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

Challenges and Achievements in Prevention and Treatment of Smallpox

Declaration of smallpox eradication by the WHO in 1980 led to discontinuation of the worldwide vaccination campaign. The increasing percentage of unvaccinated individuals, the existence of its causative infectious agent variola virus (VARV), and the recent synthetic achievements increase the threat of intentional or accidental release and reemergence of smallpox. Control of smallpox would require an emergency vaccination campaign, as no other protective measure has been approved to achieve eradication and ensure worldwide protection. Experimental data in surrogate animal models support the assumption, based on anecdotal, uncontrolled historical data, that vaccination up to 4 days postexposure confers effective protection. The long incubation period, and the uncertainty of the exposure status in the surrounding population, call for the development and evaluation of safe and effective methods enabling extension of the therapeutic window, and to reduce the disease manifestations and vaccine adverse reactions. To achieve these goals, we need to evaluate the efficacy of novel and already licensed vaccines as a sole treatment, or in conjunction with immune modulators and antiviral drugs. In this review, we address the available data, recent achievements, and open questions

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

The Role of Heparanase in Lymph Node Metastatic Dissemination: Dynamic Contrast-Enhanced MRI of Eb Lymphoma in Mice

Heparanase expression has been linked to increased tumor invasion, metastasis, and angiogenesis and with poor prognosis. The aim of the study was to monitor the effect of heparanase expression on lymph node metastasis, in heparanase-overexpressing subcutaneous Eb mouse T-lymphoma tumors, and their draining lymph node. Dynamic contrast-enhanced magnetic resonance imaging (MRI) using biotin-BSA-GdDTPA-FAM/ROX was applied for analysis of blood volume, vascular permeability, and interstitial convection, and for detection of very early stages of such metastatic dissemination. Eb tumors increased extravasation, interstitial convection, and lymphatic drain of the contrast material. Interstitial flow directions were mapped by showing radial outflow interrupted in some tumors by directional flow toward the popliteal lymph node. Heparanase expression significantly increased contrast enhancement of the popliteal lymph node but not of the primary tumor. Changes in MR contrast enhancement preceded the formation of pathologically detectable metastases, and were detectable when only a few enhanced green fluorescent protein (EGFP)-expressing Eb cells were found near and within the nodes. These results demonstrate very early, heparanase-dependent vascular changes in lymph nodes that were visible by MRI following administration of biotin-BSA-GdDTPA-FAM/ROX, and can be used for studying the initial stages of lymph node infiltration

Influence of Poly(I:C) treatment on viral dissemination evaluated by <i>in-vivo</i> bioluminescence imaging.

<p>BALB/c mice were infected with 2 i.n. ECTV-Luc LD₅₀ and left untreated (n = 5) or treated with poly(I:C) on day 3 p.e. (n = 3). (A) Infected untreated mice 8 days p.e. (B) Infected mice treated on day 3 p.e. with poly(I:C) and imaged on day 8 p.e. (C) Poly(I:C) treated group on day 3 p.e. imaged on day 16 p.e. Bioluminescent images were obtained using an f/stop of 1, binning factor of 4, and acquisition time of 1 sec (A, B) or 40 sec (C). Relative photon flux expression is represented by a pseudocolor heat map. (D) Morbidity, based on weight change (lines, left Y axis) and bioluminescence signal on days 7, 8, 14 and 16 p.e. (bars, right Y axis) of the groups shown in panels A–C (red for infected untreated, black for poly(I:C) treated on day 3 p.e.). Bioluminescent signal intensity as total photon flux (photon/s/cm2/sr), was calculated by region of interest (ROI) analysis on the chest and abdomen area marked by a white box on the right mouse in panel (A). Same ROI was used for all mice examined. Asterisk denote for significant reduction in photon flux (n = 3–5 in each group, P<0.05). Dagger represent dead mice.</p

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

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

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