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1.
J Infect Dis ; 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31301137

RESUMO

BACKGROUND: Monoclonal antibodies can mediate protection against Ebola virus (EBOV) infection through direct neutralization as well as through the recruitment of innate immune effector functions. However, the antibody functional response following survival of acute EBOV disease has not been well characterized. METHODS: Serum antibodies from EVD survivors from Sierra Leone were profiled to capture variation in overall subclass/isotype abundance, neutralizing activity, and innate immune effector functions. RESULTS: Antibodies from EVD survivors exhibit robust innate immune effector functions, mediated primarily by IgG1 and IgA1. CONCLUSIONS: Development of functional antibodies follows survival of acute EVD.

2.
Sci Immunol ; 4(32)2019 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-30796092

RESUMO

Chikungunya virus (CHIKV) is an emerging mosquito-borne virus that has caused explosive outbreaks worldwide. Although neutralizing monoclonal antibodies (mAbs) against CHIKV inhibit infection in animals, the contribution of Fc effector functions to protection remains unknown. Here, we evaluated the activity of therapeutic mAbs that had or lacked the ability to engage complement and Fcγ receptors (FcγR). When administered as post-exposure therapy in mice, the Fc effector functions of mAbs promoted virus clearance from infected cells and reduced joint swelling-results that were corroborated in antibody-treated transgenic animals lacking activating FcγR. The control of CHIKV infection by antibody-FcγR engagement was associated with an accelerated influx of monocytes. A series of immune cell depletions revealed that therapeutic mAbs required monocytes for efficient clearance of CHIKV infection. Overall, our study suggests that in mice, FcγR expression on monocytes is required for optimal therapeutic activity of antibodies against CHIKV and likely other related viruses.

3.
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
4.
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
5.
J Virol ; 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518655

RESUMO

Ebolaviruses Zaire (EBOV), Bundibugyo (BDBV) and Sudan (SUDV) cause human disease with high case fatality rates. Experimental monovalent vaccines, which all utilize the sole envelope glycoprotein (GP), do not protect against heterologous ebolaviruses. Human parainfluenza virus type 3-vectored vaccines offer benefits including needle-free administration and induction of mucosal responses in the respiratory tract. Multiple approaches were taken to induce a broad protection against the three ebolaviruses. While GP consensus-based antigens failed to elicit neutralizing antibodies, polyvalent immunization induced neutralizing responses to all three ebolaviruses and protected animals from death and disease caused by EBOV, SUDV and BDBV. As immunization with a cocktail of antigenically-related antigens can skew the responses and change the epitope hierarchy, we performed comparative analysis of antibody repertoire and Fc-mediated protective mechanisms in animals immunized with monovalent versus polyvalent vaccines. Compared to the monovalent vaccines, sera from trivalent vaccinated guinea pigs bound and neutralized EBOV and SUDV at equivalent levels and BDBV at only slightly reduced level. Peptide microarrays revealed a preponderance of binding to amino acids 389-403, 397-415 and 477-493, representing three linear epitopes in the mucin-like domain known to induce a protective antibody response. Competition binding assays with monoclonal antibodies isolated from human ebolavirus survivors demonstrated that the immune sera block binding of antibodies specific for the GP glycan cap, GP1-GP2 interface, the mucin-like domain, and the membrane-proximal external region. Thus, cocktail administration of three ebolavirus vaccines induces a desirable broad antibody response, without skewing of the response toward preferential recognition of a single virus.IMPORTANCE Symptoms of the disease caused by ebolaviruses Ebola, Bundibugyo and Sudan are similar, and their endemic areas overlap. However, because of the limited antigenic relatedness of ebolavirus glycoprotein (GP) used in all candidate vaccines against these viruses, they protect only against homologous but not heterologous ebolaviruses. Therefore, a broadly specific pan-ebolavirus vaccine is required, which might be achieved by administration of a cocktail of vaccines. The effects of cocktail administration of ebolavirus vaccines on the antibody repertoire remain unknown. Here in-depth analysis of the antibody responses to cocktail administration of human parainfluenza type 3-vectored vaccines against individual ebolaviruses was performed, which included analysis of binding to GP, neutralization of individual ebolaviruses, epitope specificity, Fc-mediated functions, and protection against the three ebolaviruses. The results demonstrated potent and balanced responses against individual ebolaviruses and no significant reduction of the responses, compared to that induced by individual vaccines.

6.
Nat Immunol ; 19(11): 1169-1178, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30333617

RESUMO

Recent Ebola virus disease epidemics have highlighted the need for effective vaccines and therapeutics to prevent future outbreaks. Antibodies are clearly critical for control of this deadly disease; however, the specific mechanisms of action of protective antibodies have yet to be defined. In this Perspective we discuss the antibody features that correlate with in vivo protection during infection with Ebola virus, based on the results of a systematic and comprehensive study of antibodies directed against this virus. Although neutralization activity mediated by the Fab domains of the antibody is strongly correlated with protection, recruitment of immune effector functions by the Fc domain has also emerged as a complementary, and sometimes alternative, route to protection. For a subset of antibodies, Fc-mediated clearance and killing of infected cells seems to be the main driver of protection after exposure and mirrors observations in vaccination studies. Continued analysis of antibodies that achieve protection partially or wholly through Fc-mediated functions, the precise functions required, the intersection with specificity and the importance of these functions in different animal models is needed to identify and begin to capitalize on Fc-mediated protection in vaccines and therapeutics alike.

7.
PLoS Pathog ; 14(8): e1007204, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30138408

RESUMO

Recent studies suggest that some monoclonal antibodies (mAbs) specific for ebolavirus glycoprotein (GP) can protect experimental animals against infections. Most mAbs isolated from ebolavirus survivors appeared to target the glycan cap or the stalk region of the viral GP, which is the envelope protein and the only antigen inducing virus-neutralizing antibody response. Some of the mAbs were demonstrated to be protective in vivo. Here, a panel of mAbs from four individual survivors of ebolavirus infection that target the glycan cap or stem region were selected for investigation of the mechanisms of their antiviral effect. Comparative characterization of the inhibiting effects on multiple steps of viral replication was performed, including attachment, post-attachment, entry, binding at low pH, post-cleavage neutralization of virions, viral trafficking to endosomes, cell-to-cell transmission, viral egress, and inhibition when added early at various time points post-infection. In addition, Fc-domain related properties were characterized, including activation and degranulation of NK cells, antibody-dependent cellular phagocytosis and glycan content. The two groups of mAbs (glycan cap versus stem) demonstrated very different profiles of activities suggesting usage of mAbs with different epitope specificity could coordinate inhibition of multiple steps of filovirus infection through Fab- and Fc-mediated mechanisms, and provide a reliable therapeutic approach.

8.
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.

9.
Cell ; 174(4): 938-952.e13, 2018 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-30096313

RESUMO

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

10.
Immunity ; 49(2): 363-374.e10, 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30029854

RESUMO

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.

11.
J Virol ; 2018 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-29643249

RESUMO

A vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here we report the immunogenicity of longitudinal prime/boost vaccination regimens over a period of 200 weeks in guinea pigs with a panel of HIV-1 envelope (Env) gp140 protein immunogens. We assessed vaccine regimens that included a monovalent clade C gp140 regimen (C97), a tetravalent regimen consisting of four clade C gp140s (4C), and a tetravalent regimen consisting of a clade A, B, C, and mosaic gp140 (ABCM). We found that the 4C and ABCM prime/boost regimens were capable of eliciting a greater magnitude and breadth of binding antibodies targeting variable loop 2 (V2) over time, compared to the monovalent C97 only regimen. The longitudinal boosting regimen conducted over more than two years increased the magnitude of certain tier 1 NAbs, but did not increase the magnitude or breadth of heterologous tier 2 NAbs. These data suggest that additional immunogen design strategies are needed to induce broad, high titer tier 2 NAbs.IMPORTANCE The elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study we explored the use of a long-term vaccination regimen with differing immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than two years increased tier 1 NAbs but did not increase the magnitude and breadth of tier 2 NAbs. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses.

12.
Virology ; 515: 250-260, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29324290

RESUMO

Mannose binding lectin (MBL) generally plays a protective role during viral infection, yet MBL-mediated complement activation promotes Ross River virus (RRV)-induced inflammatory tissue destruction, contributing to arthritis and myositis. As MBL binds to carbohydrates, we hypothesized that N-linked glycans on the RRV envelope glycoproteins act as ligands for MBL. Using a panel of RRV mutants lacking the envelope N-linked glycans, we found that MBL deposition onto infected cells was dependent on the E2 glycans. Moreover, the glycan-deficient viruses exhibited reduced disease and tissue damage in a mouse model of RRV-induced myositis compared to wild-type RRV, despite similar viral load and inflammatory infiltrates within the skeletal muscle. Instead, the reduced disease induced by glycan-deficient viruses was linked to decreased MBL deposition and complement activation within inflamed tissues. These results demonstrate that the viral N-linked glycans promote MBL deposition and complement activation onto RRV-infected cells, contributing to the development of RRV-induced myositis.


Assuntos
Infecções por Alphavirus/imunologia , Proteínas do Sistema Complemento/imunologia , Polissacarídeos/imunologia , Vírus do Rio Ross/imunologia , Proteínas do Envelope Viral/imunologia , Infecções por Alphavirus/virologia , Animais , Ativação do Complemento , Modelos Animais de Doenças , Humanos , Lectina de Ligação a Manose/imunologia , Camundongos Endogâmicos C57BL , Polissacarídeos/química , Vírus do Rio Ross/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
13.
Trends Mol Med ; 22(11): 969-982, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27756530

RESUMO

Induction of pathogen-specific binding antibodies has long been considered a signature of protective immunity following vaccination and infection. The humoral immune response is a complex network of antibodies that target different specificities and drive different functions, collectively acting to limit and clear infection either directly, via pathogen neutralization, or indirectly, via pathogen clearance by the innate immune system. Emerging data suggest that not all antibody responses are equal, and qualitative features of antibodies may be key to defining protective immune profiles. Here, we review the most recent advances in our understanding of protective functional antibody responses in natural infection, vaccination, and monoclonal antibody therapeutics. Moreover, we highlight opportunities to augment or modulate antibody-mediated protection through enhancement of antibody functionality.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Doenças Transmissíveis/imunologia , Imunidade Humoral , Imunidade Inata , Vacinação/métodos , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Afinidade de Anticorpos , Controle de Doenças Transmissíveis/métodos , Humanos
14.
J Virol ; 88(7): 3719-32, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24429363

RESUMO

UNLABELLED: Ross River virus (RRV) is one of a group of mosquito-transmitted alphaviruses that cause debilitating, and often chronic, musculoskeletal disease in humans. Previously, we reported that replacement of the nonstructural protein 1 (nsP1) gene of the mouse-virulent RRV strain T48 with that from the mouse-avirulent strain DC5692 generated a virus that was attenuated in a mouse model of disease. Here we find that the six nsP1 nonsynonymous nucleotide differences between strains T48 and DC5692 are determinants of RRV virulence, and we identify two nonsynonymous nucleotide changes as sufficient for the attenuated phenotype. RRV T48 carrying the six nonsynonymous DC5692 nucleotide differences (RRV-T48-nsP1(6M)) was attenuated in both wild-type and Rag1(-/-) mice. Despite the attenuated phenotype, RRV T48 and RRV-T48-nsP1(6M) loads in tissues of wild-type and Rag1(-/-) mice were indistinguishable from 1 to 3 days postinoculation. RRV-T48-nsP1(6M) loads in skeletal muscle tissue, but not in other tissues, decreased dramatically by 5 days postinoculation in both wild-type and Rag1(-/-) mice, suggesting that the RRV-T48-nsP1(6M) mutant is more sensitive to innate antiviral effectors than RRV T48 in a tissue-specific manner. In vitro, we found that the attenuating mutations in nsP1 conferred enhanced sensitivity to type I interferon. In agreement with these findings, RRV T48 and RRV-T48-nsP1(6M) loads were similar in mice deficient in the type I interferon receptor. Our findings suggest that the type I IFN response controls RRV infection in a tissue-specific manner and that specific amino acid changes in nsP1 are determinants of RRV virulence by regulating the sensitivity of RRV to interferon. IMPORTANCE: Arthritogenic alphaviruses, including Ross River virus (RRV), infect humans and cause debilitating pain and inflammation of the musculoskeletal system. In this study, we identified coding changes in the RRV nsP1 gene that control the virulence of RRV and its sensitivity to the antiviral type I interferon response, a major component of antiviral defense in mammals. Furthermore, our studies revealed that the effects of these attenuating mutations are tissue specific. These findings suggest that these mutations in nsP1 influence the sensitivity of RRV to type I interferon only in specific host tissues. The new knowledge gained from these studies contributes to our understanding of host responses that control alphavirus infection and viral determinants that counteract these responses.


Assuntos
Infecções por Alphavirus/virologia , Interações Hospedeiro-Patógeno , Interferon Tipo I/imunologia , Mutação de Sentido Incorreto , Vírus do Rio Ross/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Infecções por Alphavirus/patologia , Estruturas Animais/virologia , Animais , Análise Mutacional de DNA , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Vírus do Rio Ross/imunologia , Carga Viral , Proteínas não Estruturais Virais/genética , Virulência , Fatores de Virulência/genética
15.
PLoS Pathog ; 8(3): e1002586, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22457620

RESUMO

Mosquito-borne alphaviruses such as chikungunya virus and Ross River virus (RRV) are emerging pathogens capable of causing large-scale epidemics of virus-induced arthritis and myositis. The pathology of RRV-induced disease in both humans and mice is associated with induction of the host inflammatory response within the muscle and joints, and prior studies have demonstrated that the host complement system contributes to development of disease. In this study, we have used a mouse model of RRV-induced disease to identify and characterize which complement activation pathways mediate disease progression after infection, and we have identified the mannose binding lectin (MBL) pathway, but not the classical or alternative complement activation pathways, as essential for development of RRV-induced disease. MBL deposition was enhanced in RRV infected muscle tissue from wild type mice and RRV infected MBL deficient mice exhibited reduced disease, tissue damage, and complement deposition compared to wild-type mice. In contrast, mice deficient for key components of the classical or alternative complement activation pathways still developed severe RRV-induced disease. Further characterization of MBL deficient mice demonstrated that similar to C3(-/-) mice, viral replication and inflammatory cell recruitment were equivalent to wild type animals, suggesting that RRV-mediated induction of complement dependent immune pathology is largely MBL dependent. Consistent with these findings, human patients diagnosed with RRV disease had elevated serum MBL levels compared to healthy controls, and MBL levels in the serum and synovial fluid correlated with severity of disease. These findings demonstrate a role for MBL in promoting RRV-induced disease in both mice and humans and suggest that the MBL pathway of complement activation may be an effective target for therapeutic intervention for humans suffering from RRV-induced arthritis and myositis.


Assuntos
Infecções por Alphavirus/complicações , Artrite Reativa/virologia , Lectina de Ligação a Manose/metabolismo , Miosite/virologia , Vírus do Rio Ross/fisiologia , Infecções por Alphavirus/metabolismo , Infecções por Alphavirus/patologia , Animais , Artrite Reativa/metabolismo , Artrite Reativa/patologia , Ativação do Complemento , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/virologia , Miosite/metabolismo , Miosite/patologia , Vírus do Rio Ross/patogenicidade , Líquido Sinovial/metabolismo , Replicação Viral
16.
Am J Pathol ; 178(1): 32-40, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21224040

RESUMO

Chikungunya virus (CHIKV), an emerging mosquito-borne Alphavirus, causes debilitating rheumatic disease in humans that can last for weeks to months. Starting in 2004, a CHIKV outbreak in the Indian Ocean region affected millions of people, and infected travelers introduced CHIKV to new regions. The pathogenesis of CHIKV is poorly understood, and no approved vaccines or specific therapies exist. A major challenge to the study of CHIKV disease is the lack of a small animal model that recapitulates the major outcomes of human infection. In this study, the pathogenesis of CHIKV in C57BL/6J mice was investigated using biological and molecular clones of CHIKV isolated from human serum (CHIKV SL15649). After 14-day-old mice were inoculated with CHIKV SL15649 in the footpad, they displayed reduced weight gain and swelling of the inoculated limb. Histologic analysis of hind limb sections revealed severe necrotizing myositis, mixed inflammatory cell arthritis, chronic active tenosynovitis, and multifocal vasculitis. Interestingly, these disease signs and viral RNA persisted in musculoskeletal tissues for at least 3 weeks after inoculation. This work demonstrates the development of a mouse model of CHIKV infection with clinical manifestations and histopathologic findings that are consistent with the disease signs of CHIKV-infected humans, providing a useful tool for studying viral and host factors that drive CHIKV pathogenesis and for evaluating potential therapeutics against this emerging viral disease.


Assuntos
Artrite Reumatoide/virologia , Vírus Chikungunya , Modelos Animais de Doenças , Camundongos , Miosite/virologia , Tenossinovite/virologia , Infecções por Alphavirus/patologia , Animais , Artrite Reumatoide/patologia , Febre de Chikungunya , Membro Posterior/patologia , Humanos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Miosite/patologia , Tenossinovite/patologia
17.
Virology ; 410(1): 216-27, 2011 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-21131014

RESUMO

The viral determinants of alphavirus-induced rheumatic disease have not been elucidated. We identified an RRV strain (DC5692) which, in contrast to the T48 strain, does not induce musculoskeletal inflammation in a mouse model of RRV disease. Substitution of the RRV T48 strain nonstructural protein 1 (nsP1) coding sequence with that from strain DC5692 generated a virus that was attenuated in vivo despite similar viral loads in tissues. In contrast, substitution of the T48 PE2 coding region with the PE2 coding region from DC5692 resulted in attenuation in vivo and reduced viral loads in tissues. In gain of virulence experiments, substitution of the DC5692 strain nsP1 and PE2 coding regions with those from the T48 strain was sufficient to restore full virulence to the DC5692 strain. These findings indicate that determinants in both nsP1 and PE2 have critical and distinct roles in the pathogenesis of RRV-induced musculoskeletal inflammatory disease in mice.


Assuntos
Infecções por Alphavirus/patologia , Infecções por Alphavirus/virologia , Doenças Musculoesqueléticas/virologia , Vírus do Rio Ross/genética , Proteínas Virais/metabolismo , Animais , Sequência de Bases , Cricetinae , Regulação Viral da Expressão Gênica/fisiologia , Inflamação/virologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Músculo Esquelético/patologia , Músculo Esquelético/virologia , Doenças Musculoesqueléticas/patologia , Mutação , RNA Viral , Vírus Reordenados , Vírus do Rio Ross/patogenicidade , Proteínas Virais/genética , Virulência , Replicação Viral
18.
Infect Immun ; 78(6): 2529-43, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20308296

RESUMO

A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal DeltaasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain chi9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/P(cro)) (P(R)), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P(BAD) c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of beta-lactamase, and cloned into pYA4534 under the control of the P(trc) promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain chi9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.


Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Vetores Genéticos , Peste/prevenção & controle , Proteínas Citotóxicas Formadoras de Poros/imunologia , Salmonella typhimurium/imunologia , Yersinia pestis/imunologia , Animais , Antígenos de Bactérias/genética , Vacinas Bacterianas/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Proteínas Citotóxicas Formadoras de Poros/genética , Salmonella typhimurium/genética , Análise de Sobrevida , Células Th1/imunologia , Células Th2/imunologia , Yersinia pestis/genética
19.
Clin Vaccine Immunol ; 17(3): 363-71, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20053873

RESUMO

We are developing a Salmonella vectored vaccine to prevent infant pneumonia and other diseases caused by Streptococcus pneumoniae. One prerequisite for achieving this goal is to construct and evaluate new recombinant attenuated Salmonella vaccine (RASV) strains suitable for use in neonates and infants. Salmonella enterica serovar Typhimurium strain chi9558(pYA4088) specifies delivery of the pneumococcal protective antigen PspA and can protect adult mice from challenge with S. pneumoniae. This strain is completely safe for oral delivery to day-old and infant mice. Here we assess the colonizing ability, immunogenicity, and protective efficacy of chi9558(pYA4088) in neonatal mice. Colonization was assessed in mice 0, 2, 4, or 7 days of age after oral inoculation. In the presence of maternal antibodies, the colonization of lymphoid tissues was delayed, but the immune responses were enhanced in mice born to immunized mothers. Both oral and intranasal routes were used to assess immunogenicity. All orally or intranasally immunized neonatal and infant mice born to either immunized or naïve mothers developed PspA-specific mucosal and systemic immune responses. Mice born to immunized mothers produced higher titers of PspA-specific antibodies in the blood and mucosa and greater numbers of PspA-specific interleukin-4 (IL-4)-secreting cells than mice born to naïve mothers. More importantly, mice born to immune mothers showed a significant increase in protection against S. pneumoniae challenge. These results suggest that strain chi9558(pYA4088) can circumvent some of the limitations of the immature immune system in neonatal and infant mice, generating enhanced protective immune responses in the presence of maternal antibodies.


Assuntos
Animais Recém-Nascidos/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Choque Térmico/imunologia , Vacinas contra Salmonella/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Infecções por Salmonella/prevenção & controle , Vacinas contra Salmonella/administração & dosagem , Salmonella typhimurium , Vacinas Sintéticas
20.
Clin Vaccine Immunol ; 17(3): 354-62, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20053874

RESUMO

Recombinant bacterial vaccines must be safe, efficacious, and well tolerated, especially when administered to newborns and infants to prevent diseases of early childhood. Many means of attenuation have been shown to render vaccine strains susceptible to host defenses or unable to colonize lymphoid tissue effectively, thus decreasing their immunogenicity. We have constructed recombinant attenuated Salmonella vaccine strains that display high levels of attenuation while retaining the ability to induce high levels of immunogenicity and are well tolerated in high doses when administered to infant mice as young as 24 h old. The strains contain three means of regulated delayed attenuation, as well as a constellation of additional mutations that aid in enhancing safety, regulate antigen expression, and reduce disease symptoms commonly associated with Salmonella infection. The vaccine strains are well tolerated when orally administered to infant mice 24 h old at doses as high as 3.5 x 10(8) CFU.


Assuntos
Animais Recém-Nascidos/imunologia , Vacinas contra Salmonella/imunologia , Salmonella typhimurium/imunologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Feminino , Genes Bacterianos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Salmonella typhimurium/genética , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
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