Studies on the Role of Necroptosis Proteins RIPK3 and MLKL in Resistance to Viral Infection and on the Role of CD8 T-Cells Induced by mRNA-LNP Vaccines in Protection from SARS-CoV-2 in Mice
Receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL) are proteins critical for necroptosis, a mechanism of programmed cell death that is activated when apoptosis is inhibited and is thought to be antiviral. Here, we investigated the role of RIPK3 and MLKL in controlling the Orthopoxvirus ectromelia virus (ECTV), a natural pathogen of the mouse. We found that C57BL/6 (B6) mice deficient in RIPK3 (Ripk3-/-) or MLKL (Mlkl-/-) were as susceptible as wild-type (WT) B6 mice to ECTV lethality after low-dose intraperitoneal infection and were as resistant as WT B6 mice after ECTV infection through the natural footpad route. Additionally, after footpad infection, Mlkl-/- but not Rip3-/- mice endured lower viral titers than WT mice in the draining lymph node (dLN) at three and the spleen or the liver at seven days post-infection. Despite the improved viral control, Mlkl-/- mice did not differ from WT in the expression of interferons or interferon-stimulated genes or the recruitment of Natural Killer (NK) cells and inflammatory monocytes (iMOs) to the dLN. Additionally, The CD8 T-cell responses in Mlkl-/- and WT mice were similar even though in the dLNs of Mlkl-/- mice, professional antigen-presenting cells were more heavily infected. Finally, the histopathology in the livers of Mlkl-/- mice and WT mice at 7 dpi did not differ. Thus, the mechanism of the increased virus control by Mlkl-/- mice remains to be defined. Vaccines exploit the adaptive immune system’s ability to recognize and respond to antigens from pathogens to quickly resolve subsequent infections. The encapsulation of mRNA encoding an antigen of interest in lipid nanoparticles (mRNA-LNP) has recently emerged as a very effective vaccine technology. The SARS-CoV-2 mRNA-LNP vaccines, which carry mRNA encoding the Spike (S) protein stabilized in the pre-fusion conformation by means of two proline mutations (S-2P), have been instrumental in combating the SARS-CoV-2 pandemic. The main correlate of protection induced by the S-2P SARS-CoV-2 mRNA-LNP vaccines is the induction of antibodies (Abs). S-2P SARS-CoV-2 mRNA-LNP vaccines also induce CD8 T-cell responses, but their role in acquired protection against COVID-19 remains unknown. The S protein contains the peptide VNFNFNGL, which is a strong CD8 T-cell epitope in C57BL/6 (B6) mice. We show that VNFNFNGL mRNA-LNPs, carrying just a VNFNFNGL-encoding mini-mRNA, is as efficient as S-2P mRNA-LNP at inducing VNFNFNGL-specific CD8 T-cell responses. Using two mouse models of SARS-CoV-2 infection, we show that the CD8 T-cells induced by VNFNFNGL mRNA-LNP immunization protected mice from SARS-CoV-2 lethality but, different from S-2P mRNA-LNP, not from weight loss. Depletion of CD8 T-cells in mice immunized with VNFNFNGL, but not with S-2P mRNA-LNP, ablated protection, indicating that CD8 T-cells can protect, but are unnecessary when protective Abs are present.
Montoya, Brian, "Studies on the Role of Necroptosis Proteins RIPK3 and MLKL in Resistance to Viral Infection and on the Role of CD8 T-Cells Induced by mRNA-LNP Vaccines in Protection from SARS-CoV-2 in Mice" (2023). ProQuest ETD Collection - Thomas Jefferson University. AAI30423052.