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

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.The live, attenuated yellow fever virus (YFV) strain 17D vaccine has been used safely and effectively in over 500 million individuals over the past 70 years. This excellent safety record also underpins its potential application as a platform for chimeric vaccines against other medically important flaviviruses (dengue, Japanese encephalitis, and West Nile viruses), which are currently in clinical trials (reviewed in reference 13). Despite knowledge of the complete genome sequences of 17D (33) and its virulent parent (10), the Asibi strain isolated in Africa in 1927, the in vivo mechanism(s) and molecular determinants for virulence attenuation of the YFV vaccine are still not completely resolved. YFV strain 17D was the first human vaccine derived from repeated passage in tissue culture (reviewed in reference 39). More than 230 passages in embryonic mouse tissue (18 passages), chicken embryo tissue (50 passages), and chicken embryo tissue from which the head and spinal chord had been removed (152 passages) separate the 17D from the Asibi strain. The adaptation of 17D to growth in mouse and chicken embryonal tissues culture has resulted in loss of viscerotropism, a property which accounts for the major disease manifestations of yellow fever in primates: high viremia, hepatitis, renal dysfunction, hemorrhagic fever, and circulatory shock (reviewed in reference 28); it also resulted in reduced neurotropism and loss of tropism for mosquitoes, the obligatory vector in the natural transmission cycle of YFV. The complex passage history of YFV strain 17D has given rise to as many as 68 nucleotide mutations and 32 amino acid changes in the viral RNA genome (10). However, when the Asibi genome is compared to the consensus sequence of different 17D vaccine strains derived from it, the number of differences potentially contributing to the attenuated virulence phenotype can be reduced to 20 amino acids and 4 nucleotides in the 3Ј untranslated region...

Spectrum of disease outcomes in mice infected with YFV-17D

“…Attenuation of YFV-17D has been attributed to mutations in the YFV-17D envelope protein, which may hinder spread of the virus to visceral tissues, and was recently linked to reduced quasispecies diversity (Beck et al, 2014;Hahn et al, 1987;Lee & Lobigs, 2008). Considering that YFV-17D is currently being used in chimeric vaccines to JEV, and for the development of vaccines to WNV and DENV, a better understanding of its attenuation is imperative (Guirakhoo et al, 2006;Guy et al, 2010;Monath et al, , 2006Pugachev et al, 2005).…”

“…YFV can cause a viscerotropic disease resembling human disease, but only in peripherally injected immunodeficient animals such as mice deficient for IFN-a/b receptors (Meier et al, 2009). Peripheral injection of YFV-17D in adult mice lacking both IFN-a/b receptors and IFN-c receptors can cause a lethal neurotropic infection with transient viral infection of visceral organs and no observed tissue damage (Meier et al, 2009;Lee & Lobigs, 2008;Thibodeaux et al, 2012). Using a genetically marked pool of viruses, we previously found that YFV-17D encounters no major barriers that restrict viral diversity or dissemination in mice that lack IFN-a/b receptors (Erickson & Pfeiffer, 2013).…”

“…Laboratory rodents have varying degrees of susceptibility to YFV and YFV-17D infection, with disease outcome dependent on age, viral strain, immune status of the host, inoculation route and viral inoculum titre (Barrett & Gould, 1986;Fitzgeorge & Bradish, 1980;Horsfall, 1965;Lee & Lobigs, 2008;Meier et al, 2009;Theiler, 1930;Thibodeaux et al, 2012;Zisman et al, 1971). Infant mice are susceptible to fatal infection with intravenously or intraperitoneally injected virulent strains of YFV, including YFV-Asibi, as well as attenuated strains of YFV, such as YFV-17D (Fitzgeorge & Bradish, 1980;Horsfall, 1965;Zisman et al, 1971).…”

Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

“…route. Previous studies with engineered or selected mutants of WNV and some other JEV complex flaviviruses have identified single amino acid mutations that can attenuate mouse neuroinvasiveness (13,15,16,74,75) by multiple mechanisms that may be associated with direct or indirect effects on receptor binding or with increased glycosaminoglycan binding due to charge changes. Although it is possible that the reductions in the virulence of the EIII chimeras may have occurred due to negative effects on virion assembly, structure, or stability, the lack of obvious effects on in vitro behavior suggests that specific characteristics of the particular EIII sequence used likely play a more significant role in determining viral pathogenesis in mice.…”

The implication of amino acid mutations at flavivirus NS1-2A cleavage site on NS1’ protein production

“…The NS1 protein was preceded by its authentic signal peptide and the NS2A protein was C-terminally fused to an influenza virus hemagglutinin (HA) epitope tag (Pro-TyrAsp-Val-Pro-Asp-Tyr-Ala-Gly), which allows recovery of NS2A by immunoprecipitation with monoclonal antibody (mAb), 12CA5 (Wilson et al, 1984). To introduce amino acid substitutions in the octapeptide region, a fusion polymerase chain reaction (PCR) approach was employed (Lee and Lobigs, 2008) using the upstream primer, 5'-ACTGGATTGAGAGTGGACTCAATG-3', and downstream primer, 5'-CTGATCAGCGAGCTCTAGCATTTAAGGTGA-3', in combination with the corresponding mutagenesis primers (sequences will be provided upon request). Construction of mutant derivatives from plasmid, pRc/CMV.NS1-2A.HA, was by double digestion of mutagenized fragments (932 bp) and the corresponding ns1-ns2A gene region in the wild-type (wt) plasmid with restriction enzymes, PpuMI and XbaI, and replacement of the wt with a mutated cDNA fragment.…”