RESUMO
Venezuelan (VEE), eastern (EEE), and western (WEE) equine encephalitis viruses are encephalitic New World alphaviruses that cause periodic epizootic and epidemic outbreaks in horses and humans that may cause severe morbidity and mortality. Currently there are no FDA-licensed vaccines or effective antiviral therapies. Each year, there are a limited number of human cases of encephalitic alphaviruses; thus, licensure of a vaccine or therapeutic would require approval under the FDA animal rule. Approval under the FDA animal rule requires the disease observed in the animal model to recapitulate what is observed in humans. Currently, initial testing of vaccines and therapeutics is performed in the mouse model. Unfortunately, alphavirus disease manifestations in a mouse do not faithfully recapitulate human disease; the VEEV mouse model is lethal whereas in humans VEEV is rarely lethal. In an effort to identify a more appropriate small animal model, we evaluated hamsters in an aerosol exposure model of encephalitic alphavirus infection. The pathology, lethality, and viremia observed in the infected hamsters was inconsistent with what is observed in NHP models and humans. These data suggest that hamsters are not an appropriate model for encephalitic alphaviruses to test vaccines or potential antiviral therapies.
RESUMO
Venezuelan equine encephalitis virus (VEEV) outbreaks occur sporadically. Additionally, VEEV has a history of development as a biothreat agent. Yet, no FDA-approved vaccine or therapeutic exists for VEEV disease. The sporadic outbreaks present a challenge for testing medical countermeasures (MCMs) in humans; therefore, well-defined animal models are needed for FDA Animal Rule licensure. The cynomolgus macaque (CM) model has been studied extensively at high challenge doses of the VEEV Trinidad donkey strain (>1.0 × 108 plaque-forming units [PFU]), doses that are too high to be a representative human dose. Based on viremia of two subtypes of VEEV, IC, and IAB, we found the CM infectious dose fifty (ID50) to be low, 12 PFU, and 6.7 PFU, respectively. Additionally, we characterized the pattern of three clinical parameters (viremia, temperature, and lymphopenia) across a range of doses to identify a challenge dose producing consistent signs of infection. Based on these studies, we propose a shift to using a lower challenge dose of 1.0 × 103 PFU in the aerosol CM model of VEEV disease. At this dose, NHPs had the highest viremia, demonstrated a fever response, and had a measurable reduction in complete lymphocyte counts-biomarkers that can demonstrate MCM efficacy.
Assuntos
Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Vacinas Virais , Animais , Cavalos , Humanos , Macaca fascicularis , Viremia/tratamento farmacológico , Modelos Animais de DoençasRESUMO
The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.B79) is essential for viral replication. High throughput in silico/in vitro screening using a focused set of known cysteine protease inhibitors identified two epoxysuccinyl prodrugs, E64d and CA074 methyl ester (CA074me) and a reversible oxindole inhibitor. Here, we determined the X-ray crystal structure of the CA074-inhibited nsP2 protease and compared it with our E64d-inhibited structure. We found that the two inhibitors occupy different locations in the protease. We designed hybrid inhibitors with improved potency. Virus yield reduction assays confirmed that the viral titer was reduced by >5 logs with CA074me. Cell-based assays showed reductions in viral replication for CHIKV, VEEV, and WEEV, and weaker inhibition of EEEV by the hybrid inhibitors. The most potent was NCGC00488909-01 which had an EC50 of 1.76 µM in VEEV-Trd-infected cells; the second most potent was NCGC00484087 with an EC50 = 7.90 µM. Other compounds from the NCATS libraries such as the H1 antihistamine oxatomide (>5-log reduction), emetine, amsacrine an intercalator (NCGC0015113), MLS003116111-01, NCGC00247785-13, and MLS00699295-01 were found to effectively reduce VEEV viral replication in plaque assays. Kinetic methods demonstrated time-dependent inhibition by the hybrid inhibitors of the protease with NCGC00488909-01 (Ki = 3 µM) and NCGC00484087 (Ki = 5 µM). Rates of inactivation by CA074 in the presence of 6 mM CaCl2, MnCl2, or MgCl2 were measured with varying concentrations of inhibitor, Mg2+ and Mn2+ slightly enhanced inhibitor binding (3 to 6-fold). CA074 inhibited not only the VEEV nsP2 protease but also that of CHIKV and WEEV.
Assuntos
Cisteína Proteases , Vírus da Encefalite Equina Venezuelana , Animais , Cavalos , Replicação Viral , Inibidores de Cisteína Proteinase/farmacologiaRESUMO
The purpose of this study was to evaluate the effects of the route of administration on the immunogenicity and efficacy of a combined western, eastern, and Venezuelan equine encephalitis (WEVEE) virus-like replicon particle (VRP) vaccine in cynomolgus macaques. The vaccine consisted of equal amounts of WEEV, EEEV, and VEEV VRPs. Thirty-three animals were randomly assigned to five treatment or control groups. Animals were vaccinated with two doses of WEVEE VRPs or the control 28 days apart. Blood was collected 28 days following primary vaccination and 21 days following boost vaccination for analysis of the immune response to the WEVEE VRP vaccine. NHPs were challenged by aerosol 28 or 29 days following second vaccination with WEEV CBA87. Vaccination with two doses of WEVEE VRP was immunogenic and resulted in neutralizing antibody responses specific for VEEV, EEEV and WEEV. None of the vaccinated animals met euthanasia criteria following aerosol exposure to WEEV CBA87. However, one NHP control (total of 11 controls) met euthanasia criteria after infection with WEEV CBA87. Statistically significant differences in median fever hours were noted in control NHPs compared to vaccinated NHPs, providing a quantitative measure of infection and efficacy of the vaccine against a WEEV challenge. Alterations in lymphocytes, monocytes, and neutrophils were observed. Lymphopenia was observed in control NHPs.
Assuntos
Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Vacinas Virais , Aerossóis , Animais , Anticorpos Antivirais , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/prevenção & controle , Cavalos , Macaca fascicularis , RepliconRESUMO
Several ebolaviruses cause outbreaks of severe disease. Vaccines and monoclonal antibody cocktails are available to treat Ebola virus (EBOV) infections, but not Sudan virus (SUDV) or other ebolaviruses. Current cocktails contain antibodies that cross-react with the secreted soluble glycoprotein (sGP) that absorbs virus-neutralizing antibodies. By sorting memory B cells from EBOV infection survivors, we isolated two broadly reactive anti-GP monoclonal antibodies, 1C3 and 1C11, that potently neutralize, protect rodents from disease, and lack sGP cross-reactivity. Both antibodies recognize quaternary epitopes in trimeric ebolavirus GP. 1C11 bridges adjacent protomers via the fusion loop. 1C3 has a tripartite epitope in the center of the trimer apex. One 1C3 antigen-binding fragment anchors simultaneously to the three receptor-binding sites in the GP trimer, and separate 1C3 paratope regions interact differently with identical residues on the three protomers. A cocktail of both antibodies completely protected nonhuman primates from EBOV and SUDV infections, indicating their potential clinical value.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Ebolavirus , Doença pelo Vírus Ebola , Animais , Epitopos , Glicoproteínas/química , Subunidades ProteicasRESUMO
Venezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Encefalomielite Equina Venezuelana/virologia , Doenças dos Cavalos/prevenção & controle , Doenças dos Cavalos/virologia , Anticorpos de Domínio Único/uso terapêutico , Animais , Anticorpos Neutralizantes/farmacologia , Cavalos , Humanos , Engenharia de Proteínas , Anticorpos de Domínio Único/farmacologiaRESUMO
The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are classified by the Centers for Disease Control and Prevention (CDC) as biothreat agents. Currently, no licensed medical countermeasures (MCMs) against these viruses are available for humans. Neutralizing antibodies (NAbs) are fast-acting and highly effective MCMs for use in both pre- and post-exposure settings against biothreat agents. While significant work has been done to identify anti-VEEV NAbs, less has been done to identify NAbs against EEEV and WEEV. In order to develop anti-EEEV or -WEEV NAbs, mice were immunized using complementary strategies with a variety of different EEEV or WEEV immunogens to maximize the generation of NAbs to each of these viruses. Of the hybridomas generated, three anti-EEEV and seven anti-WEEV monoclonal antibodies were identified with in vitro neutralization activity. The most potent neutralizers (two anti-EEEV NAbs and three anti-WEEV NAbs) were further evaluated for neutralization activity against additional strains of EEEV, a single strain of Madariaga virus (formerly South American EEEV), or WEEV. Of these, G1-2-H4 and G1-4-C3 neutralized all three EEEV strains and the Madariaga virus strain, whereas G8-2-H9 and 12 WA neutralized six out of eight WEEV strains. To determine the protective efficacy of these NAbs, the five most potent neutralizers were evaluated in respective mouse aerosol challenge models. All five NAbs demonstrated various levels of protection when administered at doses of 2.5 mg/kg or 10 mg/kg 24 h before the respective virus exposure via the aerosol route. Of these, anti-EEEV NAb G1-4-C3 and anti-WEEV NAb 8C2 provided 100% protection at both doses and all surviving mice were free of clinical signs throughout the study. Additionally, no virus was detected in the brain 14 days post virus exposure. Taken together, efficacious NAbs were developed that demonstrate the potential for the development of cross-strain antibody-based MCMs against EEEV and WEEV infections.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Vírus da Encefalite Equina do Leste/imunologia , Vírus da Encefalite Equina do Oeste/imunologia , Encefalomielite Equina/prevenção & controle , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/imunologia , Proteção Cruzada , Modelos Animais de Doenças , Imunização , Camundongos , Testes de NeutralizaçãoRESUMO
Most alphaviruses are mosquito-borne and can cause severe disease in humans and domesticated animals. In North America, eastern equine encephalitis virus (EEEV) is an important human pathogen with case fatality rates of 30-90%. Currently, there are no therapeutics or vaccines to treat and/or prevent human infection. One critical impediment in countermeasure development is the lack of insight into clinically relevant parameters in a susceptible animal model. This study examined the disease course of EEEV in a cynomolgus macaque model utilizing advanced telemetry technology to continuously and simultaneously measure temperature, respiration, activity, heart rate, blood pressure, electrocardiogram (ECG), and electroencephalography (EEG) following an aerosol challenge at 7.0 log10 PFU. Following challenge, all parameters were rapidly and substantially altered with peak alterations from baseline ranged as follows: temperature (+3.0-4.2°C), respiration rate (+56-128%), activity (-15-76% daytime and +5-22% nighttime), heart rate (+67-190%), systolic (+44-67%) and diastolic blood pressure (+45-80%). Cardiac abnormalities comprised of alterations in QRS and PR duration, QTc Bazett, T wave morphology, amplitude of the QRS complex, and sinoatrial arrest. An unexpected finding of the study was the first documented evidence of a critical cardiac event as an immediate cause of euthanasia in one NHP. All brain waves were rapidly (~12-24 hpi) and profoundly altered with increases of up to 6,800% and severe diffuse slowing of all waves with decreases of ~99%. Lastly, all NHPs exhibited disruption of the circadian rhythm, sleep, and food/fluid intake. Accordingly, all NHPs met the euthanasia criteria by ~106-140 hpi. This is the first of its kind study utilizing state of the art telemetry to investigate multiple clinical parameters relevant to human EEEV infection in a susceptible cynomolgus macaque model. The study provides critical insights into EEEV pathogenesis and the parameters identified will improve animal model development to facilitate rapid evaluation of vaccines and therapeutics.
Assuntos
Infecções por Alphavirus/virologia , Modelos Animais de Doenças , Eletroencefalografia , Vírus da Encefalite Equina do Leste , Monitorização Fisiológica/instrumentação , Telemetria/instrumentação , Aerossóis , Infecções por Alphavirus/patologia , Animais , Pressão Sanguínea , Temperatura Corporal , Chlorocebus aethiops , Feminino , Frequência Cardíaca , Humanos , Macaca fascicularis , Masculino , Monitorização Fisiológica/métodos , Atividade Motora , Fenômenos Fisiológicos Respiratórios , Telemetria/métodos , Células VeroRESUMO
A single domain antibody (clone CC3) previously found to neutralize a vaccine strain of the chikungunya virus (PRNT50 = 2. 5 ng/mL) was found to be broadly neutralizing. Clone CC3 is not only able to neutralize a wild-type (WT) strain of chikungunya virus (CHIKV), but also neutralizes WT strains of Mayaro virus (MAYV) and Ross River virus (RRV); both arthralgic, Old World alphaviruses. Interestingly, CC3 also demonstrated a degree of neutralizing activity against the New World alphavirus, Venezuelan equine encephalitis virus (VEEV); albeit both the vaccine strain, TC-83, and the parental, WT Trinidad donkey strain had PRNT50 values ~1,000-fold higher than that of CHIKV. However, no neutralization activity was observed with Western equine encephalitis virus (WEEV). Ten CC3 variants designed to possess a range of isoelectric points, both higher and lower, were constructed. This approach successfully identified several lower pI mutants which possessed improved thermal stabilities by as much as 10°C over the original CC3 (Tm = 62°C), and excellent refolding abilities while maintaining their capacity to bind and neutralize CHIKV.
RESUMO
Neglected diseases caused by arenaviruses such as Lassa virus (LASV) and filoviruses like Ebola virus (EBOV) primarily afflict resource-limited countries, where antiviral drug development is often minimal. Previous studies have shown that many approved drugs developed for other clinical indications inhibit EBOV and LASV and that combinations of these drugs provide synergistic suppression of EBOV, often by blocking discrete steps in virus entry. We hypothesize that repurposing of combinations of orally administered approved drugs provides effective suppression of arenaviruses. In this report, we demonstrate that arbidol, an approved influenza antiviral previously shown to inhibit EBOV, LASV, and many other viruses, inhibits murine leukemia virus (MLV) reporter viruses pseudotyped with the fusion glycoproteins (GPs) of other arenaviruses (Junin virus [JUNV], lymphocytic choriomeningitis virus [LCMV], and Pichinde virus [PICV]). Arbidol and other approved drugs, including aripiprazole, amodiaquine, sertraline, and niclosamide, also inhibit infection of cells by infectious PICV, and arbidol, sertraline, and niclosamide inhibit infectious LASV. Combining arbidol with aripiprazole or sertraline results in the synergistic suppression of LASV and JUNV GP-bearing pseudoviruses. This proof-of-concept study shows that arenavirus infection in vitro can be synergistically inhibited by combinations of approved drugs. This approach may lead to a proactive strategy with which to prepare for and control known and new arenavirus outbreaks.
Assuntos
Antivirais/uso terapêutico , Infecções por Arenaviridae/tratamento farmacológico , Arenavirus/efeitos dos fármacos , Administração Oral , Animais , Infecções por Arenaviridae/virologia , Linhagem Celular , Chlorocebus aethiops , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Células HEK293 , Humanos , Camundongos , Estudo de Prova de Conceito , Células VeroRESUMO
Introduction: Failure of an existing effluent decontamination system (EDS) prompted the consideration of commercial off-the-shelf solutions for decontamination of containment laboratory waste. A bleach-based chemical EDS was purchased to serve as an interim solution. Methods: Studies were conducted in the laboratory to validate inactivation of Bacillus spores with bleach in complex matrices containing organic simulants including fetal bovine serum, humic acid, and animal room sanitation effluent. Results: These studies demonstrated effective decontamination of >106 spores at a free chlorine concentration of ≥5700 parts per million with a 2-hour contact time. Translation of these results to biological validation of the bleach-based chemical EDS required some modifications to the system and its operation. Discussion: The chemical EDS was validated for the treatment of biosafety levels 3 and 4 waste effluent using laboratory-prepared spore packets along with commercial biological indicators; however, several issues and lessons learned identified during the process of onboarding are also discussed, including bleach product source, method of validation, dechlorination, and treated waste disposal.
RESUMO
The human immune response to eastern equine encephalitis virus (EEEV) infection is poorly characterized due to the rarity of infection. We examined the humoral and cellular immune response to EEEV acquired from an infected donor via liver transplantation. Both binding and highly neutralizing antibodies to EEEV as well as a robust EEEV-specific IgG memory B cell response were generated. Despite triple-drug immunosuppressive therapy, a virus-specific CD4+ T cell response, predominated by interferon-γ production, was generated. T cell epitopes on the E2 envelope protein were identified by interferon-γ ELISpot. Although these results are from a single person who acquired EEEV by a non-traditional mechanism, to our knowledge this work represents the first analysis of the human cellular immune response to EEEV.
RESUMO
Recent studies highlight that infection with Coxsackievirus B3, Venezuelan equine encephalitis virus (VEEV), Marburg virus, or stimulation using poly I:C (dsRNA), upregulates the signaling adaptor protein MyD88 and impairs the host antiviral type I interferon (IFN) responses. In contrast, MyD88 deficiency (MyD88-/-) increases the type I IFN and survivability of mice implying that MyD88 up regulation limits the type I IFN response. Reasoning that MyD88 inhibition in a virus-like manner may increase type I IFN responses, our studies revealed lipopolysaccharide stimulation of U937 cells or poly I:C stimulation of HEK293-TLR3, THP1 or U87 cells in the presence of a previously reported MyD88 inhibitor (compound 4210) augmented IFN-ß and RANTES production. Consistent with these results, overexpression of MyD88 decreased IFN-ß, whereas MyD88 inhibition rescued IFN-ß production concomitant with increased IRF3 phosphorylation, suggesting IRF-mediated downstream signaling to the IFN-ß response. Further, compound 4210 treatment inhibited MyD88 interaction with IRF3/IRF7 indicating that MyD88 restricts type I IFN signaling through sequestration of IRF3/IRF7. In cell based infection assays, compound 4210 treatment suppressed replication of VEEV, Eastern equine encephalitis virus, Ebola virus (EBOV), Rift Valley Fever virus, Lassa virus, and Dengue virus with IC50 values ranging from 11 to 42 µM. Notably, administration of compound 4210 improved survival, weight change, and clinical disease scores in mice following challenge with VEEV TC-83 and EBOV. Collectively, these results provide evidence that viral infections responsive to MyD88 inhibition lead to activation of IRF3/IRF7 and promoted a type I IFN response, thus, raising the prospect of an approach of host-directed antiviral therapy.
Assuntos
Antivirais/farmacologia , Interferon Tipo I/genética , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Vírus/efeitos dos fármacos , Animais , Células HEK293 , Humanos , Concentração Inibidora 50 , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fosforilação , Poli I-C/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Células THP-1 , Viroses/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Vírus/classificaçãoRESUMO
We sequenced the complete coding genome of the western equine encephalitis virus (WEEV) strain Fleming. This strain was originally isolated in 1938 from a human WEEV case.
RESUMO
There are no FDA licensed vaccines or therapeutics for Venezuelan equine encephalitis virus (VEEV) which causes a debilitating acute febrile illness in humans that can progress to encephalitis. Previous studies demonstrated that murine and macaque monoclonal antibodies (mAbs) provide prophylactic and therapeutic efficacy against VEEV peripheral and aerosol challenge in mice. Additionally, humanized versions of two neutralizing mAbs specific for the E2 glycoprotein, 1A3B-7 and 1A4A-1, administered singly protected mice against aerosolized VEEV. However, no studies have demonstrated protection in nonhuman primate (NHP) models of VEEV infection. Here, we evaluated a chimeric antibody 1A3B-7 (c1A3B-7) containing mouse variable regions on a human IgG framework and a humanized antibody 1A4A-1 containing a serum half-life extension modification (Hu-1A4A-1-YTE) for their post-exposure efficacy in NHPs exposed to aerosolized VEEV. Approximately 24 hours after exposure, NHPs were administered a single bolus intravenous mAb. Control NHPs had typical biomarkers of VEEV infection including measurable viremia, fever, and lymphopenia. In contrast, c1A3B-7 treated NHPs had significant reductions in viremia and lymphopenia and on average approximately 50% reduction in fever. Although not statistically significant, Hu-1A4A-1-YTE administration did result in reductions in viremia and fever duration. Delay of treatment with c1A3B-7 to 48 hours post-exposure still provided NHPs protection from severe VEE disease through reductions in viremia and fever. These results demonstrate that post-exposure administration of c1A3B-7 protected macaques from development of severe VEE disease even when administered 48 hours following aerosol exposure and describe the first evaluations of VEEV-specific mAbs for post-exposure prophylactic use in NHPs. Viral mutations were identified in one NHP after c1A3B-7 treatment administered 24 hrs after virus exposure. This suggests that a cocktail-based therapy, or an alternative mAb against an epitope that cannot mutate without resulting in loss of viral fitness may be necessary for a highly effective therapeutic.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Neutralizantes/farmacologia , Encefalomielite Equina Venezuelana/imunologia , Vacinas Virais/farmacologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/imunologia , Modelos Animais de Doenças , Encefalomielite Equina Venezuelana/prevenção & controle , Humanos , Macaca fascicularis , Vacinas Virais/imunologiaRESUMO
Licensure of a vaccine to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires use of the U.S. Food and Drug Administration (FDA) Animal Rule to assess vaccine efficacy as human studies are not feasible or ethical. An approach to selecting VEEV challenge strains for use under the Animal Rule was developed, taking into account Department of Defense (DOD) vaccine requirements, FDA Animal Rule guidelines, strain availability, and lessons learned from the generation of filovirus challenge agents within the Filovirus Animal Nonclinical Group (FANG). Initial down-selection to VEEV IAB and IC epizootic varieties was based on the DOD objective for vaccine protection in a bioterrorism event. The subsequent down-selection of VEEV IAB and IC isolates was based on isolate availability, origin, virulence, culture and animal passage history, known disease progression in animal models, relevancy to human disease, and ability to generate sufficient challenge material. Methods for the propagation of viral stocks (use of uncloned (wild-type), plaque-cloned, versus cDNA-cloned virus) to minimize variability in the potency of the resulting challenge materials were also reviewed. The presented processes for VEEV strain selection and the propagation of viral stocks may serve as a template for animal model development product testing under the Animal Rule to other viral vaccine programs. This manuscript is based on the culmination of work presented at the "Alphavirus Workshop" organized and hosted by the Joint Vaccine Acquisition Program (JVAP) on 15 December 2014 at Fort Detrick, Maryland, USA.
Assuntos
Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Vacinas Virais/uso terapêutico , Animais , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/virologia , Guias como Assunto , Humanos , Programas de Imunização/métodos , Programas de Imunização/normas , Virologia/métodosRESUMO
Ebola virus (EBOV) continues to pose significant threats to global public health, requiring ongoing development of multiple strategies for disease control. To date, numerous monoclonal antibodies (mAbs) that target the EBOV glycoprotein (GP) have demonstrated potent protective activity in animal disease models and are thus promising candidates for the control of EBOV. However, recent work in a variety of virus diseases has highlighted the importance of coupling Fab neutralization with Fc effector activity for effective antibody-mediated protection. To determine the contribution of Fc effector activity to the protective function of mAbs to EBOV GP, we selected anti-GP mAbs targeting representative, protective epitopes and characterized their Fc receptor (FcγR) dependence in vivo in FcγR humanized mouse challenge models of EBOV disease. In contrast to previous studies, we find that anti-GP mAbs exhibited differential requirements for FcγR engagement in mediating their protective activity independent of their distance from the viral membrane. Anti-GP mAbs targeting membrane proximal epitopes or the GP mucin domain do not rely on Fc-FcγR interactions to confer activity, whereas antibodies against the GP chalice bowl and the fusion loop require FcγR engagement for optimal in vivo antiviral activity. This complexity of antibody-mediated protection from EBOV disease highlights the structural constraints of FcγR binding for specific viral epitopes and has important implications for the development of mAb-based immunotherapeutics with optimal potency and efficacy.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Receptores de IgG/metabolismo , Animais , Afinidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Imunoglobulina G/imunologia , Camundongos , Mucinas/antagonistas & inibidores , Mucinas/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de IgG/químicaRESUMO
Western, Eastern, and Venezuelan equine encephalitis viruses (WEEV, EEEV, and VEEV, respectively) are important mosquito-borne agents that pose public health and bioterrorism threats. Despite considerable advances in understanding alphavirus replication, there are currently no available effective vaccines or antiviral treatments against these highly lethal pathogens. To develop a potential countermeasure for viral encephalitis, we generated a trivalent, or three-component, EEV vaccine composed of virus-like particles (VLPs). Monovalent VLPs elicited neutralizing antibody responses and protected mice and nonhuman primates (NHPs) against homologous challenges, but they were not cross-protective. In contrast, NHPs immunized with trivalent VLPs were completely protected against aerosol challenge by each of these three EEVs. Passive transfer of IgG from immunized NHPs protected mice against aerosolized EEV challenge, demonstrating that the mechanism of protection was humoral. Because they are replication incompetent, these trivalent VLPs represent a potentially safe and effective vaccine that can protect against diverse encephalitis viruses.
Assuntos
Vírus da Encefalite/imunologia , Encefalite por Arbovirus/imunologia , Encefalite por Arbovirus/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Encefalite por Arbovirus/patologia , Encefalite por Arbovirus/virologia , Imunização , Imunoglobulina G/imunologia , Macaca fascicularis , Camundongos Endogâmicos BALB C , Vacinas de Partículas Semelhantes a Vírus/ultraestruturaRESUMO
Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.