaps with the VKNPTDTGHGT epitope.So, the whole VKNPTDTGHGT epitope was taken for further analysis. The VKNPTDTGHGT epitope showed 96.67% conservancy and also possesses flexibility, hydrophilicity and non-toxicity. Therefore, VKNPTDTGHGT can be regarded as a potential vaccine candidate against Yellow fever virus with further in vitro and in vivo validation. Bangladesh J Microbiol, Volume 35 Number 1 June 2018, pp 27-35

Download Full-text

Molecular determinants of Yellow Fever Virus pathogenicity in Syrian Golden Hamsters: one mutation away from virulence

10.1101/249383 ◽

2018 ◽

Author(s):

Raphaëlle Klitting ◽

Laura Roth ◽

Félix A. Rey ◽

Xavier de Lamballerie

Keyword(s):

Yellow Fever ◽

Reverse Genetics ◽

Yellow Fever Virus ◽

Virulent Strain ◽

Fever Virus ◽

Viral Loads ◽

Synonymous Mutations ◽

E Protein ◽

Α Helix

ABSTRACTYellow fever virus (Flavivirusgenus) is an arthropod-borne pathogen which can infect humans, causing a severe viscerotropic disease with a high mortality rate. Adapted viral strains allow the reproduction of yellow fever disease in hamsters with features similar to the human disease. Here, we used the Infectious Subgenomic Amplicons reverse genetics method to produce an equivalent to the hamster-virulent strain, Yellow FeverAp7, by introducing a set of 4 synonymous and 6 non-synonymous mutations into a single subgenomic amplicon, derived from the sequence of theAsibistrain. The resulting strain, Yellow FeverAp7M, induced a disease similar to that described forAp7in terms of symptoms, weight evolution, viral loads in the liver and lethality. Using the same methodology, we produced mutant strains derived from eitherAp7MorAsibiviruses and investigated the role of each ofAp7Mnon-synonymous mutations in itsin vivophenotype. This allowed identifying key components of the virulence mechanism in hamsters. InAp7Mvirus, the reversion of either E/Q27H or E/D155A mutations, led to an important reduction of both virulence andin vivoreplicative fitness. In addition, the introduction of the single D155AAp7Mmutation within the E protein of theAsibivirus was sufficient to drastically modify its phenotype in hamsters towards both a greater replication efficiency and virulence. Finally, inspection of theAsibistrain E protein structure combined toin vivotesting revealed the importance of an exposed α-helix in domain I, containing residues 154 and 155, forAp7Mvirulence in hamsters.

Download Full-text