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Principal Investigator: Andrea Marzi
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2022
Award: $8,179,925
Funding agency: National Institute of Allergy and Infectious Diseases
Filovirus pathogenesis
1. Tai Forest virus (TAFV) causes severe disease and lethality in cynomolgus macaques
TAFV is a lesser-known ebolavirus that has caused one reported human case of infection in Ivory Coast in 1994. The patient developed dengue-like symptoms including fever and rash and was evacuated to Basel, Switzerland. There, the disease was treated, and the patient survived without sequelae. TAFV has also caused lethal disease outbreaks in chimpanzees in the Ta Forest in West Africa. Limited research has been performed with this human-pathogenic virus. With the recent emergence of Ebola virus (EBOV) and Marburg virus (MARV) in West Africa, we wanted to fill knowledge gaps regarding this virus to inform outbreak response preparedness.
TAFV disease and pathogenesis was assessed after intramuscular (IM) infection with 1,000 PFU, the standard filovirus infection dose and route for NHPs. We had access to two different TAFV stock preparations (distinctive in their cell culture passaging history) which were derived from the Swiss TAFV isolate. One stock (TAFV-RML) had a confirmed 7U genotype whereas the other one (TAFV-UTMB) presented with a G insertion at the 7U editing sit). Both stocks grew to similar titers in cell culture and were used to infect groups of NHPs. Unexpectedly, infection with the TAFV-RML stock caused no disease despite the 7U genotype, yet the NHPs seroconverted to TAFV GP. In contrast, the TAFV-UTMB stock caused severe disease and lethality in NHPs within 8-11 days (Fletcher et al., in preparation). Histopathologic analysis of key tissue samples revealed severe damage in TAFV-UTMB-infected NHPs. Sequence analysis of terminal blood samples from these NHPs revealed a uniform reversion to a 7U genotype. Currently, host transcriptomics and TAFV RNA sequences are being analyzed in longitudinal blood samples in collaboration with Prof. Ilhem Messaoudi (U Kentucky). This data is providing valuable insight into the human pathogenic potential of this virus.
2. Blocking CD47 signaling does not impact Ebola virus infection in mice
In collaboration with Dr. Kim Hasenkrug (NIAID) we tested if anti-CD47 antibody treatment results in a benefit against EBOV infection in mice. Upregulation of CD47 is well-established as a mechanism of inhibiting macrophage-mediated phagocytosis and antigen presentation, possibly to prevent immune overactivation. We found that mice treated with an anti-CD47 antibody exhibited increased activation of B cells and increases in recently cytolytic CD8 T cells. However, anti-CD47-treated mice also exhibited significant weight loss, higher virus titers, and increased inflammatory cytokine expression before succumbing more rapidly to disease. Significantly, these results demonstrate that in the context of a rapidly progressing hemorrhagic viral disease, CD47 attenuates immunopathology and disease severity (Rao et al., Antiviral Research 2021).
3. Pathogenesis of novel filoviruses in IFNAR-/- in mice
Filovirus sequences have been found in bats in Sierra Leone, Hungary and other places. We received Bombali virus (BOMV; recovered using reverse genetics) and Lloviou virus (LLOV; isolated from bats in Hungary) from collaborators and propagated these viruses at RML. 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., unpublished data). Because of the limitations of this mouse model, further pathogenesis studies in ferrets and NHPs are planned.
Filovirus vaccine development
1. VSV-MARV rapidly protects NHPs from lethal disease
MARV is on the priority list of the WHO for countermeasure development. Cases of MARV disease (MVD) identified in Guinea (August 2021) and Ghana (July 2022) highlighted its potential impact on regional public health. We previously developed a VSV-based vaccine expressing the MARV Angola GP (VSV-MARV) and demonstrated uniform protection of NHPs with a single dose within 28 days. In this project, we investigated the fast-acting potential of this vaccine by challenging NHPs by the IM route with MARV 14, 7 or 3 days after a single IM vaccination with 107 PFU VSV-MARV. We found that 100% of the animals survived when vaccinated at 7 or 14 days and 75% of the animal survived when vaccinated 3 days prior to lethal MARV challenge. Transcriptional analysis of longitudinal whole blood samples indicated activation of B cells and antiviral defense pathways after VSV-MARV vaccination. Only the 14-day vaccination group (d-14) had detectable MARV GP-specific IgM at the time of challenge, likely contributing to the limited transcriptional changes after challenge that were detected in these NHPs in comparison to baseline samples. In the 7-day vaccination group (d-7), we detected gene expression profiles indicative of a recall response on day 9 post-challenge but limited transcriptional changes at all other time points analyzed when compared to baseline samples. In the 3-day vaccination group (d-3), transcriptional analysis of samples from surviving NHPs revealed strong innate immune activation. In contrast, the animal that succumbed to disease in this group lacked signatures of antiviral immunity, likely contributing to disease outcome (Marzi et al., Frontiers in Immunology 2021). This data highlights the applicability of the VSV-MARV vaccine in outbreak situations and the development for human use is underway.
2. A single dose of VSV-TAFV uniformly protects NHPs from disease
Because ebolaviruses vary in their antigenicity, we wanted to expand our collection to include a VSV-based vaccine for every species with demonstrated potential to cause human disease. We developed a TAFV-specific VSV vaccine (VSV-TAFV) expressing the TAFV GP as viral antigen to investigate protection against TAFV infection. The immunogenicity of a single dose of 107 PFU VSV-TAFV was assessed in cynomolgus macaques vaccinated one month prior to IM TAFV challenge. Vaccinated NHPs developed antigen-specific IgG reaching peak levels 14 days after vaccination and were uniformly protected from disease after challenge. TAFV RNA was not detected within the vaccinated NHPs in any sample collected throughout the study. In contrast, all unvaccinated NHPs succumbed between day 8 and 11 after challenge presenting with fever, increasing viremia and thrombocytopenia starting on day 6 after challenge. Analysis of serum cytokine levels, antigen-specific B and T cell responses in cryopreserved PBMC preparations is ongoing. In addition, host transcriptomics will be investigated in longitudinal blood samples. These data demonstrate that the VSV-TAFV is a viable single-dose vaccine ideal for use in outbreaks and should be developed for human use (Fletcher et al., in preparation).
3. A molecular clamp vaccine against EBOV
In a collaboration with Drs. Daniel Watterson and Keith Chappell (U Queensland, Australia) we characterized a subunit-based clamp vaccine against EBOV in the hamster model. This subunit vaccine platform technology, the molecular clamp, was applied to four viruses from divergent taxonomic families in this project: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), EBOV, Lassa virus (LASV) and Nipah virus (NiV). Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge (Young et al., Frontiers in Immunology - accepted
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virus><Viral><Viral Antigens><Viral Diseases><Viral Pathogenesis><Viremia><Virulence Factors><Virus><Virus Diseases><Weight Loss><Weight Reduction><Whole Blood><activated B cells><alpha Subunit Stimulatory GTP-Binding Protein><alpha-Gs><anti-viral immunity><antibody based therapies><antibody treatment><antibody-based therapeutics><antibody-based treatment><antiviral immunity><base><biological signal transduction><blood loss><body weight loss><cell culture><cell cultures><cold preservation><cold storage><cytokine><develop a vaccine><develop vaccines><development of a vaccine><developmental><disease severity><ebolavirus><evaluate vaccines><febrile><febris><filovirus infections><forest><frontier><genetic approach><genetic strategy><host response><human disease><immune activation><immune system response><immunogen><immunogenicity><immunopathology><immunoresponse><improved><innovate><innovation><innovative><insight><intraperitoneal><longitudinal analysis><macrophage><model of animal><model 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