Recombinant Modified Vaccinia Virus Ankara Expressing a Glycosylation Mutant of Dengue Virus NS1 Induces Specific Antibody and T-Cell Responses in Mice.

The four serotypes of dengue virus (DENV1-4) continue to pose a major public health threat. The first licenced dengue vaccine, which expresses the surface proteins of DENV1-4, has performed poorly in immunologically naïve individuals, sensitising them to antibody-enhanced dengue disease. DENV non-structural protein 1 (NS1) can directly induce vascular leakage, the hallmark of severe dengue disease, which is blocked by NS1-specific antibodies, making it an attractive target for vaccine development. However, the intrinsic ability of NS1 to trigger vascular leakage is a potential drawback of its use as a vaccine antigen. Here, we modified DENV2 NS1 by mutating an N-linked glycosylation site associated with NS1-induced endothelial hyperpermeability and used modified vaccinia virus Ankara (MVA) as a vector for its delivery. The resulting construct, rMVA-D2-NS1-N207Q, displayed high genetic stability and drove efficient secretion of NS1-N207Q from infected cells. Secreted NS1-N207Q was composed of dimers and lacked N-linked glycosylation at position 207. Prime-boost immunisation of C57BL/6J mice induced high levels of NS1-specific antibodies binding various conformations of NS1 and elicited NS1-specific CD4+ T-cell responses. Our findings support rMVA-D2-NS1-N207Q as a promising and potentially safer alternative to existing NS1-based vaccine candidates, warranting further pre-clinical testing in a relevant mouse model of DENV infection.

Transient Blockade of Type I Interferon Signalling Promotes Replication of Dengue Virus Strain D2Y98P in Adult Wild-Type Mice.

Dengue virus serotypes 1 to 4 (DENV1-4) place nearly half the global population at risk of infection and the licenced tetravalent dengue vaccine fails to protect individuals who have not previously been exposed to DENV. The development of intervention strategies had long been hampered by the lack of a suitable small animal model. DENV does not replicate in wild-type mice due to its inability to antagonise the mouse type I interferon (IFN) response. Mice deficient in type I IFN signalling (Ifnar1-/- mice) are highly susceptible to DENV infection, but their immunocompromised status makes it difficult to interpret immune responses elicited by experimental vaccines. To develop an alternative mouse model for vaccine testing, we treated adult wild-type mice with MAR1-5A3-an IFNAR1-blocking, non-cell-depleting antibody-prior to infection with the DENV2 strain D2Y98P. This approach would allow for vaccination of immunocompetent mice and subsequent inhibition of type I IFN signalling prior to challenge infection. While Ifnar1-/- mice quickly succumbed to infection, MAR1-5A3-treated mice did not show any signs of illness but eventually seroconverted. Infectious virus was recovered from the sera and visceral organs of Ifnar1-/- mice, but not from those of mice treated with MAR1-5A3. However, high levels of viral RNA were detected in the samples of MAR1-5A3-treated mice, indicating productive viral replication and dissemination. This transiently immunocompromised mouse model of DENV2 infection will aid the pre-clinical assessment of next-generation vaccines as well as novel antiviral treatments.

FDA-approved Abl/EGFR/PDGFR kinase inhibitors show potent efficacy against pandemic and seasonal influenza A virus infections of human lung explants.

Influenza viruses (IVs) cause substantial global morbidity and mortality. Given the limited range of licensed antiviral drugs and their reduced efficacy due to resistance mutations, repurposing FDA-approved kinase inhibitors as fast-tracked host-targeted antivirals is an attractive strategy. We identified six FDA-approved non-receptor tyrosine kinase-inhibitors (NRTKIs) as potent inhibitors of viral replication of pandemic and seasonal IVs in vitro. We validated their efficacy in a biologically and clinically relevant ex vivo model of human precision-cut lung slices. We identified steps of the virus infection cycle affected by these inhibitors and assessed their effect(s) on host responses. Their overlapping targets suggest crosstalk between Abl, EGFR, and PDGFR pathways during IAV infection. Our data and established safety profiles of these NRTKIs provide compelling evidence for further clinical investigations and repurposing as host-targeted influenza antivirals. Moreover, these NRTKIs have broad-spectrum antiviral potential given that their kinase/pathway targets are critical for the replication of many viruses.

FDA-Approved Inhibitors of RTK/Raf Signaling Potently Impair Multiple Steps of In Vitro and Ex Vivo Influenza A Virus Infections.

Influenza virus (IV) infections pose a burden on global public health with significant morbidity and mortality. The limited range of currently licensed IV antiviral drugs is susceptible to the rapid rise of resistant viruses. In contrast, FDA-approved kinase inhibitors can be repurposed as fast-tracked host-targeted antivirals with a higher barrier of resistance. Extending our recent studies, we screened 21 FDA-approved small-molecule kinase inhibitors (SMKIs) and identified seven candidates as potent inhibitors of pandemic and seasonal IV infections. These SMKIs were further validated in a biologically and clinically relevant ex vivo model of human precision-cut lung slices. We identified steps of the virus infection cycle affected by these inhibitors (entry, replication, egress) and found that most SMKIs affected both entry and egress. Based on defined and overlapping targets of these inhibitors, the candidate SMKIs target receptor tyrosine kinase (RTK)-mediated activation of Raf/MEK/ERK pathways to limit influenza A virus infection. Our data and the established safety profiles of these SMKIs support further clinical investigations and repurposing of these SMKIs as host-targeted influenza therapeutics.