Induction of cell-cell fusion by ectromelia virus is not inhibited by its fusion inhibitory complex

<p>Abstract</p> <p>Background</p> <p>Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes). This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium.</p> <p>Results</p> <p>We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within") or by infection with a high amount of virus particles per cell (fusion "from without"). Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection.</p> <p>Conclusion</p> <p>Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.</p

TLR3 and TLR9 agonists improve postexposure vaccination efficacy of live smallpox vaccines.

Eradication of smallpox and discontinuation of the vaccination campaign resulted in an increase in the percentage of unvaccinated individuals, highlighting the need for postexposure efficient countermeasures in case of accidental or deliberate viral release. Intranasal infection of mice with ectromelia virus (ECTV), a model for human smallpox, is curable by vaccination with a high vaccine dose given up to 3 days postexposure. To further extend this protective window and to reduce morbidity, mice were vaccinated postexposure with Vaccinia-Lister, the conventional smallpox vaccine or Modified Vaccinia Ankara, a highly attenuated vaccine in conjunction with TLR3 or TLR9 agonists. We show that co-administration of the TLR3 agonist poly(I:C) even 5 days postexposure conferred protection, avoiding the need to increase the vaccination dose. Efficacious treatments prevented death, ameliorated disease symptoms, reduced viral load and maintained tissue integrity of target organs. Protection was associated with significant elevation of serum IFNα and anti-vaccinia IgM antibodies, modulation of IFNγ response, and balanced activation of NK and T cells. TLR9 agonists (CpG ODNs) were less protective than the TLR3 agonist poly(I:C). We show that activation of type 1 IFN by poly(I:C) and protection is achievable even without co-vaccination, requiring sufficient amount of the viral antigens of the infective agent or the vaccine. This study demonstrated the therapeutic potential of postexposure immune modulation by TLR activation, allowing to alleviate the disease symptoms and to further extend the protective window of postexposure vaccination

Induction of Innate Immune Response by TLR3 Agonist Protects Mice against SARS-CoV-2 Infection

SARS-CoV-2, a member of the coronavirus family, is the causative agent of the COVID-19 pandemic. Currently, there is still an urgent need in developing an efficient therapeutic intervention. In this study, we aimed at evaluating the therapeutic effect of a single intranasal treatment of the TLR3/MDA5 synthetic agonist Poly(I:C) against a lethal dose of SARS-CoV-2 in K18-hACE2 transgenic mice. We demonstrate here that early Poly(I:C) treatment acts synergistically with SARS-CoV-2 to induce an intense, immediate and transient upregulation of innate immunity-related genes in lungs. This effect is accompanied by viral load reduction, lung and brain cytokine storms prevention and increased levels of macrophages and NK cells, resulting in 83% mice survival, concomitantly with long-term immunization. Thus, priming the lung innate immunity by Poly(I:C) or alike may provide an immediate, efficient and safe protective measure against SARS-CoV-2 infection

Matrix Metalloproteinases Expression Is Associated with SARS-CoV-2-Induced Lung Pathology and Extracellular-Matrix Remodeling in K18-hACE2 Mice

The COVID-19 pandemic caused by the SARS-CoV-2 infection induced lung inflammation characterized by cytokine storm and fulminant immune response of both resident and migrated immune cells, accelerating alveolar damage. In this work we identified members of the matrix metalloprotease (MMPs) family associated with lung extra-cellular matrix (ECM) destruction using K18-hACE2-transgenic mice (K18-hACE2) infected intranasally with SARS-CoV-2. Five days post infection, the lungs exhibited overall alveolar damage of epithelial cells and massive leukocytes infiltration. A substantial pulmonary increase in MMP8, MMP9, and MMP14 in the lungs post SARS-CoV-2 infection was associated with degradation of ECM components including collagen, laminin, and proteoglycans. The process of tissue damage and ECM degradation during SARS-CoV-2 lung infection is suggested to be associated with activity of members of the MMPs family, which in turn may be used as a therapeutic intervention

Correlation between IFNγ and viral load.

<p>(A, C, E) Viral loads and IFNγ levels in BALB/c mice infected with 18 i.n. ECTV LD₅₀, untreated (n = 6) or single treated on day 2 or 3 with: poly(I:C) with or without VACV-Lister, CpG-ODNs 1585 and 1826 with or without VACV-Lister and VACV-Lister only (n = 3/group). (B, D, F) Viral loads and IFNγ levels in C57BL/6j mice infected with 2–3 i.n. ECTV LD₅₀, untreated (n = 6) or single treated on day 0 with: poly(I:C) with or without VACV-Lister, VACV-Lister or placebo; day 1: poly(I:C) with or without VACV-Lister or MVA, VACV-Lister or MVA, placebo; day 2: poly(I:C) with or without MVA (n = 3/group), MVA or placebo (n = 3/group). (A, B) spleens; (C, D) livers and (E, F) lungs. Green dots - examined mice from groups in which the survival rate was 50% and above. Red dots - examined mice from groups in which the survival rate was less than 50%, (n = 48 for either BALB/c or C57BL/6j).</p

Morbidity based on weight change following post exposure (p.e.) treatments of BALB/c mice.

<p>Mice were infected with 4–20 i.n. ECTV LD₅₀. (A, B) CPGs treatments with or without VACV-Lister on day 2 (A) and 3 (B) p.e. (C) Poly(I:C) treatments with or without VACV-Lister or VACV-Lister alone on day 2 p.e. (D) Poly(I:C) treatments with or without VACV-Lister or MVA and only vaccines treatments on day 3 p.e. (E, F) Poly(I:C) treatments with or without VACV-Lister or MVA and only MVA on day 4 (E) and 5 (F) p.e. Asterisk denote for significant difference in the area-under-the curve of weight changes along the entire experiment of the treated groups vs. the infected untreated group (* P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, <i>t</i>-test). Data collection for each treatment (weight change (mean, SE)) is indicated. Morbidity of Infected untreated mice from corresponding relevant experiments is shown. The number of mice succumbed to the infection in each time point is outlined color coded in a box above each graph. Survivals out of the total mice in each group are designated color coded next to the legend.</p

Viral load following postexposure treatments.

<p>Viral loads were determined by plaque assay from BALB/c mice 8 days postexposure (p.e.) following infection with 18 i.n. ECTV LD₅₀. (A, C, E) viral load in livers, spleens and lungs of mice treated on day 2 p.e. (B, D, E) viral load in livers, spleens and lungs of mice treated on day 3 p.e. Horizontal lines represent the geometric mean of each group. Survival proportions of each group are designated. Asterisk denote for significant reduction in viral load (n = 3 in each treated group) compared to the infected untreated group (n = 6, P<0.05).</p

Survival Table – C57BL/6j mice.

<p>Infected untreated C57BL/6j mice i.n. infected with ECTV (survived/total (n)): Exp.1 2LD₅₀ (0/3); Exp.2 3LD₅₀ (0/8).</p><p>*P<0.01, **P<0.001, ***P<0.0001 Log-rank (Mantel-Cox test) vs. infected untreated (0/11).</p><p>N.D. – not determined; p.e. – postexposure.</p><p>Survival Table – C57BL/6j mice.</p

Cellular-immune response following p.e. treatment.

<p>BALB/c mice were infected with 4 i.n. ECTV LD₅₀, left untreated or treated on day 3 p.e. and their spleens were photographed (A) and analyzed by flow-cytometry for the distribution and activation (intracellular IFNγ) of various cell populations. (B) Counting of viable lymphocytes was performed under light microscope. (C–H) Number of total and activated cells of the different cell populations: NK (C, D), of CD3⁺ CD4⁺ (E, F) and CD3⁺ CD8⁺ (G, H) cells, respectively.</p