mRNA-LNP vaccine-induced CD8+ T cells protect mice from lethal SARS-CoV-2 infection in the absence of specific antibodies.

The role of CD8+ T cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.

Resistance To Poxvirus Lethality Does Not Require the Necroptosis Proteins RIPK3 or MLKL.

Receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL) are proteins that are critical for necroptosis, a mechanism of programmed cell death that is both activated when apoptosis is inhibited and 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-/- mice, but not Ripk3-/- mice, endured lower viral titers than WT mice in the draining lymph node (dLN) at three days postinfection and in the spleen or in the liver at seven days postinfection. Despite the improved viral control, Mlkl-/- mice did not differ from WT mice in the expression of interferons or interferon-stimulated genes or in 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-/- and WT mice at 7 dpi did not differ. Thus, the mechanism of the increased virus control by Mlkl-/- mice remains to be defined. IMPORTANCE The molecules RIPK3 and MLKL are required for necroptotic cell death, which is widely thought of as an antiviral mechanism. Here we show that C57BL/6 (B6) mice deficient in RIPK3 or MLKL are as susceptible as WT B6 mice to ECTV lethality after a low-dose intraperitoneal infection and are as resistant as WT B6 mice after ECTV infection through the natural footpad route. Mice deficient in MLKL are more efficient than WT mice at controlling virus loads in various organs. This improved viral control is not due to enhanced interferon, natural killer cell, or CD8 T-cell responses. Overall, the data indicate that deficiencies in the molecules that are critical to necroptosis do not necessarily result in worse outcomes following viral infection and may improve virus control.

The roles of CD4+ T cell help, sex, and dose in the induction of protective CD8+ T cells against a lethal poxvirus by mRNA-LNP vaccines.

The role of CD4+ T cells in the induction of protective CD8+ T cells by mRNA lipid nanoparticle (LNP) vaccines is unknown. We used B6 or Tlr9 -/- mice depleted or not of CD4+ T cells and LNP vaccines loaded with mRNAs encoding the ectromelia virus (ECTV) MHC class I H-2 Kb-restricted immunodominant CD8+ T cell epitope TSYKFESV (TSYKFESV mRNA-LNPs) or the ECTV EVM158 protein, which contains TSYKFESV (EVM-158 mRNA-LNPs). Following prime and boost with 10 µg of either vaccine, Kb-TSYKFESV-specific CD8+ T cells fully protected male and female mice from ECTV at 29 (both mRNA-LNPs) or 90 days (EVM158 mRNA-LNPs) post boost (dpb) independently of CD4+ T cells. However, at 29 dpb with 1 µg mRNA-LNPs, males had lower frequencies of Kb-TSYKFESV-specific CD8+ T cells and were much less well protected than females from ECTV, also independently of CD4+ T cells. At 90 dpb with 1 µg EVM158 mRNA-LNPs, the frequencies of Kb-TSYKFESV-specific CD8+ T cells in males and females were similar, and both were similarly partially protected from ECTV, independently of CD4+ T cells. Therefore, at optimal or suboptimal doses of mRNA-LNP vaccines, CD4+ T cell help is unnecessary to induce protective anti-poxvirus CD8+ T cells specific to a dominant epitope. At suboptimal doses, protection of males requires more time to develop.

Altered cleavage of human factor VIII at the B-domain and acidic region 3 interface enhances expression after gene therapy in hemophilia A mice.

BACKGROUND: Variants of human factor VIII (hFVIII) have been developed to further understand the structure and function of hFVIII and improve gene-based therapeutics. We have previously characterized several hFVIII variants of the furin cleavage site (1645-1648) with improved secretion. We have also identified a second cleavage site in the acidic region 3 (a3) (1657-1658) that becomes the primary hFVIII intracellular cleavage position in the absence of the furin site. We tested a hypothesis that modification of this site may confer additional functional advantages to hFVIII. OBJECTIVES: The aim of this study was to conduct the biochemical and functional characterization of hFVIII variants of the furin cleavage site, the a3 cleavage site, or in combination, both in vitro and in vivo after AAV mediated gene therapy. METHODS: Recombinant hFVIII variants of the furin cleavage site (hFVIII-Δ3), the a3 cleavage site (hFVIII-S1657P/D1658E [SP/DE]), or in combination (hFVIII-Δ3-SP/DE) were purified and characterized in vitro and in vivo. RESULTS: Recombinant hFVIII-Δ3, hFVIII-SP/DE, and hFVIII-Δ3-SP/DE variants all had comparable specific activity to B-domain deleted (BDD) hFVIII. Hemophilia A mice tolerant to hFVIII did not develop immune responses to hFVIII after protein challenge with these variants or after adeno-associated virus (AAV) delivery. Following AAV delivery, hFVIII-Δ3-SP/DE resulted in expression levels that were 2- to 5-fold higher than those with hFVIII-BDD in hemophilia A mice. CONCLUSION: The novel hFVIII-Δ3-SP/DE variant of the furin and a3 cleavage sites significantly improved secretion compared with hFVIII-BDD. This key feature of the Δ3-SP/DE variant provides a unique strategy that can be combined with other approaches to further improve factor VIII expression to achieve superior efficacy in AAV-based gene therapy for hemophilia A.

Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice.

Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.

A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.

Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9-11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.