Sudan virus (SUDV) has caused multiple outbreaks of human disease with case fatality rates ranging from 41 to 100%. We have previously shown that a single vaccination with a recombinant vesicular stomatitis virus-based vaccine expressing the SUDV-Gulu glycoprotein (VSV-SUDV) prevented clinical and fatal disease from lethal SUDV challenge in cynomolgus macaques. With the high probability of future outbreaks, it is critical to determine the molecular mechanisms of VSV-SUDV-mediated protection and the ability to impart rapid protection against SUDV infection. In this study, RNA from whole blood samples obtained from nine cynomolgus macaques that were challenged with SUDV-Gulu post-vaccination with either VSV-EBOV (28 days before challenge, n = 3) or VSV-SUDV (28 or 7 days before challenge, n = 3/group) was subjected to bulk RNA sequencing. EdgeR, STEM, MaSigPro, and CIBERSORTx were used to assess longitudinal transcriptional changes elicited by vaccination and challenge. Our analysis revealed that VSV-SUDV and VSV-EBOV elicited distinct transcriptional responses. Moreover, NHPs vaccinated with VSV-EBOV (non-protective) generated a transcriptional response following SUDV challenge indicative of dysregulated inflammation. In contrast, NHPs that received VSV-SUDV vaccine generated a transcriptional response indicative of a recall adaptive immune response. Finally, in-silico deconvolution methods indicated changes in immune cell frequency consistent with immune response and resolution in the VSV-SUDV-vaccinated NHPs that are not observed with VSV-EBOV-vaccinated NHPs. These data indicate that VSV-SUDV vaccination results in a protective humoral response as late as 7 days before challenge despite transcriptional evidence of subclinical features of infection.

Griffithsin (GRFT), a lectin derived from the red algae Griffithsia, is known as an antiviral agent that binds to high-mannose glycans on viral envelope glycoproteins (GPs). In this study, we evaluated the antiviral activity of GRFT against Ebola virus (EBOV), Marburg virus (MARV), Lassa virus (LASV), Lujo virus (LUJV), and Crimean-Congo hemorrhagic fever virus (CCHFV), all of which cause severe and often fatal hemorrhagic fevers in humans. GRFT effectively blocked the entry of these viruses into cells, with stronger inhibition observed against LASV, LUJV, and CCHFV than against EBOV and MARV. This inhibitory effect depended on the lectin activity of GRFT, as its mutant lacking glycan-binding function completely lost the ability to inhibit the entry of these viruses into cells. We found that GRFT induced aggregation of virus-like particles (VLPs) derived from EBOV and MARV, whereas this activity was not observed with LASV- and LUJV-derived VLPs. Most of the escape mutants of LASV and LUJV GPs exhibited amino acid substitutions that likely disrupted GRFT binding by removing glycosylation sites at positions 79 and 73, respectively. Interestingly, other substitutions close to these positions, although unrelated to glycosylation, also contributed to reduced inhibitory activity of GRFT, suggesting a unique recognition mode in which both sugar chains and adjacent amino acid residues constitute the binding site structure for GRFT. Overall, our results provide insights into mechanisms underlying the antiviral effects of GRFT and highlight lectin-related activities as a key feature for broad-spectrum antiviral agents targeting highly glycosylated envelope GPs. IMPORTANCE:Emerging and re-emerging infectious diseases, including viral hemorrhagic fevers, pose a major global health threat due to their potential for widespread outbreaks. Currently, treatment options for such diseases are limited and often ineffective against newly emerging viruses. Here, we demonstrate that Griffithsin (GRFT), a naturally derived lectin from red algae, inhibits the entry of multiple hemorrhagic fever viruses into host cells. By blocking key processes of the viral entry mediated by envelope glycoproteins, lectins such as GRFT can exhibit broad antiviral activity that could potentially overcome the limitations of existing treatments against emerging viruses. Our findings emphasize that targeting sugar chains and adjacent structures on viral glycoproteins with GRFT could provide a therapeutic strategy against diverse viral species. Such an approach is particularly valuable for newly emerging viruses for which specific countermeasures have not yet been established.

Since the West African Ebola virus (EBOV) epidemic in 2014-2016, recurrent outbreaks of the EBOV-Makona variant have been driven by recrudescence and human-to-human transmission emphasizing the need for effective vaccination strategies. A live-attenuated recombinant vesicular stomatitis virus (VSV)-based vaccine expressing the EBOV-Kikwit variant glycoprotein (VSV-Kik) received FDA approval in December 2019 and provides complete, rapid protection against EBOV-Makona as early as 7 days post-vaccination (DPV). During the 2018-2020 Ebola outbreak, the VSV-Kik vaccine, known as ERVEBO, was administered to lower-risk individuals at a 5-fold dose reduction of the standard 2 × 107 PFU to provide broader population protection. Identification of a protective lower dose providing rapid protection would ease supply burdens during future outbreaks and enhance vaccine coverage. We previously generated a VSV-based vaccine expressing the glycoprotein of the Makona variant (VSV-Mak) which provided complete protection against homologous challenge 28 DPV at as low as 1 × 101 PFU. However, the transcriptional responses engendered by VSV-Mak and VSV-Kik vaccines in the context of early EBOV-Makona challenge have not yet been evaluated. In the current study, we compared transcriptional responses following a low dose (1 × 104 PFU) of lab-grade VSV-Mak or GMP-grade VSV-Kik and subsequent EBOV-Makona challenge 10 DPV. VSV-Kik provided complete protection against heterologous challenge and elicited rapid antiviral transcriptional changes followed by the activation of adaptive immunity. On the other hand, VSV-Mak only provided partial protection and induced minimal transcriptional response. These results highlight a glycoprotein-specific transcriptional response after vaccination despite the high EBOV variant homology.

Despite advances in vaccine and antiviral drug development, the prevention of respiratory viral infection and transmission remains a substantial challenge worldwide. One obvious limitation of these approaches is that they do not provide robust protection at the initial site of infection, which is the respiratory mucosa. Currently, strategies to enhance mucosal immunity against respiratory pathogens remain lacking. Here we engineered mucus-tethering bispecific nanobodies designed to provide the simultaneous neutralization of viruses by binding to their surface proteins and the entrapment of viruses within the mucus by securing them to mucin. Compared with conventional non-mucus-tethering nanobodies, these mucus-tethering bispecific nanobodies demonstrated increased retention in the respiratory tract, provided enhanced protection against influenza viral infection in mice and reduced SARS-CoV-2 transmission in hamsters. Together, our findings represent a promising strategy for enhancing mucosal defences against respiratory viruses by blocking viral entry and limiting onward transmission. Engineered mucus-tethering bispecific nanobodies neutralize and entrap viruses to enhance mucosal immunity, preventing influenza infection and limiting SARS-CoV-2 transmission.

Ebola virus (EBOV) disease (EVD) is a hemorrhagic disease caused by EBOV infection. EVD outcomes in pregnant women are similar to non-pregnant women however, EVD is associated with negative fetal outcomes in ∼99% of cases. There is a critical need for a tractable small animal model to study maternal/fetal transmission of EBOV. We utilized interferon α/β receptor knock out mice infected and authentic EBOV or the model virus, recombinant vesicular stomatitis virus encoding EBOV glycoprotein (rVSV/EBOV). Infection with either virus during late pregnancy resulted in placental infection and vertical transmission to the fetus within 2-3 days. Robust levels of maternal and fetal proinflammatory cytokines were evident by day 5 after EBOV infection. Within the placenta, trophoblasts and endothelial cells were viral antigen positive. Elimination of the endosomal receptor NPC1 in junctional zone trophoblasts reduced placental infection and virus transmission to the fetus. These studies establish an infectious model that provides EBOV trafficking and pathogenesis insights during pregnancy. Teaser:This model provides key insights into how viral trafficking and maternal immune responses drive adverse fetal outcomes during gestational Ebola virus infection.

In the decade since the West African Ebola virus (EBOV) epidemic, several medical countermeasures against this often-fatal hemorrhagic disease have been approved by regulatory authorities for human use. This includes monoclonal antibody-based therapies and vaccines which have been stockpiled in limited quantities. One of the vaccines is based on vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) as the immunogen. The vaccine is stockpiled in limited quantity for emergency use. A single high dose has been shown to rapidly protect humans within 10 days. We developed an updated version of this vaccine expressing the GP from the 2015 EBOV-Makona isolate. Here, we wanted to determine the protective efficacy within 10 days of a single moderate dose (10-fold and 1,000-fold dilution) of the updated vaccine in nonhuman primates (NHPs). As a comparator we included a 1000-fold dilution dose group of the approved vaccine expressing the EBOV-Kikwit GP. While we achieved uniform protection with the approved vaccine at the moderate dose, only 50% of the NHPs receiving the same dose of the updated vaccine expressing the EBOV-Makona GP were protected. This study highlights the importance of evaluating VSV-based vaccine stocks expressing different filovirus GPs in preclinical models prior to progression with clinical development. Our study also highlights that rapid vaccination with reduced doses still leads to protection but at the cost of “sterile” immunity raising concerns regarding EBOV persistence and potential downstream transmission. Therefore, lower vaccine doses should only be considered in cases of severe vaccine shortage.

Bundibugyo virus (BDBV), a member of the orthoebolaviruses in the Filoviridae family, causes severe hemorrhagic disease with high case-fatality rates. Currently, there are no medical countermeasures approved for human use hampering the response to the large ongoing outbreak in the Democratic Republic of the Congo and Uganda. While vesicular stomatitis virus (VSV)-based vaccines have demonstrated fast-acting prophylactic single-dose efficacy against multiple filoviruses, it has yet to be defined for VSV-BDBV. Here, we evaluated the rapid protection by a single-dose vaccination of VSV-BDBV in nonhuman primates (NHPs). Vaccination elicited rapid innate and early adaptive immune responses and conferred complete protection from clinical disease against BDBV challenge within 3 days. Vaccinated NHPs exhibited minimal clinical signs, limited systemic inflammation, and no infectious virus was isolated from the blood at any time. Protection correlated with neutralizing antibodies and Fc effector functionality of the humoral immune response. These findings establish VSV-BDBV as a fast-acting vaccine candidate suitable for outbreak response and highlight immune mechanisms underlying rapid protection.

ABSTRACT Ebola virus disease (EVD) is a highly pathogenic and lethal disease caused by Ebola virus (EBOV). Endemic to Sub-Saharan Africa, EBOV has been causing global health concerns during infrequent EVD outbreaks since 1976, particularly during the 2013 –2016 epidemic in West Africa. Spread by contact of contaminated bodily fluids with mucous membranes or breaks in the skin, EBOV yields an incredibly high mortality rate and, as a result, has been designated a priority pathogen and a select agent to be studied exclusively in maximum containment laboratories. EBOV has historically been studied in animal models that accurately recapitulate disease progression, symptoms, and outcomes as seen in humans via more artificial infection routes such as intramuscular (IM) and intraperitoneal (IP) inoculation. There has recently been a concerted effort to characterize disease in animal models with mucosal inoculation routes with the goal to more accurately replicate human infection and development of EVD. This review aims to summarize and characterize the different inoculation routes used in the various animal models applied in EBOV research. We also outline the differences in disease progression between “artificial” and mucosal infection routes, comparing them to the course of EVD as seen in humans to highlight similarities and differences.

The Filoviridae family encompasses Ebola virus (EBOV) and Marburg virus (MARV), some of the most lethal viruses known to cause sporadic, recurring outbreaks of severe hemorrhagic fever mainly throughout central Africa. However, other lesser-known viruses also belong to the filovirus family as they are closely related, such as Bundibugyo, Reston and Taï Forest virus. These viruses differ in their virulence in humans significantly: while EBOV and MARV show lethality in humans of up to 90 %, Reston virus appears to be avirulent in humans. Here, underlying molecular factors leading to differences in virulence via changes in filovirus entry, replication and immune evasion strategies are summarized and assessed. While the filovirus glycoprotein contributes towards virulence by facilitating entry into a wide variety of tissues, differences in virus-host interactions and replication efficacies lead to measurable variances of progeny virus production. Additionally, immune evasion strategies lead to alterations in replication efficacy thus changing who has the upper hand between the virus and the host. Understanding and unraveling the contributions of these molecular determinants on filovirus virulence provide insights into the processes causing the underlying pathogenesis. It will further help to assess the pathogenicity of newly discovered filoviruses. Finally, these molecular determinants and processes present attractive targets for therapeutic intervention and development of novel antiviral countermeasures.

The Sudan virus (SUDV) outbreaks in Uganda in 2022 and 2025 created public health concerns in-country and the entire East African region. There are currently no licensed countermeasures against SUDV. We developed a SUDV vaccine candidate based on a nanocarrier (LIONTM) complexed with an alphavirus-based replicon RNA. Here, we compare the protective efficacy of the LION-SUDV vaccine either encoding the SUDV glycoprotein (GP) alone or in combination with the Ebola virus (EBOV) GP (LION-Combination). A LION-EBOV vaccine which is protective against EBOV was also included to determine the potential for cross-protection against SUDV infection. Single-dose vaccinations were conducted three weeks before challenge with a lethal dose of guinea pig-adapted SUDV using a female guinea pig disease model. We demonstrate 100% survival and protection with the LION-SUDV and the LION-Combination vaccines, while the LION-EBOV vaccine achieved 50% protection. Antigen-specific humoral responses correlate with decreased virus replication and survival. This result warrants further studies in larger animal species to ensure that protective efficacy is maintained with the single-dose LION-SUDV vaccine.

Ebola virus (EBOV) and Marburg virus (MARV) are zoonotic filoviruses that cause hemorrhagic fever in humans. Correlative data implicate bats as natural EBOV hosts, but neither a full-length genome nor an EBOV isolate has been found in any bats sampled. Here, we model filovirus infection in the Jamaican fruit bat (JFB), Artibeus jamaicensis, by inoculation with either EBOV or MARV through a combination of oral, intranasal, and subcutaneous routes. Infection with EBOV results in systemic virus replication and oral shedding of infectious virus. MARV replication is transient and does not shed. In vitro, JFB cells replicate EBOV more efficiently than MARV, and MARV infection induces innate antiviral responses that EBOV efficiently suppresses. Experiments using VSV pseudoparticles or replicating VSV expressing the EBOV or MARV glycoprotein demonstrate an advantage for EBOV entry and replication early, respectively, in JFB cells. Overall, this study describes filovirus species-specific phenotypes for both JFB and their cells.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes a severe, sometimes fatal hemorrhagic fever (CCHF) in humans. Currently, there are no approved therapies against CCHF. In this study we used the recombinant vesicular stomatitis virus (VSV) platform to generate live-attenuated recombinant CCHF vaccine candidates expressing the CCHFV nucleoprotein (NP) and glycoprotein precursor (GPC). As one approach, we utilized the established VSV expressing the full-length Ebola virus glycoprotein (VSV-EBOV) or a truncated version of the EBOV glycoprotein and added the CCHFV-NP (VSV-CCHFnp1 or VSV-CCHFnp2, respectively). Additionally, we prepared a vaccine candidate, VSV-CCHFgpc, in which the VSV glycoprotein was replaced with the CCHFV-GPC. Vaccine constructs induced CCHFV-specific IgG antibodies comprising largely IgG2c subclass. Only, the VSV-CCHFgpc vaccine candidate induced significant T cell immune responses directed against epitopes in the CCHFV-NSm and Gc proteins. Efficacy of the vaccine candidates was evaluated using a prime-only approach in a transiently immune-suppressed mouse model. Animals vaccinated with VSV-CCHFnp2 succumbed to lethal CCHFV challenge, while the VSV-CCHFgpc vaccine candidate afforded partial protection. In contrast, vaccination with VSV-CCHFnp1 uniformly protected animals against death. Our results demonstrate the promise of VSV-CCHFnp1 as a vaccine candidate for CCHFV and warrant continued development.

Wild-type filoviruses including Marburg virus (MARV) cause disease in humans, nonhuman primates, and some immunodeficient mouse strains but generally not in immunocompetent rodents and ferrets. However, disease in immunocompetent rodents can be achieved by serial passaging of the virus as demonstrated by the mouse-, hamster-, and guinea pig-adapted strains of MARV, which often cause lethal disease in the respective rodent species. These disease models present valuable first screening models for medical countermeasure evaluation against MARV, including monoclonal antibody therapies and vaccines. The MARV hamster disease model is of particular interest since the infected hamsters display almost all of the clinical signs of Marburg virus disease observed in nonhuman primates and humans, including petechial rash, hemorrhages, coagulation disorder, and dysregulated immune responses. This chapter describes a protocol using the hamster-adapted MARV in Syrian Golden hamsters for studies investigating viral pathogenesis or evaluating the efficacy of medical countermeasures.

Development of pan-orthoebolavirus vaccines to prevent Ebola disease and to induce durable protective immunity remains an important unmet need. Current Ebola virus (EBOV) vaccines have been reported to protect humans only against EBOV, one of the four pathogenic orthoebolaviruses. Their protective efficacy against Sudan virus (SUDV), Bundibugyo virus (BDBV) and Taï forest virus (TAFV) has not been tested in humans yet. Data from animal studies show that EBOV glycoprotein (GP)-based vaccines protect against EBOV, BDBV and TAFV, but not against SUDV, suggesting that vaccines with GPs from EBOV and SUDV will likely afford pan-orthoebolavirus protection. Here we report the development and pre-clinical evaluation of a bivalent EBOV/SUDV GPs virus-like particle (VLP) vaccine that elicited strong and durable immune responses in rhesus macaques, responses that cross-reacted with and neutralized all pathogenic orthoebolaviruses in GP-based Ebola pseudovirion assays. In a mouse model of Ebola virus disease (EVD), a single shot of this vaccine afforded 100% protection against a lethal dose of mouse-adapted EBOV, protection and lack of EVD signs and symptoms that were comparable to those after vaccination with the FDA-approved rVSV-ZEBOV vaccine. Strong pre-clinical data reported here pave the way for a detailed mechanistic study before advancing this promising pan-orthoebolavirus vaccine into clinical trials. The study was funded, in part, by the Intramural Research Program, NIAID, NIH. Supported by (1) Cincinnati Children’s Innovation Fund, (2) the Intramural Research Program, NIAID, NIH. Vaccines and Immunotherapy (VAC)

Background:The Sudan virus (SUDV) outbreaks in recent years including the ongoing outbreak in Uganda created a public health emergency beyond the Eastern Africa region. Currently, there are licensed countermeasures for Ebola virus (EBOV); however, there are no licensed vaccines or therapeutics against SUDV. Methods:We developed a vesicular stomatitis virus (VSV)-based vaccine expressing the SUDV glycoprotein. Cynomolgus macaques were vaccinated intramuscularly with a single dose of VSV-SUDV either one month or one week prior to SUDV challenge. A third group was vaccinated with a single dose of VSV-EBOV one month prior to SUDV challenge to assess its cross-protective potential, and a control group received an unrelated VSV-based vaccine. Results:All vaccinated nonhuman primates (NHPs) developed antigen-specific IgG within 2 weeks of vaccination, including cross-reactive responses. After challenge with a lethal dose of SUDV, all VSV-SUDV-vaccinated NHPs were uniformly protected from disease. In contrast, the VSV-EBOV-vaccinated and control NHPs succumbed to disease between day 5 and 7 after challenge presenting with classical signs of Sudan virus disease associated with high titer viremia, high viral organ load, dysregulated cytokine profiles and typical pathological changes. The humoral immune response in the NHPs vaccinated with VSV-SUDV one month before challenge resulted in a profound and sustained antibody response with a diverse functionality profile which was not observed to the same extend in NHPs vaccinated one week before challenge. Interpretation:We demonstrated that a single dose of VSV-SUDV protected NHPs from lethal SUDV infection within one week. The fast-acting nature makes VSV-SUDV an ideal countermeasure for ring vaccination during outbreaks of Sudan virus disease. In contrast, VSV-EBOV provided no relevant protection against SUDV infection in NHPs highlighting the need for species-specific filovirus vaccines.

The filovirus, Ebola virus (EBOV), causes outbreaks of EBOV disease (EVD) throughout equatorial Africa. ERVEBO is a replication-competent recombinant vesicular stomatitis virus-vectored vaccine encoding the EBOV glycoprotein (recombinant vesicular stomatitis virus [rVSV]/EBOV), which is licensed to control EVD outbreaks. EVD outbreaks occur in regions endemic for Plasmodium-caused malaria. Plasmodium infections persist due in part to the parasite's ability to evade sterilizing immunity, which also dampens immune responses to heterologous vaccines. Acute murine Plasmodium infection at the time of rVSV/EBOV vaccination reduced vaccine-mediated protection against mouse-adapted EBOV (ma-EBOV) challenge. Decreased protection was associated with a Plasmodium-induced interferon gamma-mediated decrease of rVSV/EBOV replication in lymph node macrophages, resulting in reduced primary anti-EBOV glycoprotein antibody responses. Higher doses of rVSV/EBOV partially overcame the antibody deficits and elicited protective responses. Evidence of the negative impact of Plasmodium on the efficacy of low-dose rVSV/EBOV vaccine protocols supports the use of high antigen loads in the effective management of EVD outbreaks. IMPORTANCE:We show that a blood-stage murine Plasmodium infection negatively impacts the primary antibody response elicited by low-dose recombinant vesicular stomatitis virus (rVSV)/Ebola virus (EBOV) vaccination and results in reduced protection against a lethal dose of mouse-adapted EBOV. This defect occurs within the draining lymph node due to the elevation of interferon gamma elicited in Plasmodium yoelii (Py)-infected mice. The Py-imposed decrease in vaccine-mediated protection can be overcome with higher doses of rVSV/EBOV. While the strong protection conferred by rVSV/EBOV and significant side effects known to be associated with this vaccine have led to the suggestion that the vaccine dosage be reduced, our studies provide a rationale for maintaining the current higher dose.

The filovirus Taï Forest virus (TAFV) caused a single human case of infection originating from a chimpanzee outbreak, demonstrating that humans are susceptible to TAFV infection. Existing animal disease models use intramuscular (IM) infection; however, natural filovirus infection likely occurs mucosal. We aimed to develop a ferret disease model by inoculation of TAFV by the IM, intranasal (IN), or aerosol routes. The IM group showed minimal signs of disease while IN and aerosol inoculations resulted in moderate to severe disease and partial lethality. The surviving IN or IM TAFV-infected ferrets were rechallenged IM or IN with Ebola virus (EBOV) as a pilot study assessing the cross-protection potential between these closely related viruses. Only ferrets IN-inoculated with TAFV and IN-inoculated with EBOV were protected from disease, all others succumbed to disease after EBOV infection. This data shows that ferrets are a feasible model to assess TAFV pathogenicity by mucosal exposure routes and that possible cross-protection between TAFV and EBOV may be achieved upon mucosal exposure.

Sudan virus (SUDV) causes highly lethal outbreaks of hemorrhagic disease throughout Africa, but there has yet to be an approved vaccine or therapeutic to combat this public health threat. The most common route of natural exposure to filoviruses is through mucosal contact which greatly impacts initial viral replication. Historically, SUDV animal models used an intramuscular infection route. Here, we sought to further characterize an animal model using mucosal challenge routes and compared the impact that intramuscular, intranasal, or aerosol exposure had on SUDV pathogenicity in a ferret model. We determined that the route of infection did not significantly impact overall SUDV pathogenicity; only subtle changes were detected in magnitude of viremia and oral viral shedding. Additionally, we sought to determine if preexisting Lloviu virus (LLOV) immunity could protect ferrets from lethal SUDV infection. We found that the previous immunity elicited by LLOV infection was not sufficient to protect ferrets from lethal SUDV disease. In conclusion, our results indicate that the infection route has minimal effect on overall pathogenicity of SUDV in ferrets and that prior LLOV infection does not elicit a cross-protective immune response to SUDV.

Vesicular stomatitis virus (VSV)-based vaccination has shown protective efficacy against filovirus infection. Following the approval of a VSV-based vaccine against Ebola virus, there have been efforts toward applying the same platform for other filoviruses, including Marburg virus (MARV) and Sudan virus. Because these vaccines express filovirus glycoproteins, they are also a valuable tool to study filovirus entry under biosafety level 2 conditions. In the protocol described below, we outline how to genetically manipulate a full-length VSV vector by removing the native VSV glycoprotein and replacing it with the surface-expressed MARV glycoprotein. In addition, we describe the recovery procedure of these recombinant, full-length VSVs and detail the necessary steps of virus propagation.

Filoviruses continue to re-emerge in Africa, causing localized public health emergencies. Although vaccination has slowly been implemented for Ebola virus, not for other filoviruses, holistic approaches are needed to broadly protect against filovirus threats.