Immunobiology, molecular virology and countermeasures of highly pathogenic viruses

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

1. Investigate protective immune responses after vaccination and challenge, and develop pre- and post-exposure vaccines for emerging viruses We developed a vesicular stomatitis virus (VSV)-based vaccine protective against Marburg virus (MARV) following the example of the FDA-approved Ebola virus (EBOV) vaccine VSV-EBOV. Similar to the VSV-EBOV, the VSV-MARV is fast-acting and uniformly protected NHPs against challenge within 7 days. Transcriptomic analysis in collaboration with Ilhem MEssaoudi (UC Irvine) revealed early antiviral responses by the vaccine (Marzi et al., in preparation). An ongoing study addresses if a low dose vaccination with VSV-MARV can protect within this short time (ODonnell et al., unpublished data). After the insightful data revealed by the transcriptional analysis of the MARV NHP samples, we subjected samples from previous NHP studies using EBOV and VSV-EBOV to this same analysis. One study characterized the differences in transcriptional responses after VSV-EBOV vaccination with different doses which resulted in confirmation that vaccination significantly attenuated responses associated with fatal outcome (Pinksi et al., Frontiers in Virology - under review). We found that infection with early or late isolates from the 2013-2016 West Africa EBOV epidemic do not suggest attenuated pathogenicity as a result of genetic variation (Maroney et al., Frontiers in Microbiology 2021). Additionally, we compared transcriptional responses to fatal EBOV infection in cynomolgus and rhesus macaques and found that there are indeed species-specific factors mediating the immune responses during disease progression (Pinski et al., Emerging Microbes & Infection 2021). We have also used the VSV-EBOV as a basis vector for vaccines against other infectious viral diseases. In order to improve the parental VSV-EBOV 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 (Bhatia et al., Vaccines 2021). 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 et al, 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. In collaboration with Kim Hasenkrug (NIAID) we tested if anti-CD47 antibody treatment results in a benefit against EBOV infection in mice. Unfortunately, this was not the case (Rao et al., in preparation), 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 (Furuyama et al., Microorganisms 2020) to determine biologically relevant levels in animal serum samples. In collaboration with Dr. Chertow (NIAID) we have analyzed human specimen and found evidence of sGP in the patients serum in the absence of viremia (Furuyama et al., in preparation). This assay could potentially be developed into a diagnostic tool for EBOV. We also 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 the liver in mice. Preliminary data analyzing innate immune pathways indicate that the MAP kinase pathway might play a role in the increased EBOV replication after sGP addition (Furuyama et al, Plos Pathogens in revision). We are generating a so-called minigenome system for MARV in order to decipher differences in gene functionality between EBOV and MARV in BSL2 labs (Shifflett et al., unpublished data). 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 infectivity and pathogenesis in vitro and in vivo (Fletcher et al., unpublished data). 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. 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.