Viral Hemorrhagic Fevers: Disease Modeling and Transmission

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Mammarenaviruses Introduction: Lassa virus (LASV) is an enveloped, bisegmented, ambisense RNA virus and the cause of Lassa fever (LF) in humans. The natural reservoir of LASV is a small peri-domestic rodent, Mastomys natalensis. LASV is found across sub-Saharan West Africa with Guinea, Sierra Leone, Liberia and Nigeria historically reporting the vast majority of all LF cases. For this WHO priority pathogen there are no licensed vaccines or treatment options. (review Hansen et al., Microorganism 2021) Findings: We evaluated peptide-conjugated morpholino oligomer (PPMO) as a treatment option for LASV infection. Like siRNA, these oligomers are designed to bind mRNA and thus block translation of viral proteins. We have successfully tested PPMOs against LASV infection in cell culture and are preparing to test the efficacy in the guinea pig model Rosenke et al., unpublished. We have continued the use of the cynomolgus macaque model of LF to test both therapeutics and vaccines against LASV infection. A high dose of favipiravir was evaluated as a treatment for LASV and completely protected cynomolgous macaques against disease. Surprisingly, all animals treated with the anti-viral ribavirin succumbed to disease (Lingas et al., PLoS Comp Bio 2021). We have also tested a LASV entry inhibitor (LHF-535) in collaboration with Kineta Inc. These studies resulted in a near 70% survival in cynomolgus macaques infected with a lethal dose of LASV (Rosenke et al., unpublished). We continued to characterize Mastomys natalensis as a laboratory model for LASV. We have performed LASV infection studies to determine immune responses, viral kinetics, and transmission in Mastomys (Safronetz et al., Viruses 2021; Smith et al., Lab Anim 2021; Tang-Huau et al., Viruses 2021). We have continued to characterize the VSV-LASV vaccine in preclinical studies. A GMP produced seed stock showed complete protection against lethal LASV challenge in the cynomolgus macaque model (Rosenke et al., unpublished). We are currently evaluating a cytomegalovirus (CMV) based LASV vaccine platform to be used as a disseminating vaccine in the reservoir species Mastomys natalensis. Studies are underway to establish protection against LASV infection though dissemination of the vaccine among Mastomys Jarvis et al., unpublished. Orthonairoviruses Introduction: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tri-segmented, negative sense virus in the Bunyavirales order. The principal vector and reservoir are ticks of the Hyalomma genus and the wide geographic distribution of CCHFV follows the geographic range of these ticks. The primary routes of exposure are tick-bites and handling of infected livestock, although human-to-human spread is reported. Clinically, CCHF presents initially as a non-specific febrile illness that can rapidly progress to a severe, sometimes fatal hemorrhagic disease. The host and viral determinants of CCHFV pathogenesis are poorly understood and there are no approved vaccines or antivirals. (reviews Leventhal et al., Viruses 2021; Sergio et al., Antiviral Res 2022) Findings: We developed the first immunocompetent mouse model for CCHF based on a mouse-adapted variant (MA-CCHFV) isolated using a serial passage approach. In this model, infection of wild-type C57BL/6 mice results in disease similar to humans with high viral loads, liver pathology and inflammatory responses (Hawman et al., eLife 2021). We evaluated candidate vaccines for CCHFV in both rodents and NHPs. In collaboration with our European partners we evaluated a DNA-based vaccine for CCHFV in the macaque model. We found that prime-boost-boost vaccination with plasmids expressing the nucleoprotein (NP) and glycoprotein precursor (GPC) of CCHFV conferred substantial protection against CCHFV infection with significantly reduced viral loads and protection from liver pathology and clinical disease (Hawman et al., Nat Microbiol 2021). In a follow-up study we accelerated the vaccine approach to a two-dose vaccination regimen. Our data show that the DNA vaccine confers robust protection against CCHFV and suggest that both humoral and cellular immunity contribute to optimal vaccine-mediated protection (Hawman et al., under review). More recently, we have developed a replicating RNA vaccine expressing NP and GPC that mediated complete protection against clinical disease. Interestingly, vaccination resulted in antibodies against NP and cellular immunity against the virion-exposed GPC (Leventhal et al., eBioMed 2022). In our mouse model recapitulating human convalescence we identified T-cells and IFN as essential for controlling the infection. We also determined that prior infection with CCHFV protected against reinfection with a distinct CCHFV strain (Hawman et al., Microorganisms 2021). In our MA-CCHFV model, we identified that both type I IFN and adaptive immunity are required for control of the virus. Interestingly, we also identified a significant sex-linked bias with female mice largely resistant to severe disease. The distinct disease outcomes in male versus female mice correlates with similar signs as is reported in human CCHF cases (Hawman et al., eLife 2021). Filoviruses Introduction: Infections with ebolaviruses and marburgviruses, family Filoviridae, cause Ebola (EVD) and Marburg virus disease (MVD), respectively, with high case fatality rates. The natural reservoirs for filoviruses are likely different bat species. Filovirus are enveloped, non-segmented, negative-stranded RNA viruses expressing seven structural proteins. The virion surface displays glycoprotein (GP) trimers that facilitate virus entry by receptor binding and fusion with target cells. The West African EVD outbreak has led to the licensure of vaccines treatments making EVD a success story in the field of neglected tropical diseases. (review Hansen et al., Expert Opin Investig Drugs 2021) Findings: We continued our efforts to study filovirus replication and protein function and their role in immunology and pathogenesis (Bhatia et al., Vaccines 2021). We achieved a milestone by establishing a disease model for Reston virus (RESTV) where young pigs developed a respiratory disease that was rapidly fatal (Haddock et al., Proc Natl Acad Sci USA 2021). The model will be crucial to gain further insight into the pathogenesis and for countermeasure development. Furthermore, we could assign a role for the EBOV soluble glycoprotein in pathogenesis by activating the MAP kinase signaling pathway (Furuyama et al., PLoS Pathog 2021). We have refined our filovirus VSV vaccines. Regarding vaccine safety, we tested the VSV-EBOV vector in pigs (VSV-susceptible livestock species). In collaboration with Kansas State University we could demonstrate that high-dose VSV-EBOV can indeed cause vesicular disease in pigs, but horizontal transmission was not observed providing further safety data for this vaccine (Morzonov et al., Emerg Micobes Infect 2021). We developed second-generation VSV vectors based of the licensed VSV-EBOV. This followed the concept of utilizing the EBOV GP entry mechanism to deliver a second immunogen to favorable immune cells. To avoid skewing the immune response towards the EBOV GP, we determined the domains needed for entry deleting highly immunogenic domains on the EBOV GP (Bhatia et al., Vaccines 2021). Based on this data, we produced a bivalent VSV-EBOV-KFDV(preM/E) vector that provided efficient protection against KFDV challenge in the previously established mouse model (Bhatia et al., Emerg Microbes Infect 2021; Bhatia et al., NPJ Vaccines 2021). In collaboration we contributed to an important study that established rather minimal impact of intensive care on the outcome of EBOV infection in macaques further indicating the need of specific treatments for EVD Biondi et al., Microorganisms 2021).