Therapeutic efficacy of favipiravir against Bourbon virus in mice

Bourbon virus (BRBV) is an emerging tick-borne RNA virus in the orthomyxoviridae family that was discovered in 2014. Although fatal human cases of BRBV have been described, little is known about its pathogenesis, and no antiviral therapies or vaccines exist. We obtained serum from a fatal case in 2017 and successfully recovered the second human infectious isolate of BRBV. Next-generation sequencing of the St. Louis isolate of BRBV (BRBV-STL) showed >99% nucleotide identity to the original reference isolate. Using BRBV-STL, we developed a small animal model to study BRBV-STL tropism in vivo and evaluated the prophylactic and therapeutic efficacy of the experimental antiviral drug favipiravir against BRBV-induced disease. Infection of Ifnar1-/- mice lacking the type I interferon receptor, but not congenic wild-type animals, resulted in uniformly fatal disease 6 to 10 days after infection. RNA in situ hybridization and viral yield assays demonstrated a broad tropism of BRBV-STL with highest levels detected in liver and spleen. In vitro replication and polymerase activity of BRBV-STL were inhibited by favipiravir. Moreover, administration of favipiravir as a prophylaxis or as post-exposure therapy three days after infection prevented BRBV-STL-induced mortality in immunocompromised Ifnar1-/- mice. These results suggest that favipiravir may be a candidate treatment for humans who become infected with BRBV

A dose-dependent requirement for the proline motif of CD28 in cellular and humoral immunity revealed by a targeted knockin mutant

Activation of naive T cells requires the integration of signals through the antigen receptor and CD28. Although there is agreement on the importance of CD28, there remains controversy on the mechanism by which CD28 regulates T cell function. We have generated a gene-targeted knockin mouse expressing a mutation in the C-terminal proline-rich region of the cytoplasmic tail of CD28. Our analysis conclusively showed that this motif is essential for CD28-dependent regulation of interleukin 2 secretion and proliferation. In vivo analysis revealed that mutation of this motif-dissociated CD28-dependent regulation of cellular and humoral responses in an allergic airway inflammation model. Furthermore, we find an important gene dosage effect on the phenotype of the mutation and provide a mechanistic explanation for the conflicting data on the significance of this motif in CD28 function

A single intranasal or intramuscular immunization with chimpanzee adenovirus-vectored SARS-CoV-2 vaccine protects against pneumonia in hamsters

The development of an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), is a global priority. Here, we compare the protective capacity of intranasal and intramuscular delivery of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (chimpanzee adenovirus [ChAd]-SARS-CoV-2-S) in Golden Syrian hamsters. Although immunization with ChAd-SARS-CoV-2-S induces robust spike-protein-specific antibodies capable of neutralizing the virus, antibody levels in serum are higher in hamsters vaccinated by an intranasal compared to intramuscular route. Accordingly, against challenge with SARS-CoV-2, ChAd-SARS-CoV-2-S-immunized hamsters are protected against less weight loss and have reduced viral infection in nasal swabs and lungs, and reduced pathology and inflammatory gene expression in the lungs, compared to ChAd-control immunized hamsters. Intranasal immunization with ChAd-SARS-CoV-2-S provides superior protection against SARS-CoV-2 infection and inflammation in the upper respiratory tract. These findings support intranasal administration of the ChAd-SARS-CoV-2-S candidate vaccine to prevent SARS-CoV-2 infection, disease, and possibly transmission

Interferon induced protein 35 exacerbates H5N1 influenza disease through the expression of IL-12p40 homodimer.

Pro-inflammatory cytokinemia is a hallmark of highly pathogenic H5N1 influenza virus (IAV) disease yet little is known about the role of host proteins in modulating a pathogenic innate immune response. The host Interferon Induced Protein 35 (Ifi35) has been implicated in increased susceptibility to H5N1-IAV infection. Here, we show that Ifi35 deficiency leads to reduced morbidity in mouse models of highly pathogenic H5N1- and pandemic H1N1-IAV infection. Reduced weight loss in Ifi35-/- mice following H5N1-IAV challenge was associated with reduced cellular infiltration and decreased production of specific cytokines and chemokines including IL-12p40. Expression of Ifi35 by the hematopoietic cell compartment in bone-marrow chimeric mice contributed to increased immune cell recruitment and IL-12p40 production. In addition, Ifi35 deficient primary macrophages produce less IL-12p40 following TLR-3, TLR-4, and TLR-7 stimulation in vitro. Decreased levels of IL-12p40 and its homodimer, IL-12p80, were found in bronchoalveolar lavage fluid of H5N1-IAV infected Ifi35 deficient mice. Specific antibody blockade of IL-12p80 ameliorated weight loss and reduced cellular infiltration following H5N1-IAV infection in wild-type mice; suggesting that increased levels of IL-12p80 alters the immune response to promote inflammation and IAV disease. These data establish a role for Ifi35 in modulating cytokine production and exacerbating inflammation during IAV infection

Decreased cellular infiltration into lungs of <i>Ifi35</i> deficient mice after H5N1-VN/PR8 infection.

<p><b>(A)</b> Gating strategy for analysis of myeloid and lymphoid cells from bronchoalveolar lavage (BAL) of naïve and H5N1-VN/PR8 infected mice. Cells were gated based on CD11b, CD11c, Ly6G, Ly6C, F4/80, and MHC-II expression for myeloid populations or B220, CD3, CD4, CD8, γδ-TCR, and CD44 expression for lymphoid populations. The parental gate is shown above each plot. Representative plots are shown from a WT mouse at 3 dpi for myeloid gating or 5 dpi for lymphoid gating. <b>(B)</b> Total number of cells isolated from bronchoalveolar lavage from naïve, infected WT (black), and <i>Ifi35</i>^-/- (red) mice at indicated times post infection. <b>(C)</b> Total number of CD45⁺ alveolar macrophage (CD11c⁺, CD11b^-, MHC-II^-, F4/80⁺), neutrophil (PMNs; CD11b⁺, CD11c^-, Ly6G⁺), monocyte (CD11b⁺, Ly6G^-, Ly6C^hi), and dendritic cell (CD11c⁺, CD11b^-, MHC-II⁺) numbers in BAL. <b>(D)</b> Percentage of myeloid cell populations in BAL at 3 dpi following pre-gating on CD45⁺ cells. <b>(E)</b> Total number of CD3⁺, CD44⁺ lymphoid CD4⁺, CD8⁺, or γδ-TCR⁺ T cells in BAL. n = 8–11 for each group/time point post-infection. n = 3–4 for naïve samples. Each symbol represents an individual mouse. Bars represent the mean from at least three different experiments (± SEM). PMN, polymorphonuclear cells; n.s., not significant; *<i>P</i> < 0.01, **<i>P</i> < 0.001, ***<i>P</i> < 0.0001 using multiple student t-test.</p

<i>Ifi35</i> expression in hematopoietic cells promotes cellular recruitment into the lung and IL-12p40 production following H5N1-VN/PR8 infection.

<p><b>(A)</b> CD45.1⁺ B6-Ly5.1/Cr and CD45.2⁺ <i>Ifi35</i>^-/- mice were Busulfan treated and reconstituted with CD45.1⁺ B6-Ly5.1/Cr, CD45.2⁺ WT, or CD45.2⁺ <i>Ifi35</i>^-/- bone marrow cells (10⁷ cells). Bone marrow donor and recipient pairs for the experiment are shown. <b>(B)</b> Twelve-week-old chimeric mice were infected with 100 EID₅₀ of H5N1-VN/PR8 and BAL fluid was collected at 3 dpi and assessed for total BAL cell number or <b>(C)</b> assessed for IL-12p40 levels via ELISA. Each symbol represents an individual mouse. Bars represent the mean from at least three different experiments (± SEM). n = 8–17 per group. <b>(D)</b> IL-12p40-IRES-eYFP reporter (IL12b-yet40) mice were infected with 100 EID₅₀ of H5N1-VN/PR8 and on 0, 3, and 5 dpi whole lungs were taken for collagenase digestion and flow cytometry analysis of eYFP expression (FITC⁺) and CD45 surface staining. FITC⁺ cells were separated based on CD45 staining and enumerated per 1x10⁶ single cells collected. Bars represent the mean from at least three different experiments (± SEM). n = 4 for naïve and 11–12 for infected groups. n.s., not significant; *<i>P</i> < 0.01, **<i>P</i> < 0.001, ***<i>P</i> < 0.0001, using one-way ANOVA with multiple comparisons correction (Tukey’s comparison correction).</p

Specific cytokines and chemokines are significantly reduced in <i>Ifi35</i>^-/- mice at early time-points following H5N1-VN/PR8 infection and <i>in vivo</i> poly I:C challenge.

<p><b>(A)</b> CXCL1, <b>(B)</b> G-CSF, and <b>(C)</b> IL-12p40 concentrations (pg/mL) in bronchoalveolar lavage fluid from WT (black circle) and <i>Ifi35</i>^-/- mice (red circle) at indicated times post infection with H5N1-VN/PR8 as measured by Bio-Plex Pro Mouse Cytokine 23-Plex assay. Dotted line (red) indicates limit of detection (LOD) for individual cytokines and chemokines based on internal standard curve values. <b>(D)</b> Serum IL-12p40 concentration (pg/mL) in WT (black circle) and <i>Ifi35</i>^<i>-/-</i> (red square) mice at 3 hours post stimulation with 100μg HMW poly I:C via intraperitoneal injection. Serum cytokine levels measured using an IL-12p40 specific ELISA; dotted line indicates LOD based on standard curve values. Each symbol represents an individual mouse. Bars represent the mean from at least three different experiments (± SEM). n = 6–19 per group per time-point. *<i>P</i> < 0.01, **<i>P</i> < 0.001, ***<i>P</i> < 0.0001 using multiple student t-test.</p