The Role of Adaptive Mutations in Mouse Adapted Crimean-Congo Hemorrhagic Fever Virus

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is endemic in Europe, Asia, and Africa. The geographic distribution of CCHFV is expanding as Hyalomma ticks, the main carriers of the virus, migrate northward. Infection with CCHFV initially manifests with non-specific symptoms including fever, muscle pains, and nausea that may progress into a hemorrhagic phase characterized by severe bleeding throughout the body. The case fatality rate is reported to range between 9-50%. With increasing numbers of humans at risk, further understanding of how the virus causes disease is essential for developing effective therapeutics. Studies investigating the host and viral determinants of pathogenesis, however, have been constrained due to mouse models requiring mice to be deficient in initial innate immune responses to manifest CCHFV disease symptoms after infection. However, we have recently developed a mouse-adapted CCHFV (MA-CCHFV) which presents with disease similar to human CCHFV cases in fully immunocompetent mice. We hypothesize that adaptive mutations in MA-CCHFV have enabled the virus to overcome mouse innate immunity and cause disease in immunocompetent mice. CCHFV is an RNA virus with three genomic segments. The S segment encodes the nucleoprotein (NP) and a non-structural protein (NSs) while the M segment encodes a large multi-unit protein which is later cleaved into two structural glycoproteins and three non-structural proteins. The L segment has largely unknown function(s) but does encode a protein required for viral replication. Compared to parental strain CCHFV-Hoti, MA-CCHFV has 6 mutations which result in changes to proteins encoded by the virus. Two mutations occur in the NP, one in the NSs, two in the M segment non-structural proteins GP38 and NSm and two in the viral L protein. These mutations likely indicate key proteins CCHFV uses as virulence factors to cause severe disease. To determine the role of these adaptations, we are examining the responses of human and mouse cell lines to infection with parental and MA-CCHFV strains. In addition, we can express the viral proteins independently of the virus to isolate the specific roles of these proteins and understand how they affect the initial immune responses in mouse cells. Understanding how these mutated proteins uniquely interact with the mouse immune system will help identify the host and viral determinants of CCHFV-induced disease. This will support new avenues of focus in CCHFV research to develop effective therapeutics and vaccines. This research is funded by the Intramural Research Program, NIAID, NIH

A Look into <i>Bunyavirales</i> Genomes: Functions of Non-Structural (NS) Proteins

In 2016, the Bunyavirales order was established by the International Committee on Taxonomy of Viruses (ICTV) to incorporate the increasing number of related viruses across 13 viral families. While diverse, four of the families (Peribunyaviridae, Nairoviridae, Hantaviridae, and Phenuiviridae) contain known human pathogens and share a similar tri-segmented, negative-sense RNA genomic organization. In addition to the nucleoprotein and envelope glycoproteins encoded by the small and medium segments, respectively, many of the viruses in these families also encode for non-structural (NS) NSs and NSm proteins. The NSs of Phenuiviridae is the most extensively studied as a host interferon antagonist, functioning through a variety of mechanisms seen throughout the other three families. In addition, functions impacting cellular apoptosis, chromatin organization, and transcriptional activities, to name a few, are possessed by NSs across the families. Peribunyaviridae, Nairoviridae, and Phenuiviridae also encode an NSm, although less extensively studied than NSs, that has roles in antagonizing immune responses, promoting viral assembly and infectivity, and even maintenance of infection in host mosquito vectors. Overall, the similar and divergent roles of NS proteins of these human pathogenic Bunyavirales are of particular interest in understanding disease progression, viral pathogenesis, and developing strategies for interventions and treatments

An Intramuscular DNA Vaccine for SARS-CoV-2 Decreases Viral Lung Load but Not Lung Pathology in Syrian Hamsters

The 2019 novel coronavirus, SARS-CoV-2, first reported in December 2019, has infected over 102 million people around the world as of February 2021 and thus calls for rapid development of safe and effective interventions, namely vaccines. In our study, we evaluated a DNA vaccine against SARS-CoV-2 in the Syrian hamster model. Hamsters were vaccinated with a DNA-plasmid encoding the SARS-CoV-2 full length spike open reading frame (ORF) to induce host cells to produce spike protein and protective immune responses before exposure to infectious virus. We tested this vaccine candidate by both intranasal (IN) and intramuscular (IM) routes of administration and complexing with and without an in vivo delivery reagent. Hamsters receiving prime-boost-boost IM-only vaccinations recovered body weight quicker, had decreased lung viral loads, and increased SARS-CoV-2-specific antibody titers compared to control vaccinated animals but, surprisingly, lung pathology was as severe as sham vaccinated controls. The IM/IN combination group showed no efficacy in reducing lung virus titers or pathology. With increasing public health need for rapid and effective interventions, our data demonstrate that in some vaccine contexts, significant antibody responses and decreased viral loads may not be sufficient to prevent lung pathology

An RNA Vaccine Protects against Crimean-Congo Hemorrhagic Fever Virus Infection in Mice

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is commonly transmitted by ticks and has a wide geographic distribution, being endemic throughout southern and eastern Europe, the Middle East, Asia, and Africa. Due to climate change, this range is even expanding northward. CCHFV causes a hemorrhagic disease characterized by initial non-specific illness (fever, muscle pains, nausea) followed by severe internal and external bleeding with a case fatality rate of 5-60%. As there is no licensed therapeutics or vaccines, there is great need for highly effective countermeasures. Recently, we have developed a self-replicating RNA-based vaccine which expresses two of the viral proteins: the CCHFV nucleoprotein (repNP) and glycoprotein precursor (repGPC) and is highly protective against a lethal viral challenge in mice. We found that vaccination with this vaccine prevented disease and conferred 100% survival against a lethal CCHFV infection in mice. This vaccine significantly stimulated both antibody-producing B-cells and T-cells which kill infected cells. Our repNP vaccination primarily stimulated B-cells to produce anti-NP antibodies while repGPC primarily stimulated the T-cells. To confirm whether B-cells or T-cells are most responsible for protection, we vaccinated mice lacking B- or T-cells. While mice lacking T-cells survived, B-cell deficient mice only had ~40% survival indicating that the B-cells and antibodies are essential to confer protection. Currently, this vaccine is undergoing further preclinical testing in preparation for human clinical trials while ongoing studies are continuing to investigate how these antibodies protect against CCHFV so that we may improve additional and much needed CCHFV therapeutics. This research was funded by the Intramural Research Program, NIAID, NIH

Replicating RNA vaccination elicits an unexpected immune response that efficiently protects mice against lethal Crimean-Congo hemorrhagic fever virus challenge

Summary: Background: Crimean-Congo hemorrhagic fever virus is the cause of a severe hemorrhagic fever with cases reported throughout a wide-geographic region. Spread by the bite of infected ticks, contact with infected livestock or in the health care setting, disease begins as a non-specific febrile illness that can rapidly progress to hemorrhagic manifestations. Currently, there are no approved vaccines and antivirals such as ribavirin have unclear efficacy. Thus treatment is mostly limited to supportive care. Methods: In this report we evaluated an alphavirus-based replicon RNA vaccine expressing either the CCHFV nucleoprotein or glycoprotein precursor in a stringent, heterologous lethal challenge mouse model. Findings: Vaccination with the RNA expressing the nucleoprotein alone could confer complete protection against clinical disease, but vaccination with a combination of both the nucleoprotein and glycoprotein precursor afforded robust protection against disease and viral replication. Protection from lethal challenge required as little as a single immunization with 100ng of RNA. Unexpectedly, analysis of the immune responses elicited by the vaccine components showed that vaccination resulted in antibodies against the internal viral nucleoprotein and cellular immunity against the virion-exposed glycoproteins. Interpretation: Cumulatively this vaccine conferred robust protection against Crimean-Congo hemorrhagic fever virus and supports continued development of this vaccine candidate. Funding: This research was supported by the Intramural Research Program of the NIAID/NIH and HDT Bio

A replicating RNA vaccine confers protection in a rhesus macaque model of Crimean-Congo hemorrhagic fever

Abstract Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne febrile illness with a wide geographic distribution. In recent years the geographic range of Crimean-Congo hemorrhagic fever virus (CCHFV) and its tick vector have increased, placing an increasing number of people at risk of CCHFV infection. Currently, there are no widely available vaccines, and although the World Health Organization recommends ribavirin for treatment, its efficacy is unclear. Here we evaluate a promising replicating RNA vaccine in a rhesus macaque (Macaca mulatta) model of CCHF. This model provides an alternative to the established cynomolgus macaque model and recapitulates mild-to-moderate human disease. Rhesus macaques infected with CCHFV consistently exhibit viremia, detectable viral RNA in a multitude of tissues, and moderate pathology in the liver and spleen. We used this model to evaluate the immunogenicity and protective efficacy of a replicating RNA vaccine. Rhesus macaques vaccinated with RNAs expressing the CCHFV nucleoprotein and glycoprotein precursor developed robust non-neutralizing humoral immunity against the CCHFV nucleoprotein and had significant protection against the CCHFV challenge. Together, our data report a model of CCHF using rhesus macaques and demonstrate that our replicating RNA vaccine is immunogenic and protective in non-human primates after a prime-boost immunization