Vesicular Stomatitis Virus-Based Vaccine Protects Mice against Crimean-Congo Hemorrhagic Fever.
ABSTRACT: Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne bunyavirus, can cause a life-threatening hemorrhagic syndrome in humans but not in its animal host. The virus is widely distributed throughout southeastern Europe, the Middle East, Africa, and Asia. Disease management has proven difficult and there are no broadly licensed vaccines or therapeutics. Recombinant vesicular stomatitis viruses (rVSV) expressing foreign glycoproteins (GP) have shown promise as experimental vaccines for several viral hemorrhagic fevers. Here, we developed and assessed a replication competent rVSV vector expressing the CCHFV glycoprotein precursor (GPC), which encodes CCHFV structural glycoproteins. This construct drives strong expression of CCHFV-GP, in vitro. Using these vectors, we vaccinated STAT-1 knock-out mice, an animal model for CCHFV. The vector was tolerated and 100% efficacious against challenge from a clinical strain of CCHFV. Anti-CCHFV-GP IgG and neutralizing antibody titers were observed in surviving animals. This study demonstrates that a rVSV expressing only the CCHFV-GP has the potential to serve as a replication competent vaccine platform against CCHF infections.
Project description:Recent West African Ebola virus (EBOV) epidemics have led to testing different anti-EBOV vaccines, including a replication-defective adenovirus (RD-Ad) vector (ChAd3-EBOV) and an infectious, replication-competent recombinant vesicular stomatitis virus expressing the EBOV glycoprotein (rVSV-EBOV; also known as rVSV-ZEBOV). While RD-Ads elicit protection, when scaled up to human trials, the level of protection may be much lower than that of vaccines containing viruses that can replicate. Although a replication-competent Ad (RC-Ad) vaccine might generate a level of protection approximating that of rVSV, this infectious vector would also risk causing adenovirus disease. We recently described a "single-cycle" adenovirus (SC-Ad) vector that amplifies antigen genes like RC-Ad, but that avoids the risk of adenovirus infection. Here we have tested an SC-Ad6 vector expressing the glycoprotein (GP) from a 2014 EBOV strain in mice, hamsters, and rhesus macaques. We show that SC-Ad6-EBOV GP induces a high level of serum antibodies in all species and mediates significant protection against pseudo-challenge with rVSV-EBOV expressing luciferase in mice and hamsters. These data suggest that SC-Ad6-EBOV GP may be useful during future EBOV outbreaks.
Project description:Crimean-Congo hemorrhagic fever (CCHF) is an often lethal, acute inflammatory illness that affects a large geographic area. The disease is caused by infection with CCHF virus (CCHFV), a nairovirus from the Bunyaviridae family. Basic research on CCHFV has been severely hampered by biosafety requirements and lack of available strains and molecular tools. We report the development of a CCHF transcription- and entry-competent virus-like particle (tecVLP) system that can be used to study cell entry and viral transcription/replication over a broad dynamic range (~4 orders of magnitude). The tecVLPs are morphologically similar to authentic CCHFV. Incubation of immortalized and primary human cells with tecVLPs results in a strong reporter signal that is sensitive to treatment with neutralizing monoclonal antibodies and by small molecule inhibitors of CCHFV. We used glycoproteins and minigenomes from divergent CCHFV strains to generate tecVLPs, and in doing so, we identified a monoclonal antibody that can prevent cell entry of tecVLPs containing glycoproteins from 3 pathogenic CCHFV strains. In addition, our data suggest that different glycoprotein moieties confer different cellular entry efficiencies, and that glycoproteins from the commonly used strain IbAr10200 have up to 100-fold lower ability to enter primary human cells compared to glycoproteins from pathogenic CCHFV strains.
Project description:Severe fever with thrombocytopenia virus (SFTSV) is an emerging tick-borne phlebovirus that causes lethal human disease, for which there are no licensed antiviral vaccines or therapies. Herein, we developed a live attenuated recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate expressing the SFTSV Gn/Gc glycoproteins (rVSV-SFTSV/AH12-GP). High titers of cross-protective, broadly neutralizing antibodies were elicited by a single dose of rVSV-SFTSV/AH12-GP in both immunocompetent and immunocompromised mice against multiple strains of SFTSV and the related but distinct phlebovirus Heartland virus (HRTV). Remarkably, complete protection against lethal challenge with SFTSV was conferred in young and old immunocompromised mice irrespective of any pre-existing vector-specific immunity. Collectively, these results suggest that a rVSV vector expressing SFTSV glycoproteins is a promising candidate vaccine against two emerging phleboviruses associated with severe human diseases.
Project description:Crimean-Congo hemorrhagic fever (CCHF) is an acute, often fatal viral disease characterized by rapid onset of febrile symptoms followed by hemorrhagic manifestations. The etiologic agent, CCHF orthonairovirus (CCHFV), can infect several mammals in nature but only seems to cause clinical disease in humans. Over the past two decades there has been an increase in total number of CCHF case reports, including imported CCHF patients, and an expansion of CCHF endemic areas. Despite its increased public health burden there are currently no licensed vaccines or treatments to prevent CCHF. We here report the development and assessment of the protective efficacy of an adenovirus (Ad)-based vaccine expressing the nucleocapsid protein (N) of CCHFV (Ad-N) in a lethal immunocompromised mouse model of CCHF. The results show that Ad-N can protect mice from CCHF mortality and that this platform should be considered for future CCHFV vaccine strategies.
Project description:Crimean-Congo hemorrhagic fever virus (CCHFV) is a virus that causes severe liver dysfunctions and hemorrhagic fever, with high mortality rate. Here, we show that CCHFV infection caused a massive lipidation of LC3 in hepatocytes. This lipidation was not dependent on <i>ATG5, ATG7</i> or <i>BECN1</i>, and no signs for recruitment of the alternative ATG12-ATG3 pathway for lipidation was found. Both virus replication and protein synthesis were required for the lipidation of LC3. Despite an augmented transcription of <i>SQSTM1</i>, the amount of proteins did not show a massive and sustained increase in infected cells, indicating that degradation of SQSTM1 by macroautophagy/autophagy was still occurring. The genetic alteration of autophagy did not influence the production of CCHFV particles demonstrating that autophagy was not required for CCHFV replication. Thus, the results indicate that CCHFV multiplication imposes an overtly elevated level of LC3 mobilization that involves a possibly novel type of non-canonical lipidation. <b>Abbreviations:</b> BECN1: Beclin 1; CCHF: Crimean-Congo hemorrhagic fever; CCHFV: Crimean-Congo hemorrhagic fever virus; CHX: cycloheximide; ER: endoplasmic reticulum; GFP: green fluorescent protein; GP: glycoproteins; MAP1LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; n.i.: non-infected; NP: nucleoprotein; p.i.: post-infection; SQSTM1: sequestosome 1.
Project description:Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne orthonairovirus, causes a severe hemorrhagic disease in humans (Crimean-Congo hemorrhagic fever, CCHF). Currently, no vaccines are approved to prevent CCHF; treatment is limited to supportive care and the use of ribavirin, the therapeutic benefits of which remain unclear. CCHF is part of WHO's priority list of infectious diseases warranting further research and development. To aid in the identification of new antiviral compounds, we generated a recombinant CCHFV expressing a reporter protein, allowing us to quantify virus inhibition by measuring the reduction in fluorescence in infected cells treated with candidate compounds. The screening assay was readily adaptable to high-throughput screening (HTS) of compounds using Huh7 cells, with a signal-to-noise ratio of 50:1, and Z'-factors > 0.6 in both 96- and 384-well formats. A screen of candidate nucleoside analog compounds identified 2'-deoxy-2'-fluorocytidine (EC<sub>50</sub> = 61 ± 18 nM) as having 200 × the potency of ribavirin (EC<sub>50</sub> = 12.5 ± 2.6 μM), as well as 17 × the potency of T-705 (favipiravir), another compound with reported anti-CCHFV activity (EC<sub>50</sub> = 1.03 ± 0.16 μM). Furthermore, we also determined that 2'-deoxy-2'-fluorocytidine acts synergistically with T-705 to inhibit CCHFV replication without causing cytotoxicity. The incorporation of this reporter virus into the high-throughput screening assay described here will allow more rapid identification of effective therapeutic options to combat this emerging human pathogen.
Project description:Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis with a high case fatality rate in humans. Although the disease is widely found in Africa, Europe, and Asia, the distribution and genetic diversity of CCHF virus (CCHFV) are poorly understood in African countries. To assess the risks of CCHF in Zambia, where CCHF has never been reported, epidemiologic studies in cattle and ticks were conducted. Through an indirect immunofluorescence assay, CCHFV nucleoprotein-specific serum IgG was detected in 8.4% (88/1,047) of cattle. Among 290 Hyalomma ticks, the principal vector of CCHFV, the viral genome was detected in 11 ticks. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between African and Asian strains. This study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts.
Project description:Despite the serious public health impact of Crimean-Congo hemorrhagic fever (CCHF), the efficacy of antivirals targeting the causative agent, CCHF virus (CCHFV), remains debatable. Neutralizing monoclonal antibodies (MAbs) targeting the CCHFV glycoprotein Gc have been reported to protect mice against challenge with the prototype CCHFV strain, IbAr10200. However, due to extensive sequence diversity of CCHFV glycoproteins, it is unknown whether these MAbs neutralize other CCHFV strains. We initially used a CCHF virus-like particle (VLP) system to generate 11 VLP moieties, each possessing a glycoprotein from a genetically diverse CCHFV strain isolated in either Africa, Asia, the Middle East, or southeastern Europe. We used these VLPs in biosafety level 2 conditions to efficiently screen MAb cross-neutralization potency. Of the 16 MAbs tested, 3 (8A1, 11E7, and 30F7) demonstrated cross-neutralization activity with most CCHF VLPs, with 8A1 neutralizing all VLPs tested. Although binding studies suggest that none of the MAbs compete for the same epitope, combining 11E7, 30F7, or both 11E7 and 30F7 with 8A1 had no additive effect on increasing neutralization in this system. To confirm our findings from the VLP system, the 3 MAbs capable of strain cross-neutralization were confirmed to effectively neutralize 5 diverse CCHFV strains in vitro. Passaging CCHFV strains in the presence of sub-neutralizing concentrations of MAbs did not generate escape mutants resistant to subsequent neutralization. This study demonstrates the utility of the VLP system for screening neutralizing MAbs against multiple CCHFV strains, and provides the first evidence that a single MAb can effectively neutralize a number of diverse CCHFV strains in vitro, which may lead to development of future CCHF therapeutics.
Project description:Crimean-Congo hemorrhagic fever virus (CCHFV) is a cause of severe hemorrhagic fever. Its tick reservoir and vector are widely distributed throughout Africa, Southern and Eastern Europe, the Middle East, and Asia. Serological evidence suggests that CCHFV can productively infect a wide variety of species, but only humans develop severe, sometimes fatal disease. The role of the host adaptive immunity in control or contribution to the severe pathology seen in CCHF cases is largely unknown. Studies of adaptive immune responses to CCHFV have been limited due to lack of suitable small animal models. Wild-type mice are resistant to CCHFV infection, and type I interferon-deficient mice typically develop a rapid-onset fatal disease prior to development of adaptive immune responses. We report here a mouse model in which type I interferon-deficient mice infected with a clinical isolate of CCHFV develop a severe inflammatory disease but ultimately recover. Recovery was coincident with development of CCHFV-specific B- and T-cell responses that were sustained for weeks postinfection. We also found that recovery from a primary CCHFV infection could protect against disease following homologous or heterologous reinfection. Together this model enables study of multiple aspects of CCHFV pathogenesis, including convalescence, an important aspect of CCHF disease that existing mouse models have been unsuitable for studying.IMPORTANCE The role of antibody or virus-specific T-cell responses in control of acute Crimean-Congo hemorrhagic fever virus infection is largely unclear. This is a critical gap in our understanding of CCHF, and investigation of convalescence following severe acute CCHF has been limited by the lack of suitable small animal models. We report here a mouse model of CCHF in which infected mice develop severe disease but ultimately recover. Although mice developed an inflammatory immune response along with severe liver and spleen pathology, these mice also developed CCHFV-specific B- and T-cell responses and were protected from reinfection. This model provides a valuable tool to investigate how host immune responses control acute CCHFV infection and how these responses may contribute to the severe disease seen in CCHFV-infected humans in order to develop therapeutic interventions that promote protective immune responses.
Project description:Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the Nairoviridae family within the Bunyavirales order of viruses. Crimean-Congo hemorrhagic fever (CCHF) is the most widespread among tick-borne human viral diseases. It is endemic in many areas of Africa, Asia, the Middle East, in the Balkans, Russia and countries of the former Soviet Union. The confirmed CCHF cases were seen in Spain in 2016 to signify expansion of the virus into new geographical areas. CCHFV causes a viral human disease characterized by sudden onset of fever, headache, abdominal pain, nausea, hypotension, hemorrhage, and hepatic dysfunction with fatality rates up to 30%. Currently, there are no spesific treatments or licensed vaccines available for CCHFV. The absence of a susceptible animal model for CCHFV infection was severely hindered work on the development of vaccines. However, several animal models of CCHFV infection have been recently developed and used to assess vaccine efficacy. In this study, we have used the transiently immune-suppressed (IS) mouse model that MAb-5A3 was used to block IFN-I signaling in immune intact, wild-type mice at the time of CCHFV infection to evaluate the immune response and efficacy of the cell culture based and the mouse brain derived inactivated vaccines against CCHFV. Both vaccine preparations have provided complete protection but the cell culture based vaccine more effectively induced to CCFHV spesific antibodies and T cell responses. This is the first comparison of the cell culture based and the mouse brain derived vaccines for assessing the protective efficacy and the immunogenicity in the IS mouse CCHFV model.