Immunobiology, molecular virology and countermeasures of highly pathogenic viruses

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1. Investigate protective immune responses after vaccination and challenge, and develop pre- and post-exposure vaccines for emerging viruses We have developed a VSV-based vaccine protective against highly pathogenic avian influenza virus H5N1 based on the FDA-approved Ebola virus (ERVEBO, VSV-EBOV) vaccine. Similar to the parental vector, the vaccine is fast-acting and protected mice against challenge with the homologous H5 virus, but also with other H5 isolates (Furuyama et al., npj vaccines 2020). Importantly, we could show that, as with other influenza vaccines, protection was mainly provided by the antigen-specific IgG response. We have used the same approach for COVID-19 (see separate report; Furuyama et al., in preparation). In order to improve the parental vector for directing antigen presentation and immune responses to the 2nd antigen, we deleted the mucin-like domain and glycan cap from the EBOV GP and could demonstrate that this antigen indeed is less immunogenic (Marzi, unpublished data). We will be using this approach to optimize future vaccines based on VSV-EBOV. The VSV-EBOV is not a potent post-exposure treatment by itself, therefore, we have included micro RNAs (miRNA) in the vectors targeting different EBOV proteins. A first mouse experiment resulted in limited benefit from the miRNA expression from the vaccine when administered after challenge (ODonnell, unpublished data). We are working towards incorporating small interfering (siRNA) or monoclonal antibody (mAb) sequences instead. Expression of the mAb may provide immediate control of the virus replication and buy the time needed for the vaccine to become effective. Furthermore, we investigated in more detail the antibody-dependent enhancement of infection described for EBOV. We found that the complement factor C1q is mediating ADE for EBOV (Furuyama et al., Plos NTD in press). We are still analyzing if this phenomenon, which has only been described in vitro, might play a role for EBOV infection and pathogenesis. This would impact future vaccine design for filoviruses. In collaboration with Kim Hasenkrug we tested if anti-CD47 antibody treatment results in a benefit against EBOV infection in mice. Unfortunately, this was not the case (Marzi et al., unpublished data), however, we will continue to test of this treatment is a viable option for other emerging viruses. 2. Identification of pathogenicity factors and characterization of unknown viruses using reverse genetics approaches for filoviruses We are in the process of analyzing the contributions of the soluble glycoprotein (sGP) of EBOV to viral pathogenicity. First, we developed a sGP capture ELISA to determine biologically relevant levels in animal serum samples and human specimen provided by Dr. Chertow, NIAID (Furuyama et al, in preparation). This assay could potentially be developed into a diagnostic tool for EBOV. Next, we produced sGP from cells and generated a knock-out virus (EBOV-sGP ko) allowing us to study the effects of sGP at a biologically relevant level in cell culture and in mice. We found that sGP indeed enhances virus replication and plaque size in vitro and increases virus loads in then liver in mice (Furuyama et al, in preparation). Preliminary data analyzing innate immune pathways indicate that the MAP kinase pathway might play a role in the increased EBOV replication after sGP addition. Filovirus sequences have been found in bats in Sierra Leone, China and other places. We are in the process of employing our reverse genetics expertise to recover these viruses from plasmid and characterize in vitro and in vivo. Furthermore, in a collaborative project with Anthony van den Pol, Yale we could demonstrate that the mucin-like domain of EBOV GP enhances the oncolytic potential of the VSV-EBOV (Zhang et al., J Virol 2020). For this study we utilized the VSV vector mentioned above in objective 1 and the VSV-EBOV (approved vaccine). 3. Analysis of genetic host factors determining the susceptibility of filovirus infections Lastly, the lab is interested in identifying factors restricting the host range of filoviruses. In collaboration with Ayato Takada, Hokkaido University we could demonstrate that polymorphisms in the filovirus receptor Nieman Pick-1 determine if different bat species are susceptible to filovirus infection (Takadate et al., Cell Rep 2020). We took a bioinformatics approach using the collaborative cross (CC) mice in collaboration with Ralph Baric, UNC and identified CC strains with fatal disease or mild/no disease. We bred these 2 strains and established a F2 generation of these mice to identify a susceptibility allele for EBOV. Interestingly, we found that the trim locus is involved in EBOV susceptibility (Schaefer, Marzi et al., unpublished data). We are currently breeding trim ko mice and performing EBOV-infection studies to confirm that this host factor plays indeed a role.