Immunobiology, molecular virology and countermeasures of highly pathogenic viruses

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Filovirus pathogenesis in animal models We developed a nonhuman primate (NHP) model for Tai Forest virus (TAFV; Fletcher et al. Emerg Micobes Inf 2023). In addition, in one of our Ebola virus (EBOV) NHP studies a single NHP was euthanized on day 28 after EBOV infection having completely recovered from the acute disease and cleared EBOV from the blood. During convalescence, this NHP developed neurological signs and acute respiratory distress requiring euthanasia. We found that the organ tropism had changed with high virus titers in lungs, brain, eye, and reproductive organs but no virus in the typical target organs for acute EBOV infection (Marzi et al., J Inf Dis 2023a). The observations made with this NHP in part reflect sequelae described for EBOV survivors in West Africa albeit developing quicker after recovery from acute disease. Genomic sequences of novel filoviruses have been found in bats in Sierra Leone, Hungary, and other places. In order to assess the pathogenic potential of these viruses, we started with infection of Ifnar-/- mice, a model in which some filoviruses can cause disease. First infection studies in these mice investigating intraperitoneal and intranasal infection with a high and a low dose of these viruses did not result in disease (Fletcher et al. J Inf Dis 2023). Next, we infected ferrets with LLOV by the intranasal (IN), intramuscular (IM) or aerosol routes and observed disease progression including regular blood draws and radiographs. Interestingly, we again did not observe any clinical signs of disease and only a few of the ferrets seroconverted (Fletcher et al. manuscript in preparation). Because of the limitations of the mouse and ferret models, further pathogenesis studies in NHPs are planned. Other efforts are currently being made to develop the ferret as an animal disease model for ebolaviruses including Sudan virus (SUDV; ODonnell et al. manuscript in preparation). Studies to further investigate the pathogenesis and establish parameters as a basis for preclinical studies at our facility are being conducted. To date, animal models of EBOV infection have failed to fully characterize the pathogenesis of reproductive tract infection. Furthermore, no animal model of sexual transmission of EBOV exists. We investigated the potential of sexual transmission of EBOV using a mouse-adapted EBOV isolate in immunocompetent male mice and female Ifnar-/- mice. We showed that male mice survive acute infection and exhibit a chronic reproductive tract infection phenotype, particularly in the epididymis, with few limited reproductive changes that would result in male sterility or decreased fecundity. Additionally, we show intravaginal inoculation of female Ifnar-/- mice results in a high mortality rate associated with systemic EVD (Clancy et al. J Inf Dis 2023). In an effort to develop a diagnostic tool to detect EBOV recrudescence during convalescence, we collaborated with Drs. Daniel Chertow and Richard Davey (NIAID) and gained access to serum samples from the single EVD patient treated at the NIH Clincal Center in 2015. A previously established EBOV soluble glycoprotein (sGP) capture ELISA was used for this study (Furuyama et al. Microorg 2020). Samples from this patient were collected during acute EVD and during convalescence up to day 361 following illness onset. While blood samples were negative by RT-qPCR after recovery from acute EVD, we detected small amounts of EBOV sGP in the serum of the patient long after recovery potentially indicating viral recrudescence. As this is only observed in a single patient, additional longitudinal patient samples are needed to confirm our hypothesis that EBOV sGP may be an indicator of viral recrudescence long after recovery from acute EVD (Furuyama et al. J Inf Dis 2023). Filovirus vaccine development Marburg virus (MARV) is on the priority list of the WHO for countermeasure development. Recent cases of MARV disease (MVD) identified in Guinea (August 2021), Ghana (July 2022), Equatorial Guinea (February 2023) and Tanzania (March 2023) highlighted its potential impact on regional public health in Africa. In this project, we investigated the fast-acting potential of this vaccine by challenging NHPs by the IM route with MARV 14 or 7 days after a single low-dose IM vaccination with 103 PFU VSV-MARV. We found that 100% of the animals survived when vaccinated with this low dose at 7 or 14 days prior to lethal MARV challenge (ODonnell et al. EBioMed 2023). Transcriptional analysis of longitudinal whole blood samples indicated activation of B cells and antiviral defense pathways after VSV-MARV vaccination (Prator et al. Emerg Microbes Inf in revision). This data highlights the applicability of the VSV-MARV vaccine in outbreak situations and the development for human use is underway. We also developed a VSV-TAFV vaccine and showed that a single dose was uniformly protective from disease within 4 weeks (Fletcher et al. Emerg Microbes Inf. 2023). The most recent SUDV outbreak created a public health emergency in Uganda and the Eastern Africa region. Currently, there are licensed countermeasures for EBOV; however, there are no licensed vaccines or therapeutics against SUDV. We developed a VSV-based vaccine expressing the SUDV GP as the viral antigen and demonstrated protective efficacy with a single high-dose within 4 weeks (Marzi et al. Lancet Microbe 2023; Provisional patent filed). In addition, groups of NHPs were vaccinated 28 or 7 days prior to lethal SUDV challenge. A third group was vaccinated with a single IM dose of VSV-EBOV 28 days prior to SUDV challenge to assess its cross-protective potential since SUDV and EBOV are closely related filoviruses. In addition, there was a control group receiving an unrelated VSV-based vaccine. All vaccinated NHPs developed antigen-specific IgG within 2 weeks of vaccination, including cross-reactive responses. After challenge with a lethal dose of SUDV, all VSV-SUDV-vaccinated NHPs were uniformly protected from disease. In contrast, the VSV-EBOV-vaccinated and control NHPs succumbed to disease between day 5 and 7 after challenge presenting with classical signs of disease (Fletcher et al. manuscript in preparation). As several filoviruses have overlapping endemic areas in West and Central Africa, we investigated the impact of pre-existing immunity to the VSV vector backbone and to another filovirus GP on the efficacy of a second VSV-filovirus vaccination. In a first study, we repurposed NHPs from a successful VSV-EBOV vaccine efficacy study to demonstrate that VSV-SUDV can be used effectively in individuals previously vaccinated against EBOV. While the NHPs developed cross-reactive humoral responses to SUDV after VSV-EBOV vaccination and EBOV challenge, cross-protection was limited emphasizing the need for the development of specific countermeasures for each human-pathogenic ebolavirus (Marzi et al. Lancet Microbe 2023). In a second study, we used NHPs previously vaccinated with VSV-MARV and protected against lethal MARV challenge. After a resting period of 9 months, these NHPs were re-vaccinated with VSV-EBOV and challenged with EBOV resulting in 75% survival. Surviving NHPs developed EBOV GP-specific antibody titers and no viremia or clinical signs of disease (Marzi et al. J Inf Dis 2023b). The single vaccinated NHP succumbing to challenge showed the lowest EBOV GP-specific antibody response after challenge supporting previous findings with VSV-EBOV that antigen-specific antibodies are critical in mediating protection. In response to emerging infectious disease outbreaks we have developed VSV-EBOV-based vaccines against a number of these pathogens and conducted preclinical NHP efficacy studies. We have developed VSV-EBOV-based vaccines with preclinical NHP efficacy for Nipah virus, Kyasanur Forest disease virus and influenza A viruses.