Project description:The outbreak-causing monkeypox virus of 2022 (2022 MPXV) is classified as a clade IIb strain and phylogenetically distinct from prior endemic MPXV strains (clades I or IIa), suggesting that its virological properties may also differ. Here, we used human keratinocytes and induced pluripotent stem cell-derived colon organoids to examine the efficiency of viral growth in these cells and the MPXV infection-mediated host responses. MPXV replication was much more productive in keratinocytes than in colon organoids. We observed that MPXV infections, regardless of strain, caused cellular dysfunction and mitochondrial damage in keratinocytes. Notably, a significant increase in the expression of hypoxia-related genes was observed specifically in 2022 MPXV-infected keratinocytes. Our comparison of virological features between 2022 MPXV and prior endemic MPXV strains revealed signaling pathways potentially involved with the cellular damages caused by MPXV infections and highlights host vulnerabilities that could be utilized as protective therapeutic strategies against human mpox in the future.
Project description:Monkeypox virus (MPXV) is a zoonotic virus endemic to Africa that has recently re-emerged, becoming the first orthopoxvirus with sustained human-to-human transmission since smallpox eradication. The 2022 global epidemic of MPXV was caused by Clade IIb virus (lineage B.1) that derived from the lineage A endemic to West Africa through APOBEC3 microevolution1-3. Here, comparison of lineage B.1 virus with its closest earlier A.1 ancestor reveals that despite only 46 nucleotide differences, lineage B.1 has attenuating phenotypic changes. Both viruses replicated equivalently in cell culture, but B.1 had defects in long-range spread, which mapped to APOBEC3 mutation E353K in OPG057 (F13L). MPXV B.1 was also defective in innate immune control, inducing higher IFNβ and innate immune signalling gene expression, and showing reduced capacity to suppress interleukin-1β-mediated responses, which mapped to APOBEC mutation R48C in OPG047 (F3L). Finally, MPXV B.1 had reduced virulence in a mouse model of MPXV infection relative to the ancestral A.1 strain. Our work demonstrates that the 2022 global outbreak MPXV was intrinsically attenuated, with attenuating mutations that are absent in concurrent, more severe clade IIb outbreaks. Our data also reveal that APOBEC3 editing is a mechanism of MPXV phenotypic variation, potentially contributing to clade evolution and competition.
Project description:The soaring global monkeypox cases lead to a surge in demand for monkeypox vaccine, which far exceeds the supply. mRNA vaccine has achieved great success in prevention of coronavirus disease and holds promise against diverse pathogens. In this study, we generate a polyvalent lipid nanoparticle (LNP) mRNA vaccine candidate for monkeypox virus (MPXV) and evaluate its immunogenicity in animal models. This polyvalent MPXV mRNA vaccine candidate, MPXVac-097, encodes five 2022 MPXV targets that are important surface antigens. Three-dose (prime-boost-booster) MPXVac-097 vaccination elicits strong antibody response to A35R and E8L antigens, moderate response to M1R, but not B6R or A29, highlighting the differences in immunogenicity. Bulk T cell receptor (TCR) sequencing reveals preferential usage of VJ combinations and clonal expansion of peripheral T cells after MPXVac-097 vaccination. These data demonstrate initial feasibility of developing MPXV mRNA vaccine and pave the way for its future optimization.
Project description:Orthopoxviruses are large DNA viruses which can cause disease in numerous host species. Even though the eradication of variola virus - the causative agent of human smallpox M-bM-^@M-^S succeeded, with the end of vaccinations several other orthopoxviruses emerged as potential threat to human health. For instance, animal-borne monkeypox virus, cowpox virus and closely related vaccinia virus are all capable of establishing zoonotic infections in humans. The disease caused by each virus differs in terms of expression and severity, but we still know little about the reasons for these different phenotypes. They may be explained by the unique repertoire of host cell modulating factors encoded by each virus. In this study, we aimed at characterizing the specific modulation of the host cells gene expression profile by orthopoxvirus infection. In our study we analyzed changes in host cell gene expression of HeLa cells after infection with cowpox virus, monkeypox virus or vaccinia virus and compared these to each other and to the gene expression profile of non-infected cells using Agilent Whole Genome Microarray technology. We could identify major differences in viral modulation of host cell immune response genes, especially an induction of genes involved in leukocyte migration and Toll-like receptor signalling in cowpox and monkeypox virus infected cells. This was not observed following vaccinia virus infection. If these differences contribute to the different clinical manifestation of cowpox, monkeypox and vaccinia virus infections in certain host species remains to be elucidated. We analyzed the gene expression profile of HeLa cells wich were either mock-infected or infected with Vaccinia virus strain IHD-W, Cowpox virus strain Brighton Red or Monkeypox virus strain MSF#6 at a multiplicity of infection of 5. Experiments were performed in duplicate. At 6 h post infection total RNA was isolated from infected cells and used for microarray analysis.
Project description:Orthopox viruses, including monkeypox, multiply intracellularly and induce numerous changes in host genes expression. The virus target mainly humoral host response, and simultaneously, exploits other genes and functions to reproduce effectively. The goal of this experiment is to identify those host genes and functions that are essential for monkeypox virus replication. Mock infected control cells were treated and incubated identically to time point arms, except for virus exposure. Two time points of cells infected with monkeypox virus were harvested at 3, 7 hours post infection, and gene expression was assessed using microarray in all arms. The experiment was done in triplicate.