A small animal peripheral challenge model of yellow fever using interferon-receptor deficient mice and the 17D-204 vaccine strain.
ABSTRACT: Yellow fever virus (YFV), a member of the genus Flavivirus, is a mosquito-borne pathogen that requires wild-type (wt), virulent strains to be handled at biosafety level (BSL) 3, with HEPA-filtration of room air exhaust (BSL3+). YFV is found in tropical regions of Africa and South America and causes severe hepatic disease and death in humans. Despite the availability of effective vaccines (17D-204 or 17DD), YFV is still responsible for an estimated 200,000 cases of illness and 30,000 deaths annually. Besides vaccination, there are no other prophylactic or therapeutic strategies approved for use in human YF. Current small animal models of YF require either intra-cranial inoculation of YF vaccine to establish infection, or use of wt strains (e.g., Asibi) in order to achieve pathology. We have developed and characterized a BSL2, adult mouse peripheral challenge model for YFV infection in mice lacking receptors for interferons ?, ?, and ? (strain AG129). Intraperitoneal challenge of AG129 mice with 17D-204 is a uniformly lethal in a dose-dependent manner, and 17D-204-infected AG129 mice exhibit high viral titers in both brain and liver suggesting this infection is both neurotropic and viscerotropic. Furthermore the use of a mouse model permitted the construction of a 59-biomarker multi-analyte profile (MAP) using samples of brain, liver, and serum taken at multiple time points over the course of infection. This MAP serves as a baseline for evaluating novel therapeutics and their effect on disease progression. Changes (4-fold or greater) in serum and tissue levels of pro- and anti-inflammatory mediators as well as other factors associated with tissue damage were noted in AG129 mice infected with 17D-204 as compared to mock-infected control animals.
Project description:Mosquito-borne yellow fever virus (YFV) causes highly lethal, viscerotropic disease in humans and non-human primates. Despite the availability of efficacious live-attenuated vaccine strains, 17D-204 and 17DD, derived by serial passage of pathogenic YFV strain Asibi, YFV continues to pose a significant threat to human health. Neither the disease caused by wild-type YFV, nor the molecular determinants of vaccine attenuation and immunogenicity, have been well characterized, in large part due to the lack of a small animal model for viscerotropic YFV infection. Here, we describe a small animal model for wild-type YFV that manifests clinical disease representative of that seen in primates without adaptation of the virus to the host, which was required for the current hamster YF model. Investigation of the role of type I interferon (IFN-alpha/beta) in protection of mice from viscerotropic YFV infection revealed that mice deficient in the IFN-alpha/beta receptor (A129) or the STAT1 signaling molecule (STAT129) were highly susceptible to infection and disease, succumbing within 6-7 days. Importantly, these animals developed viscerotropic disease reminiscent of human YF, instead of the encephalitic signs typically observed in mice. Rapid viremic dissemination and extensive replication in visceral organs, spleen and liver, was associated with severe pathologies in these tissues and dramatically elevated MCP-1 and IL-6 levels, suggestive of a cytokine storm. In striking contrast, infection of A129 and STAT129 mice with the 17D-204 vaccine virus was subclinical, similar to immunization in humans. Although, like wild-type YFV, 17D-204 virus amplified within regional lymph nodes and seeded a serum viremia in A129 mice, infection of visceral organs was rarely established and rapidly cleared, possibly by type II IFN-dependent mechanisms. The ability to establish systemic infection and cause viscerotropic disease in A129 mice correlated with infectivity for A129-derived, but not WT129-derived, macrophages and dendritic cells in vitro, suggesting a role for these cells in YFV pathogenesis. We conclude that the ability of wild-type YFV to evade and/or disable components of the IFN-alpha/beta response may be primate-specific such that infection of mice with a functional IFN-alpha/beta antiviral response is attenuated. Consequently, subcutaneous YFV infection of A129 mice represents a biologically relevant model for studying viscerotropic infection and disease development following wild-type virus inoculation, as well as mechanisms of 17D-204 vaccine attenuation, without a requirement for adaptation of the virus.
Project description:Yellow fever virus (YFV), a member of the genus Flavivirus, is a mosquito-borne virus found in tropical regions of Africa and South America that causes severe hepatic disease and death in humans. Despite the availability of effective vaccines, YFV is responsible for an estimated 200,000 cases and 30,000 deaths annually. There are currently no prophylactic or therapeutic strategies approved for use in human YFV infections. Furthermore, implementation of YFV 17D-204 vaccination campaigns has become problematic due to an increase in reported post-vaccinal adverse events. We have created human/murine chimeric MAbs of a YFV-reactive murine monoclonal antibody (mMAb), 2C9, that was previously shown to protect mice from lethal YFV infection and to have therapeutic activity. The new chimeric (cMAbs) were constructed by fusion of the m2C9 IgG gene variable regions with the constant regions of human IgG and IgM and expressed in Sp2 murine myelomas. The 2C9 cMAbs (2C9-cIgG and 2C9-cIgM) reacted with 17D-204 vaccine strain in an enzyme-linked immunosorbent assay and neutralized virus in vitro similarly to the parent m2C9. Both m2C9 and 2C9-cIgG when administered prophylactically 24h prior to infection protected AG129 mice from peripheral 17D-204 challenge at antibody concentrations ?1.27 ?g/mouse; however, the 2C9-cIgM did not protect even at a dose of 127 ?g/mouse. The 17D-204 infection of AG129 mice is otherwise uniformly lethal. While the m2C9 was shown previously to be therapeutically effective in YFV-infected BALB/c mice at day 4 post-infection, the m2C9 and 2C9-cIgG demonstrated therapeutic activity only when administered 1 day post-infection in 17D-204-infected AG129 mice.
Project description:By infecting mice with the yellow fever virus vaccine strain 17D (YFV-17D; Stamaril®), the dose dependence and evolutionary consequences of neurotropic yellow fever infection was assessed. Highly susceptible AG129 mice were used to allow for a maximal/unlimited expansion of the viral populations. Infected mice uniformly developed neurotropic disease; the virus was isolated from their brains, plaque purified and sequenced. Viral RNA populations were overall rather homogenous [Shannon entropies 0-0.15]. The remaining, yet limited intra-host population diversity (0-11 nucleotide exchanges per genome) appeared to be a consequence of pre-existing clonal heterogeneities (quasispecies) of Stamaril®. In parallel, mice were infected with a molecular clone of YFV-17D which was in vivo launched from a plasmid. Such plasmid-launched YFV-17D had a further reduced and almost clonal evolution. The limited intra-host evolution during unrestricted expansion in a highly susceptible host is relevant for vaccine and drug development against flaviviruses in general. Firstly, a propensity for limited evolution even upon infection with a (very) low inoculum suggests that fractional dosing as implemented in current YF-outbreak control may pose only a limited risk of reversion to pathogenic vaccine-derived virus variants. Secondly, it also largely lowers the chance of antigenic drift and development of resistance to antivirals.
Project description:Zika virus: Zika virus chimeric vaccineZika virus (ZIKV) infection generally results in mild symptoms but can cause serious developmental abnormalities in infants born to ZIKV infected mothers. Kai Dallmeier and colleagues at the KU Leuven in Belgium, engineered a chimeric live-attenuated vaccine (YF-ZIKprM/E) by swapping the glycoprotein from the Yellow Fever vaccine YFV-17D with that of a pre-epidemic ZIKV strain. YF-ZIKprM/E is very well tolerated with no adverse effects even following high dose intracranial infection. Mice highly susceptible to ZIKV infection—including AG129 and type I interferon receptor deficient strains—vaccinated with a single dose of YF-ZIKprM/E are fully protected from lethal ZIKV challenge. Protection can be achieved within 7 days and by low doses of YF-ZIKprM/E, is durable and generally results in sterilizing immunity. YF-ZIKprM/E elicits both neutralizing antibodies and robust cellular immunity. Finally, YF-ZIKprM/E can also prevent vertical transmission of ZIKV and achieve efficient protection of pups from neurological defects following intraplacental challenge.
Project description:The first comparison of a live RNA viral vaccine strain to its wild-type parental strain by deep sequencing is presented using as a model the yellow fever virus (YFV) live vaccine strain 17D-204 and its wild-type parental strain, Asibi.The YFV 17D-204 vaccine genome was compared to that of the parental strain Asibi by massively parallel methods. Variability was compared on multiple scales of the viral genomes. A modeled exploration of small-frequency variants was performed to reconstruct plausible regions of mutational plasticity.Overt quasispecies diversity is a feature of the parental strain, whereas the live vaccine strain lacks diversity according to multiple independent measurements. A lack of attenuating mutations in the Asibi population relative to that of 17D-204 was observed, demonstrating that the vaccine strain was derived by discrete mutation of Asibi and not by selection of genomes in the wild-type population.Relative quasispecies structure is a plausible correlate of attenuation for live viral vaccines. Analyses such as these of attenuated viruses improve our understanding of the molecular basis of vaccine attenuation and provide critical information on the stability of live vaccines and the risk of reversion to virulence.
Project description:<h4>Unlabelled</h4>The 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.<h4>Importance</h4>The 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.
Project description:One paradigm to explain the complexity of viral RNA populations is that the low fidelity of the RNA-dependent RNA polymerase (RdRp) drives high mutation rates and consequently genetic diversity. Like most RNA viruses, wild-type yellow fever virus (YFV) replication is error-prone due to the lack of proofreading by the virus-encoded RdRp. However, there is evidence that replication of the live attenuated YF vaccine virus 17D, derived from wild-type strain Asibi, is less error-prone than wild-type RNA viruses. Recent studies comparing the genetic diversity of wild-type Asibi and 17D vaccine virus found that wild-type Asibi has the typical heterogeneous population of an RNA virus, while there is limited intra- and interpopulation variability of 17D vaccine virus. Utilizing chimeric and mutant infectious clone-derived viruses, we show that high and low genetic diversity profiles of wild-type Asibi virus and vaccine virus 17D, respectively, are multigenic. Introduction of either structural (pre-membrane and envelope) genes or NS2B or NS4B substitutions into the Asibi and 17D backbone resulted in altered variant population, nucleotide diversity, and mutation frequency compared to the parental viruses. Additionally, changes in genetic diversity of the chimeric and mutant viruses correlated with the phenotype of multiplication kinetics in human alveolar A549 cells. Overall, the paradigm that only the error-prone RdRp controls genetic diversity needs to be expanded to address the role of other genes in genetic diversity, and we hypothesize that it is the replication complex as a whole and not the RdRp alone that controls genetic diversity.IMPORTANCE With the advent of advanced sequencing technology, studies of RNA viruses have shown that genetic diversity can contribute to both attenuation and virulence and the paradigm is that this is controlled by the error-prone RNA-dependent RNA polymerase (RdRp). Since wild-type yellow fever virus (YFV) strain Asibi has genetic diversity typical of a wild-type RNA virus, while 17D virus vaccine has limited diversity, it provides a unique opportunity to investigate RNA population theory in the context of a well-characterized live attenuated vaccine. Utilizing infectious clone-derived viruses, we show that genetic diversity of RNA viruses is complex and that multiple genes, including structural genes and NS2B and NS4B genes also contribute to genetic diversity. We suggest that the replication complex as a whole, rather than only RdRp, drives genetic diversity, at least for YFV.
Project description:Humans infected with yellow fever virus (YFV), a mosquito-borne flavivirus, can develop illness ranging from a mild febrile disease to hemorrhagic fever and death. The 17D vaccine strain of YFV was developed in the 1930s, has been used continuously since development and has proven very effective. Genetic differences between vaccine and wild-type viruses are few, yet viral or host mechanisms associated with protection or disease are not fully understood. Over the past 20 years, a number of cases of vaccine-associated disease have been identified following vaccination with 17D; these cases have been correlated with reduced immune status at the time of vaccination. Recently, several studies have evaluated T cell responses to vaccination in both humans and non-human primates, but none have evaluated the response to wild-type virus infection. In the studies described here, monocyte-derived macrophages (MDM) and dendritic cells (MoDC) from both humans and rhesus macaques were evaluated for their ability to support infection with either wild-type Asibi virus or the 17D vaccine strain and the host cytokine and chemokine response characterized. Human MoDC and MDM were also evaluated for their ability to stimulate CD4+ T cells. It was found that MoDC and MDM supported viral replication and that there were differential cytokine responses to infection with either wild-type or vaccine viruses. Additionally, MoDCs infected with live 17D virus were able to stimulate IFN-? and IL-2 production in CD4+ T cells, while cells infected with Asibi virus were not. These data demonstrate that wild-type and vaccine YFV stimulate different responses in target antigen presenting cells and that wild-type YFV can inhibit MoDC activation of CD4+ T cells, a critical component in development of protective immunity. These data provide initial, but critical insight into regulatory capabilities of wild-type YFV in development of disease.
Project description:Yellow fever virus (YFV)-17D is an empirically developed, highly effective live-attenuated vaccine that has been administered to human beings for almost a century. YFV-17D has stood as a paradigm for a successful viral vaccine, and has been exploited as a potential virus vector for the development of recombinant vaccines against other diseases. In this study, a DNA-launched YFV-17D construct (pBeloBAC-FLYF) was explored as a new modality to the standard vaccine to combine the commendable features of both DNA vaccine and live-attenuated viral vaccine. The DNA-launched YFV-17D construct was characterized extensively both in cell culture and in mice. High titres of YFV-17D were generated upon transfection of the DNA into cells, whereas a mutant with deletion in the capsid-coding region (pBeloBAC-YF/?C) was restricted to a single round of infection, with no release of progeny virus. Homologous prime-boost immunization of AAD mice with both pBeloBAC-FLYF and pBeloBAC-YF/?C elicited specific dose-dependent cellular immune response against YFV-17D. Vaccination of A129 mice with pBeloBAC-FLYF resulted in the induction of YFV-specific neutralizing antibodies in all vaccinated subjects. These promising results underlined the potential of the DNA-launched YFV both as an alternative to standard YFV-17D vaccination and as a vaccine platform for the development of DNA-based recombinant YFV vaccines.
Project description:The yellow fever (YF) 17D vaccine is one of the most effective human vaccines ever created. The YF vaccine has been produced since 1937 in embryonated chicken eggs inoculated with the YF 17D virus. Yet, little information is available about the infection mechanism of YF 17DD virus in this biological model. To better understand this mechanism, we infected embryos of Gallus gallus domesticus and analyzed their histopathology after 72 hours of YF infection. Some embryos showed few apoptotic bodies in infected tissues, suggesting mild focal infection processes. Confocal and super-resolution microscopic analysis allowed us to identify as targets of viral infection: skeletal muscle cells, cardiomyocytes, nervous system cells, renal tubular epithelium, lung parenchyma, and fibroblasts associated with connective tissue in the perichondrium and dermis. The virus replication was heaviest in muscle tissues. In all of these specimens, RT-PCR methods confirmed the presence of replicative intermediate and genomic YF RNA. This clearer characterization of cell targets in chicken embryos paves the way for future development of a new YF vaccine based on a new cell culture system.