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1.
PLoS One ; 14(5): e0216949, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31100082

RESUMO

The development of an effective vaccine against HIV infection remains a global priority. Dendritic cell (DC)-based HIV immunotherapeutic vaccine is a promising approach which aims at optimizing the HIV-specific immune response using primed DCs to promote and enhance both the cellular and humoral arms of immunity. Since the Ebola virus envelope glycoprotein (EboGP) has strong DC-targeting ability, we investigated whether EboGP is able to direct HIV particles towards DCs efficiently and promote potent HIV-specific immune responses. Our results indicate that the incorporation of EboGP into non-replicating virus-like particles (VLPs) enhances their ability to target human monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs). Also, a mucin-like domain deleted EboGP (EboGPΔM) can further enhanced the MDDCs and MDMs-targeting ability. Furthermore, we investigated the effect of EboGP on HIV immunogenicity in mice, and the results revealed a significantly stronger HIV-specific humoral immune response when immunized with EboGP-pseudotyped HIV VLPs compared with those immunized with HIV VLPs. Splenocytes harvested from mice immunized with EboGP-pseudotyped HIV VLPs secreted increased levels of macrophage inflammatory proteins-1α (MIP-1α) and IL-4 upon stimulation with HIV Env and/or Gag peptides compared with those harvested from mice immunized with HIV VLPs. Collectively, this study provides evidence for the first time that the incorporation of EboGP in HIV VLPs can facilitate DC and macrophage targeting and induce more potent immune responses against HIV.

2.
Antiviral Res ; 167: 68-77, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30953674

RESUMO

Lassa virus (LASV) causes Lassa hemorrhagic fever in humans and poses a significant threat to public health in West Africa. Current therapeutic treatments for Lassa fever are limited, making the development of novel countermeasures an urgent priority. In this study, we identified losmapimod, a p38 mitogen-activated protein kinase (MAPK) inhibitor, from 102 screened compounds as an inhibitor of LASV infection. Losmapimod exerted its inhibitory effect against LASV after p38 MAPK down-regulation, and, interestingly, had no effect on other arenaviruses capable of causing viral hemorrhagic fever. Mechanistic studies showed that losmapimod inhibited LASV entry by affecting the stable signal peptide (SSP)-GP2 subunit interface of the LASV glycoprotein, thereby blocking pH-dependent viral fusion. As an aryl heteroaryl bis-carboxyamide derivative, losmapimod represents a novel chemical scaffold with anti-LASV activity, and it provides a new lead structure for the future development of LASV fusion inhibitors.

3.
Virol J ; 16(1): 42, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940169

RESUMO

BACKGROUND: HIV integrase (IN) and its cellular cofactors, including lens-epithelium-derived growth factor (LEDGF/p75), Ku70, p300, and Rad52, are subject to small ubiquitin-like modifier (SUMO) modification. In addition to covalent SUMOylation, SUMO paralogs can also noncovalently bind proteins through SUMO-interacting motifs (SIMs). However, little is known about whether HIV IN contains SIMs and the roles of these motifs. RESULTS: We searched for the amino acid sequence of HIV IN and investigated three putative SIMs of IN: SIM1 72VILV75, SIM2 200IVDI203 and SIM3 257IKVV260. Our mutational analysis showed that 200IVDI203 and 257IKVV260 are two bona fide SIMs that mediate IN-SUMO noncovalent interactions. Additionally, a cell-based SUMOylation assay revealed that IN SIMs negatively regulate the SUMOylation of IN, as well as the interaction between IN and SUMO E2 conjugation enzyme Ubc9. Conversely, IN SIMs are required for its interactions with LEDGF/p75 but not with Ku70. Furthermore, our study reveals that SIM2 and SIM3 are required for the nuclear localization of IN. Finally, we investigated the impact of IN SIM2 and SIM3 on HIV single cycle replication in CD4+ C8166 T cells, and the results showed that viruses carrying IN SIM mutants are replication defective at the steps of the early viral life cycle, including reverse transcription, nuclear import and integration. CONCLUSION: Our data suggested that the INSIM-SUMO interaction constitutes a new regulatory mechanism of IN functions and might be important for HIV-1 replication.


Assuntos
Integrase de HIV/metabolismo , HIV-1/fisiologia , Proteína SUMO-1/metabolismo , Sumoilação , Replicação Viral , Motivos de Aminoácidos , Células HEK293 , Integrase de HIV/genética , HIV-1/enzimologia , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Reação em Cadeia da Polimerase em Tempo Real
4.
Antiviral Res ; 165: 1-10, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30836107

RESUMO

Among the five currently recognized type viruses within the genus Ebolavirus, Reston virus (RESTV) is not known to cause disease in humans, although asymptomatic infections have been confirmed in the past. Intriguingly, despite the absence of pathogenicity in humans, RESTV is highly lethal to nonhuman primates and has been isolated from domestic pigs co-infected with other viruses in the Philippines and China. Whether infection in these animals can support the eventual emergence of a human-pathogenic RESTV remains unclear and requires further investigation. Unfortunately, there is currently no lethal small animal model available to investigate RESTV pathogenicity or pan-ebolavirus therapeutics. Here we show that wild type RESTV is uniformly lethal in ferrets. In this study, ferrets were challenged with 1260 TCID50 of wild type RESTV either intramuscularly or intranasally and monitored for clinical signs, survival, virus replication, alteration in serum biochemistry and blood cell counts. Irrespective of the route of challenge, viremia occurred in all ferrets on day 5 post-infection, and all animals succumbed to infection between days 9 and 11. Additionally, several similarities were observed between this model and the other ferret models of filovirus infection, including substantial decreases in lymphocyte and platelet counts and abnormalities in serum biochemistry indicating hepatic injury. The ferret model represents the first uniformly lethal model for RESTV infection, and it will undoubtedly prove useful for evaluating virus pathogenicity as well as pan-ebolavirus countermeasures.

5.
PLoS Pathog ; 15(2): e1007564, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30817809

RESUMO

There are a number of vaccine candidates under development against a small number of the most common outbreak filoviruses all employing the virus glycoprotein (GP) as the vaccine immunogen. However, antibodies induced by such GP vaccines are typically autologous and limited to the other members of the same species. In contrast, T-cell vaccines offer a possibility to design a single pan-filovirus vaccine protecting against all known and even likely existing, but as yet unencountered members of the family. Here, we used a cross-filovirus immunogen based on conserved regions of the filovirus nucleoprotein, matrix and polymerase to construct simian adenovirus- and poxvirus MVA-vectored vaccines, and in a proof-of-concept study demonstrated a protection of the BALB/c and C57BL/6J mice against high, lethal challenges with Ebola and Marburg viruses, two distant members of the family, by vaccine-elicited T cells in the absence of GP antibodies.


Assuntos
Filoviridae/imunologia , Linfócitos T/imunologia , Vacinas Virais/farmacologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra Ebola , Ebolavirus/patogenicidade , Feminino , Filoviridae/metabolismo , Filoviridae/patogenicidade , Doença pelo Vírus Ebola , Imunidade Celular/imunologia , Masculino , Marburgvirus/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estudo de Prova de Conceito , Linfócitos T/metabolismo
6.
Viruses ; 11(3)2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875741

RESUMO

Filoviruses cause lethal hemorrhagic fever in humans. The filovirus nucleoprotein (NP) is expressed in high abundance in infected cells and is essential for virus replication. To generate anti-filovirus monoclonal antibodies (mAbs) against the NP, mice were immunized with peptides known as B-cell epitopes corresponding to different filovirus NPs, and hybridomas were screened using FLAG-tagged filovirus NP constructs. Numerous mAbs were identified, isotyped, and characterized. The anti-NP mAbs demonstrated different ranges of binding affinities to various filovirus NPs. Most of the clones specifically detected both recombinant and wild-type NPs from different filoviruses, including Ebola (EBOV), Sudan (SUDV), Bundibugyo (BDBV), Marburg (MARV), Tai Forest (TAFV), and Reston (RESTV) viruses in western blot analysis. The mAbs were also able to detect native NPs within the cytoplasm of infected cells by immunofluorescence confocal microscopy. Thus, this panel of mAbs represents an important set of tools that may be potentially useful for diagnosing filovirus infection, characterizing virus replication, and detecting NP⁻host protein interactions.

7.
Cell Host Microbe ; 25(1): 39-48.e5, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30629917

RESUMO

Passive administration of monoclonal antibodies (mAbs) is a promising therapeutic approach for Ebola virus disease (EVD). However, all mAbs and mAb cocktails that have entered clinical development are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against outbreak-causing Bundibugyo virus (BDBV) and Sudan virus (SUDV). Here, we advance MBP134, a cocktail of two broadly neutralizing human mAbs, ADI-15878 from an EVD survivor and ADI-23774 from the same survivor but specificity-matured for SUDV GP binding affinity, as a candidate pan-ebolavirus therapeutic. MBP134 potently neutralized all ebolaviruses and demonstrated greater protective efficacy than ADI-15878 alone in EBOV-challenged guinea pigs. A second-generation cocktail, MBP134AF, engineered to effectively harness natural killer (NK) cells afforded additional improvement relative to its precursor in protective efficacy against EBOV and SUDV in guinea pigs. MBP134AF is an optimized mAb cocktail suitable for evaluation as a pan-ebolavirus therapeutic in nonhuman primates.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Bem-Estar do Animal , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/uso terapêutico , Antivirais , Modelos Animais de Doenças , Ebolavirus/patogenicidade , Epitopos/imunologia , Feminino , Filoviridae/imunologia , Cobaias , Doença pelo Vírus Ebola/virologia , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Recombinantes/imunologia , Resultado do Tratamento
8.
Cell Host Microbe ; 25(1): 49-58.e5, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30629918

RESUMO

Recent and ongoing outbreaks of Ebola virus disease (EVD) underscore the unpredictable nature of ebolavirus reemergence and the urgent need for antiviral treatments. Unfortunately, available experimental vaccines and immunotherapeutics are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against other ebolaviruses associated with EVD, including Sudan virus (SUDV) and Bundibugyo virus (BDBV). Here we show that MBP134AF, a pan-ebolavirus therapeutic comprising two broadly neutralizing human antibodies (bNAbs), affords unprecedented effectiveness and potency as a therapeutic countermeasure to antigenically diverse ebolaviruses. MBP134AF could fully protect ferrets against lethal EBOV, SUDV, and BDBV infection, and a single 25-mg/kg dose was sufficient to protect NHPs against all three viruses. The development of MBP134AF provides a successful model for the rapid discovery and translational advancement of immunotherapeutics targeting emerging infectious diseases.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Ebolavirus/patogenicidade , Furões/virologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Bem-Estar do Animal , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/administração & dosagem , Linhagem Celular , Cercopithecus aethiops , Modelos Animais de Doenças , Feminino , Filoviridae/imunologia , Infecções por Filoviridae/imunologia , Infecções por Filoviridae/prevenção & controle , Infecções por Filoviridae/virologia , Glicoproteínas/imunologia , Cobaias , Células HEK293 , Doença pelo Vírus Ebola/virologia , Humanos , Células Matadoras Naturais , Macaca , Macaca fascicularis , Masculino , Primatas , Análise de Sobrevida , Resultado do Tratamento , Proteínas Virais/imunologia
9.
Nat Commun ; 10(1): 105, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631063

RESUMO

The 2013-2016 Ebola virus (EBOV) disease epidemic demonstrated the grave consequences of filovirus epidemics in the absence of effective therapeutics. Besides EBOV, two additional ebolaviruses, Sudan (SUDV) and Bundibugyo (BDBV) viruses, as well as multiple variants of Marburg virus (MARV), have also caused high fatality epidemics. Current experimental EBOV monoclonal antibodies (mAbs) are ineffective against SUDV, BDBV, or MARV. Here, we report that a cocktail of two broadly neutralizing ebolavirus mAbs, FVM04 and CA45, protects nonhuman primates (NHPs) against EBOV and SUDV infection when delivered four days post infection. This cocktail when supplemented by the anti-MARV mAb MR191 exhibited 100% efficacy in MARV-infected NHPs. These findings provide a solid foundation for clinical development of broadly protective immunotherapeutics for use in future filovirus epidemics.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Ebolavirus/imunologia , Infecções por Filoviridae/imunologia , Marburgvirus/imunologia , Doenças dos Primatas/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Ebolavirus/classificação , Ebolavirus/efeitos dos fármacos , Ebolavirus/fisiologia , Infecções por Filoviridae/terapia , Infecções por Filoviridae/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Imunoterapia/métodos , Marburgvirus/efeitos dos fármacos , Marburgvirus/fisiologia , Doenças dos Primatas/terapia , Doenças dos Primatas/virologia , Primatas , Resultado do Tratamento
10.
J Virol ; 93(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30541860

RESUMO

Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans, with fatality rates reaching 90% and with no licensed specific therapeutics to treat ill patients. Advances over the past 5 years have firmly established monoclonal antibody (MAb)-based products as the most promising therapeutics for treating EBOV infections, but production is costly and quantities are limited; therefore, MAbs are not the best candidates for mass use in the case of an epidemic. To address this need, we generated EBOV-specific polyclonal F(ab')2 fragments from horses hyperimmunized with an EBOV vaccine. The F(ab')2 was found to potently neutralize West African and Central African EBOV in vitro Treatment of nonhuman primates (NHPs) with seven doses of 100 mg/kg F(ab')2 beginning 3 or 5 days postinfection (dpi) resulted in a 100% survival rate. Notably, NHPs for which treatment was initiated at 5 dpi were already highly viremic, with observable signs of EBOV disease, which demonstrated that F(ab')2 was still effective as a therapeutic agent even in symptomatic subjects. These results show that F(ab')2 should be advanced for clinical testing in preparation for future EBOV outbreaks and epidemics.IMPORTANCE EBOV is one of the deadliest viruses to humans. It has been over 40 years since EBOV was first reported, but no cure is available. Research breakthroughs over the past 5 years have shown that MAbs constitute an effective therapy for EBOV infections. However, MAbs are expensive and difficult to produce in large amounts and therefore may only play a limited role during an epidemic. A cheaper alternative is required, especially since EBOV is endemic in several third world countries with limited medical resources. Here, we used a standard protocol to produce large amounts of antiserum F(ab')2 fragments from horses vaccinated with an EBOV vaccine, and we tested the protectiveness in monkeys. We showed that F(ab')2 was effective in 100% of monkeys even after the animals were visibly ill with EBOV disease. Thus, F(ab')2 could be a very good option for large-scale treatments of patients and should be advanced to clinical testing.

11.
Antiviral Res ; 161: 20-27, 2018 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-30423362

RESUMO

Therapeutic vaccination is a promising strategy for controlling chronic hepatitis B virus (HBV). Here, we tested whether several novel vaccination strategies could be used to induce HBV-specific adaptive immune responses and control/eradicate HBV in a mouse model. Robust HBV antigen-specific antibody responses were elicited by several vaccination strategies using a novel particle vaccine (HBSS1), which expresses a fusion of the S (amino acids [aa] 1-223) and preS1 (aa 21-47) antigens, and/or a recombinant adenovirus rAdSS1 vaccine. However, antigen-specific cell-mediated immunity and high levels of production of multiple cytokines were elicited only by heterologous prime-boost immunization; i.e., priming with the HBSS1 vaccine followed by a rAdSS1 boost. Furthermore, the most rapid loss of serum HBsAg, HBeAg and DNA was achieved by the novel vaccination regimen (priming with HBSS1 formulated with adjuvants [alum plus PolyI:C]), which was strongly associated with more potent and functional HBsAg-specific CD4+ and CD8+ T-cell responses and increased production of interleukin (IL)-2, interferon (IFN)-γ, tumor necrosis factor-α, IL-12, and IFN-γ-induced protein (IP)-10. Thus, our novel heterogeneous prime-boost vaccine regimen shows promise as a therapeutic strategy against HBV.

12.
Cell Rep ; 25(7): 1982-1993.e4, 2018 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-30428362

RESUMO

Synthetically engineered DNA-encoded monoclonal antibodies (DMAbs) are an in vivo platform for evaluation and delivery of human mAb to control against infectious disease. Here, we engineer DMAbs encoding potent anti-Zaire ebolavirus (EBOV) glycoprotein (GP) mAbs isolated from Ebola virus disease survivors. We demonstrate the development of a human IgG1 DMAb platform for in vivo EBOV-GP mAb delivery and evaluation in a mouse model. Using this approach, we show that DMAb-11 and DMAb-34 exhibit functional and molecular profiles comparable to recombinant mAb, have a wide window of expression, and provide rapid protection against lethal mouse-adapted EBOV challenge. The DMAb platform represents a simple, rapid, and reproducible approach for evaluating the activity of mAb during clinical development. DMAbs have the potential to be a mAb delivery system, which may be advantageous for protection against highly pathogenic infectious diseases, like EBOV, in resource-limited and other challenging settings.

13.
mSphere ; 3(5)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30381349

RESUMO

Ebola virus (EBOV) has been responsible for sporadic outbreaks in Central Africa since 1976 and has the potential of causing social disruption and public panic as illustrated by the 2013-2016 epidemic in West Africa. Transmission of EBOV has been described to occur via contact with infected bodily fluids, supported by data indicating that infectious EBOV could be cultured from blood, semen, saliva, urine, and breast milk. Parameters influencing transmission of EBOV are, however, largely undefined in part due to the lack of an established animal model to study mechanisms of pathogen spread. Here, we investigated EBOV transmissibility in male and female ferrets. After intranasal challenge, an infected animal was placed in direct contact with a naive ferret and in contact with another naive ferret (separated from the infected animal by a metal mesh) that served as the indirect-contact animal. All challenged animals, male direct contacts, and one male indirect contact developed disease and died. The remaining animals were not viremic and remained asymptomatic but developed EBOV-glycoprotein IgM and/or IgG specific antibodies-indicative of virus transmission. EBOV transmission via indirect contact was frequently observed in this model but resulted in less-severe disease compared to direct contact. Interestingly, these observations are consistent with the detection of specific antibodies in humans living in areas of EBOV endemicity.IMPORTANCE Our knowledge regarding transmission of EBOV between individuals is vague and is mostly limited to spreading via direct contact with infectious bodily fluids. Studying transmission parameters such as dose and route of infection is nearly impossible in naturally acquired cases-hence the requirement for a laboratory animal model. Here, we show as a proof of concept that ferrets can be used to study EBOV transmission. We also show that transmission in the absence of direct contact is frequent, as all animals with indirect contact with the infected ferrets had detectable antibodies to the virus, and one succumbed to infection. Our report provides a new small-animal model for studying EBOV transmission that does not require adaptation of the virus, providing insight into virus transmission among humans during epidemics.

14.
J Virol ; 2018 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-30333174

RESUMO

Sequencing of Ebola virus (EBOV) genomes during the 2014-16 epidemic identified several naturally-occurring, dominant mutations potentially impacting virulence or tropism. Here, we characterized EBOV variants carrying one of the following substitutions: A82V in the glycoprotein (GP), R111C in the nucleoprotein (NP), or D759G in the RNA-dependent RNA-polymerase (L). Compared with wild-type EBOV/C07 (WT), NP and L mutants conferred a replication advantage in monkey VeroE6, human A549 and insectivorous bat Tb1.Lu cells, while L displayed a disadvantage in human Huh7 cells. GP mutant replication was significantly delayed in Tb1.Lu and similar to WT in other cells. The L mutant was less virulent, as evidenced by increased survival in mice and a significantly delayed time to death in ferrets, but increased lengths of EBOV shedding may have contributed to the prolonged epidemic. Our results show that single substitutions can have observable impacts on EBOV pathogenicity and provide a framework to study other mutations.IMPORTANCEDuring the Ebola virus (EBOV) disease outbreak in West Africa in 2014-16, it was discovered that several mutations in the virus emerged and became prevalent in the human population. This suggests that these mutations may play a role impacting viral fitness. We investigated three of these previously identified mutations (in the glycoprotein (GP), nucleoprotein (NP) or RNA-dependent RNA polymerase (L)) in cell culture as well as in mice and ferrets by generating recombinant viruses (based on an early West African EBOV strain) carrying one of these mutations. The NP and L mutations appear to decrease virulence, whereas GP slightly increases virulence but mainly impacts viral tropism. Our results show that these single mutations can impact EBOV virulence in animals and have implications for the rational design of efficacious antiviral therapies against these infections.

15.
J Infect Dis ; 2018 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-30304515

RESUMO

Background: There remains an important need for prophylactic anti-Ebola virus vaccine candidates that elicit long-lasting immune responses and can be delivered to vulnerable populations that are unable to receive live-attenuated or viral vector vaccines. Methods: We designed novel synthetic anti-Ebola virus glycoprotein (EBOV-GP) DNA vaccines as a strategy to expand protective breadth against diverse EBOV strains and evaluated the impact of vaccine dosing and route of administration on protection against lethal EBOV-Makona challenge in cynomolgus macaques. Long-term immunogenicity was monitored in nonhuman primates for >1 year, followed by a 12-month boost. Results: Multiple-injection regimens of the EBOV-GP DNA vaccine, delivered by intramuscular administration followed by electroporation, were 100% protective against lethal EBOV-Makona challenge. Impressively, 2 injections of a simple, more tolerable, and dose-sparing intradermal administration followed by electroporation generated strong immunogenicity and was 100% protective against lethal challenge. In parallel, we observed that EBOV-GP DNA vaccination induced long-term immune responses in macaques that were detectable for at least 1 year after final vaccination and generated a strong recall response after the final boost. Conclusions: These data support that this simple intradermal-administered, serology-independent approach is likely important for additional study towards the goal of induction of anti-EBOV immunity in multiple at-risk populations.

16.
Antiviral Res ; 160: 87-93, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30339847

RESUMO

The family Arenaviridae consists of numerous enveloped RNA viruses with ambisense coding strategies. Eight arenaviruses, including Lassa virus, are known to cause severe and fatal viral hemorrhagic fever (VHF) in humans, yet vaccines and treatments for disease caused by arenaviruses are very limited. In this study, we screened a natural product library consisting of 131 compounds and identified tangeretin, a polymethoxylated flavone widely present in citrus fruit peels, as a Lassa virus entry inhibitor that blocks viral fusion. Further analyses demonstrated the efficacy of tangeretin against seven other VHF-causing arenaviruses, suggesting that this compound, which has a history of medical usage, could be used to develop an effective therapeutic to treat infection and disease caused by Lassa virus and related viruses.

17.
Int Immunopharmacol ; 64: 217-222, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30199846

RESUMO

BACKGROUND: Rift Valley fever virus (RVFV) is an emerging arbovirus in Africa and the Arabian Peninsula, in which infection with RVFV poses a serious threat to humans and livestock globally. Approved treatments for RVFV infection, especially for use in humans, have not yet been developed. There is an urgent need for effective drugs to prevent RVFV disease. METHODS: In previous study, we developed RVFV virus like particles (VLPs) expressing the surface glycoproteins Gn and Gc. The morphology was shown to be similar to live RVFV under electron microscopy. In this study, we immunized horses with RVFV VLPs, prepared the immunoglobulin F(ab')2 fragments, and characterized its in vitro neutralization and in vivo efficacy in mice. RESULTS: F(ab')2 was found to potently neutralize RVFV in VeroE6 cells, and passive transfer of immunoglobulin F(ab')2 fragments resulting in reduced mortality in RVFV infected mice. CONCLUSION: Our results show that passive immunotherapy with equine immunoglobulin F(ab')2 fragments is a promising strategy to treat RVFV infections.

18.
Vaccine ; 36(41): 6053-6060, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30195490

RESUMO

Ebola virus (EBOV) disease (EVD) leads to lethal hemorrhagic fever with a case fatality rate as high as 90%, thus posing a serious global public health concern. However, while several vaccines based on the EBOV glycoprotein have been confirmed to be effective in animal experiments, no licensed vaccines or effective treatments have been approved since the first outbreak was reported in 1976. In this study, we prepared the extracellular domain of the EBOV GP protein (designated as N20) by prokaryotic expression and purification via chromatography. Using CTA1-DD (designated as H45) as a mucosal adjuvant, we evaluated the immunogenicity of N20 by intranasal administration and the associated protective efficacy against mouse-adapted EBOV challenge in mice. We found that intranasal vaccination with H45-adjuvanted N20 could stimulate humoral immunity, as supported by GP-specific IgG titers; Th1 cellular immunity, based on IgG subclasses and IFN-γ/IL-4 secreting cells; and mucosal immunity, based on the presence of anti-EBOV IgA in vaginal lavages. We also confirmed that the vaccine could completely protect mice against a lethal mouse-adapted EBOV (MA-EBOV) challenge with few side effects (based on weight loss). In comparison, mice that received N20 or H45 alone succumbed to lethal MA-EBOV challenge. Therefore, mucosal vaccination with H45-adjuvanted N20 represents a potential vaccine candidate for the prevention of EBOV in an effective, safe, and convenient manner.

19.
Clin Exp Vaccine Res ; 7(2): 119-128, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30112351

RESUMO

Purpose: The goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones. Materials and Methods: For hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus. Results: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953-7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection. Conclusion: This study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.

20.
Cell Host Microbe ; 24(2): 221-233.e5, 2018 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-30092199

RESUMO

The recent Ebola virus (EBOV) epidemic highlighted the need for effective vaccines and therapeutics to limit and prevent outbreaks. Host antibodies against EBOV are critical for controlling disease, and recombinant monoclonal antibodies (mAbs) can protect from infection. However, antibodies mediate an array of antiviral functions including neutralization as well as engagement of Fc-domain receptors on immune cells, resulting in phagocytosis or NK cell-mediated killing of infected cells. Thus, to understand the antibody features mediating EBOV protection, we examined specific Fc features associated with protection using a library of EBOV-specific mAbs. Neutralization was strongly associated with therapeutic protection against EBOV. However, several neutralizing mAbs failed to protect, while several non-neutralizing or weakly neutralizing mAbs could protect. Antibody-mediated effector functions, including phagocytosis and NK cell activation, were associated with protection, particularly for antibodies with moderate neutralizing activity. This framework identifies functional correlates that can inform therapeutic and vaccine design strategies against EBOV and other pathogens.

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