Reverse genetic systems are powerful tools in molecular virology that allow the generation of infectious recombinant virus and the manipulation of viral genomes. Reverse genetic systems enable the incorporation of reporter genes, facilitating many virological assays, including high-throughput screening. Additionally, reverse genetic systems can be used to introduce targeted mutations into the viral genome, allowing investigations of viral genetic elements and protein functions in virus pathogenesis and biology. Here we describe in detail the materials and methods required for the Crimean-Congo hemorrhagic fever virus (CCHFV) reverse genetic system. This system can be used to generate complete infectious recombinant virus, and virus-like replicon particles (VRPs) lacking the M segment but complemented with an exogenous source of glycoprotein precursor (GPC); resulting in single-round replicon particles that can be used to study components of the viral replicative cycle at a lower biosafety level.

Advancement of vaccine candidates that demonstrate protective efficacy in screening studies necessitates detailed safety and immunogenicity investigations in pre-clinical models. A non-spreading Crimean-Congo hemorrhagic fever virus (CCHFV) viral replicon particle (VRP) vaccine was developed for single-dose administration to protect against disease. To date, several studies have supported safety, immunogenicity, and efficacy of the CCHF VRP in multiple highly sensitive murine models of lethal disease, but the VRP had yet to be evaluated in large animals. Here, we performed studies in non-human primates to further evaluate clinical utility of the VRP vaccine. Twelve adult male and female rhesus macaques were vaccinated intramuscularly and followed daily for clinical monitoring. At 3, 7, 14, 21, and 28 days post vaccination, animals were sedated for more detailed clinical assessment; for quantification of vaccine presence in blood and mucosal samples; and for evaluation of hematology, plasma inflammatory markers, and immunity. Consistent with findings in mice, vaccination was well tolerated, with no clinical alterations nor indication of vaccine spread or shedding. In addition, vaccination induced both humoral and cell-mediated responses, with immune profile and kinetics also corroborating data from small animal models. These studies provide key data in non-human primates further supporting development of the VRP for human clinical use.

Ebola disease (EBOD) in humans is a severe disease caused by at least four related viruses in the genus Orthoebolavirus, most often by the eponymous Ebola virus. Due to human-to-human transmission and incomplete success in treating cases despite promising therapeutic development, EBOD is a high priority in public health research. Yet despite almost 50 years since EBOD was first described, the sources of these viruses remain undefined and much remains to be understood about the disease epidemiology and virus emergence and spread. One important approach to improve our understanding is detection of antibodies that can reveal past human infections. However, serosurveys routinely describe seroprevalences that imply infection rates much higher than those clinically observed. Proposed hypotheses to explain this difference include existence of common but less pathogenic strains or relatives of these viruses, misidentification of EBOD as something else, and a higher proportion of subclinical infections than currently appreciated. The work presented here maps B-cell epitopes in the spike protein of Ebola virus and describes a single epitope that is cross-reactive with an antigen seemingly unrelated to orthoebolaviruses. Antibodies against this epitope appear to explain most of the unexpected reactivity towards the spike, arguing against common but unidentified infections in the population. Importantly, antibodies of cross-reactive donors from within and outside the known EBOD geographic range bound the same epitope. In light of this finding, it is plausible that epitope mapping enables broadly applicable specificity improvements in the field of serology.

ABSTRACTNipah virus (NiV) is an emerging zoonotic RNA virus that can cause fatal respiratory and neurological disease in animals and humans. Accurate NiV diagnostics and surveillance tools are crucial for the identification of acute and resolved infections and to improve our understanding of NiV transmission and circulation. Here, we have developed and validated a split NanoLuc luciferase NiV glycoprotein (G) biosensor for detecting antibodies in clinical and animal samples. This assay is performed by simply mixing reagents and measuring luminescence, which depends on the complementation of the split NanoLuc luciferase G biosensor following its binding to antibodies. This anti-NiV-G "mix-and-read" assay was validated using the WHO's first international standard for anti-NiV antibodies and more than 700 serum samples from the NiV-endemic country of Bangladesh. Anti-NiV antibodies from survivors persisted for at least 8 years according to both ⍺NiV-G mix-and-read and NiV neutralization assays. The ⍺NiV-G mix-and-read assay sensitivity (98.6%) and specificity (100%) were comparable to anti-NiV IgG ELISA performance but failed to detect anti-NiV antibodies in samples collected less than a week following the appearance of symptoms. Overall, the anti-NiV-G biosensor represents a simple, fast, and reliable tool that could support the expansion of NiV surveillance and retrospective outbreak investigations.

Immunizing mice with Crimean-Congo hemorrhagic fever virus (CCHFV) nucleoprotein (NP), glycoprotein precursor (GPC), or with the GP38 domain of GPC, can be protective when the proteins are delivered with viral vectors or as a DNA or RNA vaccine. Subunit vaccines are a safe and cost-effective alternative to some vaccine platforms, but Gc and Gn glycoprotein subunit vaccines for CCHFV fail to protect despite eliciting high levels of neutralizing antibodies. Here, we investigated humoral and cellular immune responses and the protective efficacy of recombinant NP, GP38, and GP38 forms (GP85 and GP160) associated with the highly glycosylated mucin-like (MLD) domain, as well as the NP + GP38 combination. Vaccination with GP160, GP85, or GP38 did not confer protection, and vaccination with the MLD-associated GP38 forms blunted the humoral immune responses to GP38, worsened clinical chemistry, and increased viral RNA in the blood compared to the GP38 vaccination. In contrast, NP vaccination conferred 100% protection from lethal outcome and was associated with mild clinical disease, while the NP + GP38 combination conferred even more robust protection by reducing morbidity compared to mice receiving NP alone. Thus, recombinant CCHFV NP alone is a promising vaccine candidate conferring 100% survival against heterologous challenge. Moreover, incorporation of GP38 should be considered as it further enhances subunit vaccine efficacy by reducing morbidity in surviving animals.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe human disease and is considered a WHO priority pathogen due to the lack of efficacious vaccines and antivirals. A CCHF virus replicon particle (VRP) has previously shown protective efficacy in a lethal Ifnar(-/-) mouse model when administered as a single dose at least 3 days prior to challenge. Here, we determine that non-specific immune responses are not sufficient to confer short-term protection, since Lassa virus VRP vaccination 3 days prior to CCHFV challenge was not protective. We also investigate how CCHF VRP vaccination confers protective efficacy by examining viral kinetics, histopathology, clinical analytes and immunity early after challenge (3 and 6 days post infection) and compare to unvaccinated controls. We characterize how these effects differ based on vaccination period and correspond to previously reported CCHF VRP-mediated protection. Vaccinating Ifnar(-/-) mice with CCHF VRP 28, 14, 7, or 3 days prior to challenge, all known to confer complete protection, significantly reduced CCHFV viral load, mucosal shedding, and markers of clinical disease, with greater reductions associated with longer vaccination periods. Interestingly, there were no significant differences in innate immune responses, T cell activation, or antibody titers after challenge between groups of mice vaccinated a week or more before challenge, but higher anti-NP antibody avidity and effector function (ADCD) were positively associated with longer vaccination periods. These findings support the importance of antibody-mediated responses in VRP vaccine-mediated protection against CCHFV infection.

Nipah virus (NiV) is a highly pathogenic paramyxovirus. The Syrian hamster model recapitulates key features of human NiV disease and is a critical tool for evaluating antivirals and vaccines. Here we describe longitudinal humoral immune responses in NiV-infected Syrian hamsters. Samples were obtained 1-28 days after infection and analyzed by ELISA, neutralization, and Fc-mediated effector function assays. NiV infection elicited robust antibody responses against the nucleoprotein and attachment glycoprotein. Levels of neutralizing antibodies were modest and only detectable in surviving animals. Fc-mediated effector functions were mostly observed in nucleoprotein-targeting antibodies. Antibody levels and activities positively correlated with challenge dose.

Ebola disease outbreaks are major public health events because of human-to-human transmission and high mortality. These outbreaks are most often caused by Ebola virus, but at least three related viruses can also cause the disease. In 2022, Sudan virus re-emerged causing more than 160 confirmed and probable cases. This report describes generation of a recombinant Sudan virus and demonstrates its utility by quantifying antibody cross-reactivity between Ebola and Sudan virus glycoproteins after human infection or vaccination with a licensed Ebola virus vaccine.

Crimean-Congo hemorrhagic fever virus (CCHFV; family Nairoviridae) is a tick-borne pathogen that frequently causes lethal disease in humans. CCHFV has a wide geographic distribution, and cases have been reported in Africa, Asia, the Middle East, and Europe. Availability of a safe and efficacious vaccine is critical for restricting outbreaks and preventing disease in endemic countries. We previously developed a virus-like replicon particle (VRP) vaccine that provides complete protection against homologous and heterologous lethal CCHFV challenge in mice after a single dose. However, the immune responses induced by this vaccine are not well characterized, and correlates of protection remain unknown. Here we comprehensively characterized the kinetics of cell-mediated and humoral immune responses in VRP-vaccinated mice, and demonstrate that they predominantly target the nucleoprotein (NP). NP antibodies are not associated with protection through neutralizing activity, but VRP vaccination results in NP antibodies possessing Fc-mediated antibody effector functions, such as complement activation (ADCD) and antibody-mediated cellular phagocytosis (ADCP). This suggests that Fc-mediated effector functions may contribute to this vaccine's efficacy.

Nipah virus (NiV) causes a highly lethal disease in humans who present with acute respiratory or neurological signs. No vaccines against NiV have been approved to date. Here, we report on the clinical impact of a novel NiV-derived nonspreading replicon particle lacking the fusion (F) protein gene (NiVΔF) as a vaccine in three small animal models of disease. A broad antibody response was detected that included immunoglobulin G (IgG) and IgA subtypes with demonstrable Fc-mediated effector function targeting multiple viral antigens. Single-dose intranasal vaccination up to 3 days before challenge prevented clinical signs and reduced virus levels in hamsters and immunocompromised mice; decreases were seen in tissues and mucosal secretions, critically decreasing potential for virus transmission. This virus replicon particle system provides a vital tool to the field and demonstrates utility as a highly efficacious and safe vaccine candidate that can be administered parenterally or mucosally to protect against lethal Nipah disease.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) causes a life-threatening disease with up to a 40% mortality rate. With no approved medical countermeasures, CCHFV is considered a public health priority agent. The non-neutralizing mouse monoclonal antibody (mAb) 13G8 targets CCHFV glycoprotein GP38 and protects mice from lethal CCHFV challenge when administered prophylactically or therapeutically. Here, we reveal the structures of GP38 bound with a human chimeric 13G8 mAb and a newly isolated CC5-17 mAb from a human survivor. These mAbs bind overlapping epitopes with a shifted angle. The broad-spectrum potential of c13G8 and CC5-17 and the practicality of using them against Aigai virus, a closely related nairovirus were examined. Binding studies demonstrate that the presence of non-conserved amino acids in Aigai virus corresponding region prevent CCHFV mAbs from binding Aigai virus GP38. This information, coupled with in vivo efficacy, paves the way for future mAb therapeutics effective against a wide swath of CCHFV strains.