•Live-attenuated V4020 VEEV vaccine with rearranged structural genes is described.•V4020 included attenuating mutations engineered to prevent reversions.•Vaccinated mice developed neutralizing ...antibodies and survived challenge.•Intracranial injections in mice showed attenuated replication of V4020 compared to the TC83.•V4020 has safety advantage over TC83, while provides equivalent protection after VEEV challenge.
Novel live-attenuated V4020 vaccine was prepared for Venezuelan equine encephalitis virus (VEEV), an alphavirus from the Togaviridae family. The genome of V4020 virus was rearranged, with the capsid gene expressed using a duplicate subgenomic promoter downstream from the glycoprotein genes. V4020 also included both attenuating mutations from the TC83 VEEV vaccine secured by mutagenesis to prevent reversion mutations. The full-length infectious RNA of V4020 vaccine virus was expressed from pMG4020 plasmid downstream from the CMV promoter and launched replication of live-attenuated V4020 in vitro or in vivo. BALB/c mice vaccinated with a single dose of V4020 virus or with pMG4020 plasmid had no adverse reactions to vaccinations and developed high titers of neutralizing antibodies. After challenge with the wild type VEEV, vaccinated mice survived with no morbidity, while all unvaccinated controls succumbed to lethal infection. Intracranial injections in mice showed attenuated replication of V4020 vaccine virus as compared to the TC83. We conclude that V4020 vaccine has safety advantage over TC83, while provides equivalent protection in a mouse VEEV challenge model.
Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and apathogenic strains. Despite remarkable ...genetic similarity, the viscerotropic WE strain of LCMV causes a fatal LASV fever-like hepatitis in non-human primates (NHPs) while the mouse-adapted Armstrong (ARM) strain of LCMV is deeply attenuated in NHPs and can vaccinate against LCMV-WE challenge. Here, we demonstrate that internalization of WE is more sensitive to the depletion of membrane cholesterol than ARM infection while ARM infection is more reliant on endosomal acidification. LCMV-ARM induces robust NF-κB and interferon response factor (IRF) activation while LCMV-WE seems to avoid early innate sensing and failed to induce strong NF-κB and IRF responses in dual-reporter monocyte and epithelial cells. Toll-like receptor 2 (TLR-2) signaling appears to play a critical role in NF-κB activation and the silencing of TLR-2 shuts down IL-6 production in ARM but not in WE-infected cells. Pathogenic LCMV-WE infection is poorly recognized in early endosomes and failed to induce TLR-2/Mal-dependent pro-inflammatory cytokines. Following infection, Interleukin-1 receptor-associated kinase 1 (IRAK-1) expression is diminished in LCMV-ARM- but not LCMV-WE-infected cells, which indicates it is likely involved in the LCMV-ARM NF-κB activation. By confocal microscopy, ARM and WE strains have similar intracellular trafficking although LCMV-ARM infection appears to coincide with greater co-localization of early endosome marker EEA1 with TLR-2. Both strains co-localize with Rab-7, a late endosome marker, but the interaction with LCMV-WE seems to be more prolonged. These findings suggest that LCMV-ARM's intracellular trafficking pathway may facilitate interaction with innate immune sensors, which promotes the induction of effective innate and adaptive immune responses.
Junin (JUNV) and Machupo (MACV), two mammalian arenaviruses placed on the 2018 WHO watch list, are prevalent in South America causing Argentine and Bolivian hemorrhagic fevers (AHF and BHF), ...respectively. The live attenuated JUNV vaccine, Candid #1, significantly reduced the incidence of AHF. Vaccination induces neutralizing antibody (nAb) responses which effectively target GP1 (the viral attachment glycoprotein) pocket which accepts the tyrosine residue of the cellular receptor, human transferrin receptor 1 (TfR1). In spite of close genetic relationships between JUNV and MACV, variability in the GP1 receptor binding site (e.g., MACV GP1 loop 10) results in poor MACV neutralization by Candid #1-induced nAbs. Candid #1 is not recommended for vaccination of children younger than 15 years old (a growing “at risk” group), pregnant women, or other immunocompromised individuals. Candid #1’s primary reliance on limited missense mutations for attenuation, genetic heterogeneity, and potential stability concerns complicate approval of this vaccine in the US. To address these issues, we applied alphavirus RNA replicon vector technology based on the human Venezuelan equine encephalitis vaccine (VEEV) TC-83 to generate replication restricted virus-like-particles vectors (VLPVs) simultaneously expressing cellular glycoprotein precursors (GPC) of both viruses, JUNV and MACV. Resulting JV&MV VLPVs were found safe and immunogenic in guinea pigs. Immunization with VLPVs induced humoral responses which correlated with complete protection against lethal disease after challenge with pathogenic strains of JUNV (Romero) and MACV (Carvallo).
Viral hemorrhagic fevers (VHFs) encompass a group of diseases with cardinal symptoms of fever, hemorrhage, and shock. The liver is a critical mediator of VHF disease pathogenesis and high levels of ...ALT/AST transaminases in plasma correlate with poor prognosis. In fact, Lassa Fever (LF), the most prevalent VHF in Africa, was initially clinically described as hepatitis. Previous studies in non-human primate (NHP) models also correlated LF pathogenesis with a robust proliferative response in the liver. The purpose of the current study was to gain insight into the mechanism of liver injury and to determine the potential role of proliferation in LF pathogenesis. C57Bl/6J mice were infected with either the pathogenic (for NHPs) strain of lymphocytic choriomeningitis virus (LCMV, the prototypic arenavirus), LCMV-WE, or with the non-pathogenic strain, LCMV-ARM. As expected, LCMV-WE, but not ARM, caused a hepatitis-like infection. LCMV-WE also induced a robust increase in the number of actively cycling hepatocytes. Despite this increase in proliferation, there was no significant difference in liver size between LCMV-WE and LCMV-ARM, suggesting that cell cycle was incomplete. Indeed, cells appeared arrested in the G1 phase and LCMV-WE infection increased the number of hepatocytes that were simultaneously stained for proliferation and apoptosis. LCMV-WE infection also induced expression of a non-conventional virus receptor, AXL-1, from the TAM (TYRO3/AXL/MERTK) family of receptor tyrosine kinases and this expression correlated with proliferation. Taken together, these results shed new light on the mechanism of liver involvement in VHF pathogenesis. Specifically, it is hypothesized that the induction of hepatocyte proliferation contributes to expansion of the infection to parenchymal cells. Elevated levels of plasma transaminases are likely explained, at least in part, by abortive cell cycle arrest induced by the infection. These results may lead to the development of new therapies to prevent VHF progression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Lassa virus (LASV), a highly prevalent mammalian arenavirus endemic in West Africa, can cause Lassa fever (LF), which is responsible for thousands of deaths annually. LASV is transmitted to humans ...from naturally infected rodents. At present, there is not an effective vaccine nor treatment. The genetic diversity of LASV is the greatest challenge for vaccine development. The reassortant ML29 carrying the L segment from the nonpathogenic Mopeia virus (MOPV) and the S segment from LASV is a vaccine candidate under current development. ML29 demonstrated complete protection in validated animal models against a Nigerian strain from clade II, which was responsible for the worst outbreak on record in 2018. This study demonstrated that ML29 was more attenuated than MOPV in STAT1
mice, a small animal model of human LF and its sequelae. ML29 infection of these mice resulted in more than a thousand-fold reduction in viremia and viral load in tissues and strong LASV-specific adaptive T cell responses compared to MOPV-infected mice. Persistent infection of Vero cells with ML29 resulted in generation of interfering particles (IPs), which strongly interfered with the replication of LASV, MOPV and LCMV, the prototype of the Arenaviridae. ML29 IPs induced potent cell-mediated immunity and were fully attenuated in STAT1
mice. Formulation of ML29 with IPs will improve the breadth of the host's immune responses and further contribute to development of a pan-LASV vaccine with full coverage meeting the WHO requirements.
Olanzapine (OLZ), an effective treatment of schizophrenia and other disorders, causes weight gain and metabolic syndrome. Most studies to date have focused on the potential effects of OLZ on the ...central nervous system's mediation of weight; however, peripheral changes in liver or other key metabolic organs may also play a role in the systemic effects of OLZ. Thus, the purpose of this study was to investigate the effects of OLZ on hepatic metabolism in a mouse model of OLZ exposure. Female C57Bl/6J mice were administered OLZ (8 mg/kg per day) or vehicle subcutaneously by osmotic minipumps for 28 days. Liver and plasma were taken at sacrifice for biochemical analyses and for comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry metabolomics analysis. OLZ increased body weight, fat pad mass, and liver-to-body weight ratio without commensurate increase in food consumption, indicating that OLZ altered energy expenditure. Expression and biochemical analyses indicated that OLZ induced anaerobic glycolysis and caused a pseudo-fasted state, which depleted hepatic glycogen reserves. OLZ caused similar effects in cultured HepG2 cells, as determined by Seahorse analysis. Metabolomic analysis indicated that OLZ increased hepatic concentrations of amino acids that can alter metabolism via the mTOR pathway; indeed, hepatic mTOR signaling was robustly increased by OLZ. Interestingly, OLZ concomitantly activated AMP-activated protein kinase (AMPK) signaling. Taken together, these data suggest that disturbances in glucose and lipid metabolism caused by OLZ in liver may be mediated, at least in part, via simultaneous activation of both catabolic (AMPK) and anabolic (mammalian target of rapamycin) pathways, which yields new insight into the metabolic side effects of this drug.
Mammarenavirusesare single-stranded RNA viruses with a bisegmented ambisense genome. Ingestion has been shown as a natural route of transmission for both Lassa virus (LASV) and Lymphocytic ...choriomeningitis virus (LCMV). Due to the mechanism of transmission, epithelial tissues are among the first host cells to come in contact with the viruses, and as such they potentially play a role in spread of virus to naïve hosts. The role of the intestinal epithelia during arenavirus infection remains to be uncharacterized. We have utilized a well-established cell culture model, Caco-2, to investigate the role of intestinal epithelia during intragastric infection. We found that LCMV-Armstrong, LCMV-WE, and Mopeia (MOPV) release infectious progeny via similar patterns. However, the reassortant virus, ML-29, containing the L segment of MOPV and S segment of LASV, exhibits a unique pattern of viral release relative to LCMV and MOPV. Furthermore, we have determined attachment efficacy to Caco-2 cells is potentially responsible for observed replication kinetics of these viruses in a polarized Caco-2 cell model. Collectively, our data shows that viral dissemination and interaction with intestinal epithelia may be host, tissue, and viral specific.
It is known that chronic ethanol significantly impairs liver regeneration. However, the effect of acute ethanol exposure on liver regeneration remains largely unknown. To address this question, ...C57Bl6/J mice were exposed to acute ethanol (6 g/kg intragastrically) for 3 days, and partial hepatectomy (PHx) was performed 24 h after the last dose. Surprisingly, acute ethanol preexposure promoted liver regeneration. This effect of ethanol did not correlate with changes in expression of cell cycle regulatory genes (e.g., cyclin D1, p21, and p27) but did correlate with protection against the effect of PHx on indices of impaired lipid and carbohydrate metabolism. Ethanol preexposure protected against inhibition of the oxidant-sensitive mitochondrial enzyme, aconitase. The activity of aldehyde dehydrogenase 2 (ALDH2) was significantly increased by ethanol preexposure. The effect of ethanol was blocked by inhibiting (Daidzin) and was mimicked by activating (Alda-1) ALDH2. Lipid peroxides are also substrates for ALDH2; indeed, alcohol preexposure blunted the increase in lipid peroxidation (4OH-nonenal adducts) caused by PHx. Taken together, these data suggest that acute preoperative ethanol exposure "preconditions" the liver to respond more rapidly to regenerate after PHx by activating mitochondrial ALDH2, which prevents oxidative stress in this compartment.
Early studies demonstrated that whole‐body androgen receptor (AR)–knockout mice with hypogonadism exhibit insulin resistance. However, details about the mechanisms underlying how androgen/AR ...signaling regulates insulin sensitivity in individual organs remain unclear. We therefore generated hepatic AR‐knockout (H‐AR−/y) mice and found that male H‐AR−/y mice, but not female H‐AR−/− mice, fed a high‐fat diet developed hepatic steatosis and insulin resistance, and aging male H‐AR−/y mice fed chow exhibited moderate hepatic steatosis. We hypothesized that increased hepatic steatosis in obese male H‐AR−/y mice resulted from decreased fatty acid β‐oxidation, increased de novo lipid synthesis arising from decreased PPARα, increased sterol regulatory element binding protein 1c, and associated changes in target gene expression. Reduced insulin sensitivity in fat‐fed H‐AR−/y mice was associated with decreased phosphoinositide‐3 kinase activity and increased phosphenolpyruvate carboxykinase expression and correlated with increased protein‐tyrosine phosphatase 1B expression. Conclusion: Together, our results suggest that hepatic AR may play a vital role in preventing the development of insulin resistance and hepatic steatosis. AR agonists that specifically target hepatic AR might be developed to provide a better strategy for treatment of metabolic syndrome in men. (HEPATOLOGY 2008.)
The safety and genetic stability of V4020, a novel Venezuelan Equine Encephalitis Virus (VEEV) vaccine based on the investigational VEEV TC-83 strain, was evaluated in mice. V4020 was generated from ...infectious DNA, contains a stabilizing mutation in the E2-120 glycoprotein, and includes rearrangement of structural genes. After intracranial inoculation (IC), replication of V4020 was more attenuated than TC-83, as documented by low clinical scores, inflammation, viral load in brain, and earlier viral clearance. During the first 9 days post-inoculation (DPI), genes involved in inflammation, cytokine signaling, adaptive immune responses, and apoptosis were upregulated in both groups. However, the magnitude of upregulation was greater in TC-83 than V4020 mice, and this pattern persisted till 13 DPI, while V4020 gene expression profiles declined to mock-infected levels. In addition, genetic markers of macrophages, DCs, and microglia were strongly upregulated in TC-83 mice. During five serial passages in the brain, less severe clinical manifestations and a lower viral load were observed in V4020 mice and all animals survived. In contrast, 13.3% of mice met euthanasia criteria during the passages in TC-83 group. At 2 DPI, RNA-Seq analysis of brain tissues revealed that V4020 mice had lower rates of mutations throughout five passages. A higher synonymous mutation ratio was observed in the nsP4 (RdRP) gene of TC-83 compared to V4020 mice. At 2 DPI, both viruses induced different expression profiles of host genes involved in neuro-regeneration. Taken together, these results provide evidence for the improved safety and genetic stability of the experimental V4020 VEEV vaccine in a murine model.
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