Last data update: Nov 04, 2024. (Total: 48056 publications since 2009)
Records 1-30 (of 153 Records) |
Query Trace: Spiropoulou CF[original query] |
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A public, cross-reactive glycoprotein epitope confounds Ebola virus serology
Kainulainen MH , Harmon JR , Karaaslan E , Kyondo J , Whitesell A , Twongyeirwe S , Malenfant JH , Baluku J , Kofman A , Bergeron É , Waltenburg MA , Nyakarahuka L , Balinandi S , Cossaboom CM , Choi MJ , Shoemaker TR , Montgomery JM , Spiropoulou CF . J Med Virol 2024 96 (10) e29946 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. |
Optimization of Bangladesh and Malaysian genotype recombinant reporter Nipah viruses for in vitro antiviral screening and in vivo disease modeling
Lo MK , Jain S , Davies KA , Sorvillo TE , Welch SR , Coleman-McCray JD , Chatterjee P , Hotard AL , O'Neal T , Flint M , Ai H , Albariño CG , Spengler JR , Montgomery JM , Spiropoulou CF . Antiviral Res 2024 231 106013 Nipah virus (NiV) causes near-annual outbreaks of fatal encephalitis and respiratory disease in South Asia with a high mortality rate (∼70%). Since there are no approved therapeutics for NiV disease in humans, the WHO has designated NiV and henipaviral diseases priority pathogens for research and development. We generated a new recombinant green fluorescent reporter NiV of the circulating Bangladesh genotype (rNiV-B-ZsG) and optimized it alongside our previously generated Malaysian genotype reporter counterpart (rNiV-M-ZsG) for antiviral screening in primary-like human respiratory cell types. Validating our platform for rNiV-B-ZsG with a synthetic compound library directed against viral RNA-dependent RNA polymerases, we identified a hit compound and confirmed its sub-micromolar activity against wild-type NiV, green fluorescent reporter, and the newly constructed bioluminescent red fluorescent double reporter (rNiV-B-BREP) NiV. We furthermore demonstrated that rNiV-B-ZsG and rNiV-B-BREP viruses showed pathogenicity comparable to wild-type NiV-B in the Syrian golden hamster model of disease, supporting additional use of these tools for both pathogenesis and advanced pre-clinical studies in vivo. |
Streamlined detection of Nipah virus antibodies using a split nanoluc biosensor
Bergeron É , Chiang CF , Lo MK , Karaaslan E , Satter SM , Rahman MZ , Hossain ME , Aquib WR , Rahman DI , Sarwar SB , Montgomery JM , Klena JD , Spiropoulou CF . Emerg Microbes Infect 2024 2398640 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. |
Crimean Congo hemorrhagic fever virus nucleoprotein and GP38 subunit vaccine combination prevents morbidity in mice
Karaaslan E , Sorvillo TE , Scholte FEM , O'Neal TJ , Welch SR , Davies KA , Coleman-McCray JD , Harmon JR , Ritter JM , Pegan SD , Montgomery JM , Spengler JR , Spiropoulou CF , Bergeron É . NPJ Vaccines 2024 9 (1) 148 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. |
Measures to prevent and treat Nipah virus disease: research priorities for 2024-29
Moore KA , Mehr AJ , Ostrowsky JT , Ulrich AK , Moua NM , Fay PC , Hart PJ , Golding JP , Benassi V , Preziosi MP , Broder CC , de Wit E , Formenty PBH , Freiberg AN , Gurley ES , Halpin K , Luby SP , Mazzola LT , Montgomery JM , Spiropoulou CF , Mourya DT , Parveen S , Rahman M , Roth C , Wang LF , Osterholm MT . Lancet Infect Dis 2024 Nipah virus causes highly lethal disease, with case-fatality rates ranging from 40% to 100% in recognised outbreaks. No treatments or licensed vaccines are currently available for the prevention and control of Nipah virus infection. In 2019, WHO published an advanced draft of a research and development roadmap for accelerating development of medical countermeasures, including diagnostics, therapeutics, and vaccines, to enable effective and timely emergency response to Nipah virus outbreaks. This Personal View provides an update to the WHO roadmap by defining current research priorities for development of Nipah virus medical countermeasures, based primarily on literature published in the last 5 years and consensus opinion of 15 subject matter experts with broad experience in development of medical countermeasures for Nipah virus or experience in the epidemiology, ecology, or public health control of outbreaks of Nipah virus. The research priorities are organised into four main sections: cross-cutting issues (for those that apply to more than one category of medical countermeasures), diagnostics, therapeutics, and vaccines. The strategic goals and milestones identified in each section focus on key achievements that are needed over the next 6 years to ensure that the necessary tools are available for rapid response to future outbreaks of Nipah virus or related henipaviruses. |
A pan-respiratory antiviral chemotype targeting a transient host multi-protein complex
Michon M , Müller-Schiffmann A , Lingappa AF , Yu SF , Du L , Deiter F , Broce S , Mallesh S , Crabtree J , Lingappa UF , Macieik A , Müller L , Ostermann PN , Andrée M , Adams O , Schaal H , Hogan RJ , Tripp RA , Appaiah U , Anand SK , Campi TW , Ford MJ , Reed JC , Lin J , Akintunde O , Copeland K , Nichols C , Petrouski E , Moreira AR , Jiang IT , DeYarman N , Brown I , Lau S , Segal I , Goldsmith D , Hong S , Asundi V , Briggs EM , Phyo NS , Froehlich M , Onisko B , Matlack K , Dey D , Lingappa JR , Prasad DM , Kitaygorodskyy A , Solas D , Boushey H , Greenland J , Pillai S , Lo MK , Montgomery JM , Spiropoulou CF , Korth C , Selvarajah S , Paulvannan K , Lingappa VR . Open Biol 2024 14 (6) 230363 We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease. |
Identification of a macrocyclic compound targeting the Lassa virus polymerase
Aida-Ficken V , Kelly JA , Chatterjee P , Jenks MH , McMullan LK , Albariño CG , Montgomery JM , Seley-Radtke KL , Spiropoulou CF , Flint M . Antiviral Res 2024 105923 There are no approved vaccines or therapeutics for Lassa virus (LASV) infections. To identify compounds with anti-LASV activity, we conducted a cell-based screening campaign at biosafety level 4 and tested almost 60,000 compounds for activity against an infectious reporter LASV. Hits from this screen included several structurally related macrocycles. The most potent, Mac128, had a sub-micromolar EC(50) against the reporter virus, inhibited wild-type clade IV LASV, and reduced viral titers by 4 orders of magnitude. Mechanistic studies suggested that Mac128 inhibited viral replication at the level of the polymerase. |
Replicon particle vaccination induces non-neutralizing anti-nucleoprotein antibody-mediated control of Crimean-Congo hemorrhagic fever virus
Sorvillo TE , Karaaslan E , Scholte FEM , Welch SR , Coleman-McCray JD , Genzer SC , Ritter JM , Hayes HM , Jain S , Pegan SD , Bergeron É , Montgomery JM , Spiropoulou CF , Spengler JR . NPJ Vaccines 2024 9 (1) 88 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. |
Evaluation of two inoculation routes of an adenovirus-mediated viral protein inhibitor in a Crimean-Congo hemorrhagic fever mouse model
Scholte FEM , Spengler JR , Welch SR , Harmon JR , Coleman-McCray JD , Davies KA , Pegan SD , Montgomery JM , Spiropoulou CF , Bergeron É . Virus Res 2024 345 199398 Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne nairovirus with a wide geographic spread that can cause severe and lethal disease. No specific medical countermeasures are approved to combat this illness. The CCHFV L protein contains an ovarian tumor (OTU) domain with a cysteine protease thought to modulate cellular immune responses by removing ubiquitin and ISG15 post-translational modifications from host and viral proteins. Viral deubiquitinases like CCHFV OTU are attractive drug targets, as blocking their activity may enhance cellular immune responses to infection, and potentially inhibit viral replication itself. We previously demonstrated that the engineered ubiquitin variant CC4 is a potent inhibitor of CCHFV replication in vitro. A major challenge of the therapeutic use of small protein inhibitors such as CC4 is their requirement for intracellular delivery, e.g., by viral vectors. In this study, we examined the feasibility of in vivo CC4 delivery by a replication-deficient recombinant adenovirus (Ad-CC4) in a lethal CCHFV mouse model. Since the liver is a primary target of CCHFV infection, we aimed to optimize delivery to this organ by comparing intravenous (tail vein) and intraperitoneal injection of Ad-CC4. While tail vein injection is a traditional route for adenovirus delivery, in our hands intraperitoneal injection resulted in higher and more widespread levels of adenovirus genome in tissues, including, as intended, the liver. However, despite promising in vitro results, neither route of in vivo CC4 treatment resulted in protection from a lethal CCHFV infection. |
Examination of SARS-CoV-2 serological test results from multiple commercial and laboratory platforms with an in-house serum panel
Lester SN , Stumpf M , Freeman BD , Mills L , Schiffer J , Semenova V , Jia T , Desai R , Browning P , Alston B , Ategbole M , Bolcen S , Chen A , David E , Manitis P , Tatum H , Qin Y , Zellner B , Drobeniuc J , Tejada-Strop A , Chatterjee P , Shrivastava-Ranjan P , Jenks MH , McMullan LK , Flint M , Spiropoulou CF , Niemeyer GP , Werner BJ , Bean CJ , Johnson JA , Hoffmaster AR , Satheshkumar PS , Schuh AJ , Owen SM , Thornburg NJ . Access Microbiol 2024 6 (2) Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a novel human coronavirus that was identified in 2019. SARS-CoV-2 infection results in an acute, severe respiratory disease called coronavirus disease 2019 (COVID-19). The emergence and rapid spread of SARS-CoV-2 has led to a global public health crisis, which continues to affect populations across the globe. Real time reverse transcription polymerase chain reaction (rRT-PCR) is the reference standard test for COVID-19 diagnosis. Serological tests are valuable tools for serosurveillance programs and establishing correlates of protection from disease. This study evaluated the performance of one in-house enzyme linked immunosorbent assay (ELISA) utilizing the pre-fusion stabilized ectodomain of SARS-CoV-2 spike (S), two commercially available chemiluminescence assays Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack and Abbott SARS-CoV-2 IgG assay and one commercially available Surrogate Virus Neutralization Test (sVNT), GenScript USA Inc., cPass SARS-CoV-2 Neutralization Antibody Detection Kit for the detection of SARS-CoV-2 specific antibodies. Using a panel of rRT-PCR confirmed COVID-19 patients' sera and a negative control group as a reference standard, all three immunoassays demonstrated high comparable positivity rates and low discordant rates. All three immunoassays were highly sensitive with estimated sensitivities ranging from 95.4-96.6 %. ROC curve analysis indicated that all three immunoassays had high diagnostic accuracies with area under the curve (AUC) values ranging from 0.9698 to 0.9807. High positive correlation was demonstrated among the conventional microneutralization test (MNT) titers and the sVNT inhibition percent values. Our study indicates that independent evaluations are necessary to optimize the overall utility and the interpretation of the results of serological tests. Overall, we demonstrate that all serological tests evaluated in this study are suitable for the detection of SARS-CoV-2 antibodies. |
Characterization of humoral responses to Nipah virus infection in the Syrian Hamster model of disease
Scholte FEM , Rodriguez SE , Welch SR , Davies KA , Genzer SC , Coleman-McCray JD , Harmon JR , Sorvillo TE , Lo MK , Karaaslan E , Bergeron E , Montgomery JM , Spengler JR , Spiropoulou CF . J Infect Dis 2023 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. |
Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses
Davies KA , Welch SR , Sorvillo TE , Coleman-McCray JD , Martin ML , Brignone JM , Montgomery JM , Spiropoulou CF , Spengler JR . Sci Rep 2023 13 (1) 19384 Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on normalization using a suitable reference gene (RG) to minimize effects of variation due to sample collection, sample processing, or experimental set-up. Here, we investigated the suitability of nine potential RGs in laboratory animals commonly used to study viral hemorrhagic fever infection. Using tissues (liver, spleen, gonad [ovary or testis], kidney, heart, lung, eye, brain, and blood) collected from naïve animals and those infected with Crimean-Congo hemorrhagic fever (mice), Nipah (hamsters), or Lassa (guinea pigs) viruses, optimal species-specific RGs were identified based on five web-based algorithms to assess RG stability. Notably, the Ppia RG demonstrated stability across all rodent tissues tested. Optimal RG pairs that include Ppia were determined for each rodent species (Ppia and Gusb for mice; Ppia and Hrpt for hamsters; and Ppia and Gapdh for guinea pigs). These RG pair assays were multiplexed with viral targets to improve assay turnaround time and economize sample usage. Finally, a pan-rodent Ppia assay capable of detecting Ppia across multiple rodent species was developed and successfully used in ecological investigations of field-caught rodents, further supporting its pan-species utility. |
Filoviruses: Scientific gaps and prototype pathogen recommendation
Dupuy LC , Spiropoulou CF , Towner JS , Spengler JR , Sullivan NJ , Montgomery JM . J Infect Dis 2023 228 S446-s459 Viruses in the family Filoviridae, including the commonly known Ebola (EBOV) and Marburg (MARV) viruses, can cause severe hemorrhagic fever in humans and nonhuman primates. Sporadic outbreaks of filovirus disease occur in sub-Saharan Africa with reported case fatality rates ranging from 25% to 90%. The high mortality and increasing frequency and magnitude of recent outbreaks along with the increased potential for spread from rural to urban areas highlight the importance of pandemic preparedness for these viruses. Despite their designation as high-priority pathogens, numerous scientific gaps exist in critical areas. In this review, these gaps and an assessment of potential prototype pathogen candidates are presented for this important virus family. |
Recombinant Sudan virus and evaluation of humoral cross-reactivity between Ebola and Sudan virus glycoproteins after infection or rVSV-ΔG-ZEBOV-GP vaccination
Kainulainen MH , Harmon JR , Whitesell AN , Bergeron E , Karaaslan E , Cossaboom CM , Malenfant JH , Kofman A , Montgomery JM , Choi MJ , Albariño CG , Spiropoulou CF . Emerg Microbes Infect 2023 12 (2) 2265660 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. |
Development of reverse genetic tools to study Chapare and Machupo viruses
Jain S , Shrivastava-Ranjan P , Flint M , Montgomery JM , Spiropoulou CF , Albariño CG . Virology 2023 588 109888 Arenaviruses are highly pathogenic viruses that pose a serious public health threat. Chapare virus (CHAV) and Machupo virus (MACV), two New World arenaviruses, cause hemorrhagic fevers with case fatality rates of up to 45%. Research on therapeutic drug targets and vaccines for these viruses is limited because biosafety level 4 containment is required for handling them. In this study, we developed reverse genetics systems, including minigenomes and recombinant viruses, that will facilitate the study of these pathogens. The minigenome system is based on the S segment of CHAV or MACV genomes expressing the fluorescent reporter gene ZsGreen (ZsG). We also generated recombinant CHAV and MACV with and without the ZsG reporter gene. As a proof-of-concept study, we used both minigenomes and recombinant viruses to test the inhibitory effects of previously reported antiviral compounds. The new reverse genetics system described here will facilitate future therapeutic studies for these two life-threatening arenaviruses. |
Enhanced broad spectrum in vitro antiviral efficacy of 3-F-4-MeO-Bn, 3-CN, and 4-CN derivatives of lipid remdesivir nucleoside monophosphate prodrugs
McMillan RE , Lo MK , Zhang XQ , Beadle JR , Valiaeva N , Garretson AF , Clark AE , Freshman J , Murphy J , Montgomery JM , Spiropoulou CF , Schooley RT , Hostetler KY , Carlin AF . Antiviral Res 2023 219 105718 Broad spectrum oral antivirals are urgently needed for the early treatment of many RNA viruses of clinical concern. We previously described the synthesis of 1-O-octadecyl-2-O-benzyl-glycero-3-phospho-RVn (V2043), an orally bioavailable lipid prodrug of remdesivir nucleoside (RVn, GS-441524) with broad spectrum antiviral activity against viruses with pandemic potential. Here we compared the relative activity of V2043 with new RVn lipid prodrugs containing sn-1 alkyl ether or sn-2 glycerol modifications. We found that 3-F-4-MeO-Bn, 3-CN-Bn, and 4-CN-Bn sn-2 glycerol modifications improved antiviral activity compared to V2043 when tested in vitro against clinically important RNA viruses from 5 virus families. These results support the continued development of V2043 and sn-2 glycerol modified RVn lipid prodrugs for the treatment of a broad range of RNA viruses for which there are limited therapies. |
Vaccination with the Crimean-Congo hemorrhagic fever virus viral replicon vaccine induces NP-based T-cell activation and antibodies possessing Fc-mediated effector functions
Scholte FEM , Karaaslan E , O'Neal TJ , Sorvillo TE , Genzer SC , Welch SR , Coleman-McCray JD , Spengler JR , Kainulainen MH , Montgomery JM , Pegan SD , Bergeron E , Spiropoulou CF . Front Cell Infect Microbiol 2023 13 1233148 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. |
Perspectives on advancing countermeasures for filovirus disease: Report from a multi-sector meeting
Sprecher A , Cross R , Marzi A , Martins KA , Wolfe D , Montgomery JM , Spiropoulou CF , Cihlar T , Ahuka-Mundeke S , Nyhuis T , Teicher C , Crozier I , Strong J , Kobinger G , Woolsey C , Geisbert TW , Feldmann H , Muyembe JJ . J Infect Dis 2023 228 S474-S478 Although there are now approved treatments and vaccines for Ebola virus disease (EVD), the case fatality of EVD remains unacceptably high even when treated with the newly approved therapeutics; furthermore, these countermeasures are not expected to be effective against disease caused by other filoviruses. A meeting of subject matter experts from public health, research, and countermeasure development agencies and manufacturers was held during the 10th International Filovirus Symposium to discuss strategies to address these gaps, including how newer countermeasures could be advanced for field readiness. Several investigational therapeutics, vaccine candidates, and combination strategies were presented. In all, a common theme emerged: the greatest challenge to completing development was the implementation of well-designed clinical trials of safety and efficacy during filovirus disease outbreaks. These outbreaks are usually of short duration, providing but a brief opportunity for trials to be launched, and have too few cases to allow for full enrollment during a single outbreak, so clinical trials will necessarily need to span multiple outbreaks which may occur in a number of at-risk countries. Preparing for this will require agreed-upon common protocols for trials intended to bridge multiple outbreaks across all at-risk countries. A multi-national research consortium including, and led by, at-risk countries would be an ideal mechanism to negotiate agreement on protocol design and coordinate preparation. Discussion participants recommended a follow-up meeting be held in Africa with national public health and research agencies from at-risk countries to establish such a consortium. |
Single-dose mucosal replicon-particle vaccine protects against lethal Nipah virus infection up to 3 days after vaccination
Welch SR , Spengler JR , Genzer SC , Coleman-McCray JD , Harmon JR , Sorvillo TE , Scholte FEM , Rodriguez SE , O'Neal TJ , Ritter JM , Ficarra G , Davies KA , Kainulainen MH , Karaaslan E , Bergeron É , Goldsmith CS , Lo MK , Nichol ST , Montgomery JM , Spiropoulou CF . Sci Adv 2023 9 (31) eadh4057 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. |
Development of a neutralization assay using a vesicular stomatitis virus expressing Nipah virus glycoprotein and a fluorescent protein
Jain S , Lo MK , Kainulainen MH , Welch SR , Spengler JR , Satter SM , Rahman MZ , Hossain ME , Chiang CF , Klena JD , Bergeron É , Montgomery JM , Spiropoulou CF , Albariño CG . Virology 2023 587 109858 Nipah virus (NiV) is a highly pathogenic paramyxovirus with a high case fatality rate. Due to its high pathogenicity, pandemic potential, and lack of therapeutics or approved vaccines, its study requires biosafety level 4 (BSL4) containment. In this report, we developed a novel neutralization assay for use in biosafety level 2 laboratories. The assay uses a recombinant vesicular stomatitis virus expressing NiV glycoprotein and a fluorescent protein. The recombinant virus propagates as a replication-competent virus in a cell line constitutively expressing NiV fusion protein, but it is restricted to a single round of replication in wild-type cells. We used this system to evaluate the neutralization activity of monoclonal and polyclonal antibodies, plasma from NiV-infected hamsters, and serum from human patients. Therefore, this recombinant virus could be used as a surrogate for using pathogenic NiV and may constitute a powerful tool to develop therapeutics in low containment laboratories. |
Sustained replication of synthetic canine distemper virus defective genomes in vitro and in vivo (preprint)
Tilston-Lunel NL , Welch SR , Nambulli S , de Vries RD , Ho GW , Wentworth DE , Shabman R , Nichol ST , Spiropoulou CF , de Swart RL , Rennick LJ , Duprex WP . bioRxiv 2021 2021.06.11.448162 Defective interfering (DI) genomes restrict viral replication and induce type-I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity and determine their long-term effects. To address this, we generated a recombinant (r) canine distemper virus (CDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues which are a major site of CDV replication. Our established pipeline for detection, generation and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome.Importance Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes could be harnessed therapeutically. Negative strand RNA virus research has provided a rich pool of natural DI genomes over many years and they are probably the best understood in vitro. Here, we demonstrate identification, synthesis, production and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection. |
High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay (preprint)
Kainulainen MH , Bergeron E , Chatterjee P , Chapman AP , Lee J , Chida A , Tang X , Wharton RE , Mercer KB , Petway M , Jenks HM , Flietstra TD , Schuh AJ , Satheshkumar PS , Chaitram JM , Owen SM , Finn MG , Goldstein JM , Montgomery JM , Spiropoulou CF . medRxiv 2020 2020.09.16.20195446 SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.One sentence summary Protein complementation enables mix-and-read SARS-CoV-2 serology that rivals sensitivity and specificity of ELISA but excels in throughput and quantitation.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThis research was funded by the Centers for Disease Control and Prevention.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Residual specimen materials were used for diagnostics development under a protocol that was reviewed and approved by the CDC Institutional Review Board (See 45 C.F.R. part 46; 21 C.F.R. part 56)All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesNo external data links |
A Pan-respiratory Antiviral Chemotype Targeting a Transient Host Multiprotein Complex (preprint)
Muller-Schiffmann A , Michon M , Lingappa AF , Yu SF , Du L , Deiter F , Broce S , Mallesh S , Crabtree J , Lingappa UF , Macieik A , Muller L , Ostermann PN , Andree M , Adams O , Schaal H , Hogan RJ , Tripp RA , Appaiah U , Anand SK , Campi TW , Ford MJ , Reed JC , Lin J , Akintunde O , Copeland K , Nichols C , Petrouski E , Moreira AR , Jiang IT , DeYarman N , Brown I , Lau S , Segal I , Goldsmith D , Hong S , Asundi V , Briggs EM , Phyo NS , Froehlich M , Onisko B , Matlack K , Dey D , Lingappa JR , Prasad MD , Kitaygorodskyy A , Solas D , Boushey H , Greenland J , Pillai S , Lo MK , Montgomery JM , Spiropoulou CF , Korth C , Selvarajah S , Paulvannan K , Lingappa VR . bioRxiv 2021 18 We present a small molecule chemotype, identified by an orthogonal drug screen, exhibiting nanomolar activity against members of all the six viral families causing most human respiratory viral disease, with a demonstrated barrier to resistance development. Antiviral activity is shown in mammalian cells, including human primary bronchial epithelial cells cultured to an air-liquid interface and infected with SARS-CoV-2. In animals, efficacy of early compounds in the lead series is shown by survival (for a coronavirus) and viral load (for a paramyxovirus). The drug target is shown to include a subset of the protein 14-3-3 within a transient host multi-protein complex containing components implicated in viral lifecycles and in innate immunity. This multi-protein complex is modified upon viral infection and largely restored by drug treatment. Our findings suggest a new clinical therapeutic strategy for early treatment upon upper respiratory viral infection to prevent progression to lower respiratory tract or systemic disease. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. |
Differential neutralization and inhibition of SARS-CoV-2 variants by antibodies elicited by COVID-19 mRNA vaccines (preprint)
Wang L , Kainulainen MH , Jiang N , Di H , Bonenfant G , Mills L , Currier M , Shrivastava-Ranjan P , Calderon BM , Sheth M , Hossain J , Lin X , Lester S , Pusch E , Jones J , Cui D , Chatterjee P , Jenks HM , Morantz E , Larson G , Hatta M , Harcourt J , Tamin A , Li Y , Tao Y , Zhao K , Burroughs A , Wong T , Tong S , Barnes JR , Tenforde MW , Self WH , Shapiro NI , Exline MC , Files DC , Gibbs KW , Hager DN , Patel M , Laufer Halpin AS , Lee JS , Xie X , Shi PY , Davis CT , Spiropoulou CF , Thornburg NJ , Oberste MS , Dugan V , Wentworth DE , Zhou B , Batra D , Beck A , Caravas J , Cintron-Moret R , Cook PW , Gerhart J , Gulvik C , Hassell N , Howard D , Knipe K , Kondor RJ , Kovacs N , Lacek K , Mann BR , McMullan LK , Moser K , Paden CR , Martin BR , Schmerer M , Shepard S , Stanton R , Stark T , Sula E , Tymeckia K , Unoarumhi Y . bioRxiv 2021 30 The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of many new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccines. To ascertain and rank the risk of VOCs and VOIs, we analyzed their ability to escape from vaccine-induced antibodies. The variants showed differential reductions in neutralization and replication titers by post-vaccination sera. Although the Omicron variant showed the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retained moderate neutralizing activity against that variant. Therefore, vaccination remains the most effective strategy to combat the COVID-19 pandemic. |
Immunogenicity of poxvirus-based vaccines against Nipah virus
Medina-Magües ES , Lopera-Madrid J , Lo MK , Spiropoulou CF , Montgomery JM , Medina-Magües LG , Salas-Quinchucua C , Jiménez-Mora AP , Osorio JE . Sci Rep 2023 13 (1) 11384 Nipah virus (NiV), an emerging zoonotic pathogen in Southeast Asia, is transmitted from Pteropus species of fruit bats to a wide range of species, including humans, pigs, horses, dogs, and cats. NiV has killed millions of animals and caused highly fatal human outbreaks since no vaccine is commercially available. This study characterized the immunogenicity and safety of poxvirus-based Nipah vaccines that can be used in humans and species responsible for NiV transmission. Mice were vaccinated with modified vaccinia Ankara (MVA) and raccoon pox (RCN) viral vectors expressing the NiV fusion (F) and glycoprotein (G) proteins subcutaneously (SC) and intranasally (IN). Importantly, both vaccines did not induce significant weight loss or clinical signs of disease while generating high circulating neutralizing antibodies and lung-specific IgG and IgA responses. The MVA vaccine saw high phenotypic expression of effector and tissue resident memory CD8ɑ(+) T cells in lungs and splenocytes along with the expression of central memory CD8ɑ(+) T cells in lungs. The RCN vaccine generated effector memory (SC) and tissue resident (IN) CD8ɑ(+) T cells in splenocytes and tissue resident (IN) CD8ɑ(+) T cells in lung cells. These findings support MVA-FG and RCN-FG viral vectors as promising vaccine candidates to protect humans, domestic animals, and wildlife from fatal disease outcomes and to reduce the global threat of NiV. |
Late remdesivir treatment initiation partially protects African green monkeys from lethal Nipah virus infection
de Wit E , Williamson BN , Feldmann F , Goldin K , Lo MK , Okumura A , Lovaglio J , Bunyan E , Porter DP , Cihlar T , Saturday G , Spiropoulou CF , Feldmann H . Antiviral Res 2023 216 105658 Remdesivir is a nucleotide prodrug with preclinical efficacy against lethal Nipah virus infection in African green monkeys when administered 1 day post inoculation (dpi) (Lo et al., 2019). Here, we determined whether remdesivir treatment was still effective when treatment administration initiation was delayed until 3 dpi. Three groups of six African green monkeys were inoculated with a lethal dose of Nipah virus, genotype Bangladesh. On 3 dpi, one group received a loading dose of 10 mg/kg remdesivir followed by daily dosing with 5 mg/kg for 11 days, one group received 10 mg/kg on 12 consecutive days, and the remaining group received an equivalent volume of vehicle solution. Remdesivir treatment initiation on 3 dpi provided partial protection from severe Nipah virus disease that was dose dependent, with 67% of animals in the high dose group surviving the challenge. However, remdesivir treatment did not prevent clinical disease, and surviving animals showed histologic lesions in the brain. Thus, early administration seems critical for effective remdesivir treatment during Nipah virus infection. |
Fluorescent and bioluminescent reporter mouse-adapted Ebola viruses maintain pathogenicity and can be visualized in vivo
Davies KA , Welch SR , Jain S , Sorvillo TE , Coleman-McCray JD , Montgomery JM , Spiropoulou CF , Albariño C , Spengler JR . J Infect Dis 2023 228 S536-S547 Ebola virus (EBOV) causes lethal disease in humans but not in mice. Here, we generated recombinant mouse-adapted (MA)-EBOVs, including one based on the previously reported serially adapted strain (rMA-EBOV), along with single-reporter rMA-EBOVs expressing either fluorescent (ZsGreen1 [ZsG]) or bioluminescent (nano-luciferase [nLuc]) reporters, and dual-reporter rMA-EBOVs expressing both ZsG and nLuc. No detriment to viral growth in vitro was seen with inclusion of MA-associated mutations or reporter proteins. In CD-1 mice, infection with MA-EBOV, rMA-EBOV, and single-reporter rMA-EBOVs conferred 100% lethality; infection with dual-reporter rMA-EBOV resulted in 80% lethality. Bioluminescent signal from rMA-EBOV expressing nLuc was detected in vivo and ex vivo using the IVIS Spectrum CT. Fluorescent signal from rMA-EBOV expressing ZsG was detected in situ using hand-held blue-light transillumination and ex vivo through epi-illumination with the IVIS Spectrum CT. These data support the use of reporter MA-EBOV for studies of Ebola virus in animal disease models. |
Development of a novel minigenome and recombinant VSV expressing Seoul hantavirus glycoprotein-based assays to identify anti-hantavirus therapeutics
Shrivastava-Ranjan P , Jain S , Chatterjee P , Montgomery JM , Flint M , Albariño C , Spiropoulou CF . Antiviral Res 2023 214 105619 Seoul virus (SEOV) is an emerging global health threat that can cause hemorrhagic fever with renal syndrome (HFRS), which results in case fatality rates of ∼2%. There are no approved treatments for SEOV infections. We developed a cell-based assay system to identify potential antiviral compounds for SEOV and generated additional assays to characterize the mode of action of any promising antivirals. To test if candidate antivirals targeted SEOV glycoprotein-mediated entry, we developed a recombinant reporter vesicular stomatitis virus expressing SEOV glycoproteins. To facilitate the identification of candidate antiviral compounds targeting viral transcription/replication, we successfully generated the first reported minigenome system for SEOV. This SEOV minigenome (SEOV-MG) screening assay will also serve as a prototype assay for discovery of small molecules inhibiting replication of other hantaviruses, including Andes and Sin Nombre viruses. Ours is a proof-of-concept study in which we tested several compounds previously reported to have activity against other negative-strand RNA viruses using our newly developed hantavirus antiviral screening systems. These systems can be used under lower biocontainment conditions than those needed for infectious viruses, and identified several compounds with robust anti-SEOV activity. Our findings have important implications for the development of anti-hantavirus therapeutics. |
Mouse models of Ebola virus tolerance and lethality: Characterization of CD-1 mice infected with wild-type, guinea pig-adapted, or mouse-adapted variants
Spengler JR , Welch SR , Ritter JM , Harmon JR , Coleman-McCray JD , Genzer SC , Nascimento Seixas J , Scholte FEM , Davies KA , Bradfute SB , Montgomery JM , Spiropoulou CF . Antiviral Res 2022 210 105496 Development of lethal models of Ebola virus disease has been achieved by the serial passage of virus isolates from human cases in mice and guinea pigs. Use of mice infected with non-adapted virus has been limited due to the absence of overt clinical disease. In recent years, newly recognized sequelae identified in human cases has highlighted the importance of continued investigations of non-lethal infection both in humans and animal models. Here, we revisit the use of rodent-adapted and non-adapted Ebola virus (EBOV) variants in mice to investigate infection tolerance and future utility of these models in pathogenesis and therapeutic intervention studies. We found that like non-adapted wild-type EBOV, guinea pig-adapted EBOV results in widespread tissue infection, variably associated with tissue pathology, and alterations in clinical and immunological analytes in the absence of overt disease. Notably, infection with either non-lethal variant does not greatly differ from lethal mouse-adapted EBOV until near the time end-point criteria are reached in these mice, supporting use of these models of virus tolerance for continued investigations of non-lethal infection and sequelae. |
Tissue replication and mucosal swab detection of Sosuga virus in Syrian hamsters in the absence of overt tissue pathology and clinical disease
Welch SR , Ritter JM , Schuh AJ , Genzer SC , Sorvillo TE , Harmon JR , Coleman-McCray JD , Jain S , Shrivastava-Ranjan P , Seixas JN , Estetter LB , Fair PS , Towner JS , Montgomery JM , Albariño CG , Spiropoulou CF , Spengler JR . Antiviral Res 2022 209 105490 Human infection with Sosuga virus (SOSV), a recently discovered pathogenic paramyxovirus, has been reported in one individual to date. No animal models of disease are currently available for SOSV. Here, we describe initial characterization of experimental infection in Syrian hamsters, including kinetics of virus dissemination and replication, and the corresponding clinical parameters, immunological responses, and histopathology. We demonstrate susceptibility of hamsters to infection in the absence of clinical signs or significant histopathologic findings in tissues. |
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