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1.
EBioMedicine ; 46: 215-226, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31326432

RESUMO

BACKGROUND: Profiles of immunity developed in filovirus patients and survivors have begun to shed light on antigen-specific cellular immune responses that had been previously under-studied. However, our knowledge of the breadth and length of those responses and the viral targets which mediate long-term memory immunity still lags significantly behind. METHODS: We characterized antigen-specific immune responses in whole blood samples of fifteen years post-infected survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). We examined T cell and IgG responses against SUDV complete antigen and four SUDV proteins; glycoprotein (GP), nucleoprotein (NP), and viral protein 30 (VP30), and 40 (VP40). FINDINGS: We found survivors-maintained antigen-specific CD4+ T cell memory immune responses mediated mainly by the viral protein NP. In contrast, activated CD8+ T cell responses were nearly absent in SUDV survivors, regardless of the stimulating antigen used. Analysis of anti-viral humoral immunity revealed antigen-specific IgG antibodies against SUDV and SUDV proteins. Survivor IgGs mediated live SUDV neutralization in vitro and FcγRI and FcγRIII antibody Fc-dependent responses, mainly via antibodies to the viral proteins GP and VP40. INTERPRETATION: We highlight the key role of several proteins, i.e., GP, NP, and VP40, to act as mediators of distinctive and sustained cellular memory immune responses in long-term SUDV survivors. We suggest that the inclusion of these viral proteins in vaccine development may best mimic survivor native memory immune responses with the potential of protecting against viral infection. FUNDS: This research was funded by the Defense Threat Reduction Agency (CB4088) and by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI111516. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

2.
Nature ; 563(7732): 559-563, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30464266

RESUMO

The zoonotic transmission of hantaviruses from their rodent hosts to humans in North and South America is associated with a severe and frequently fatal respiratory disease, hantavirus pulmonary syndrome (HPS)1,2. No specific antiviral treatments for HPS are available, and no molecular determinants of in vivo susceptibility to hantavirus infection and HPS are known. Here we identify the human asthma-associated gene protocadherin-1 (PCDH1)3-6 as an essential determinant of entry and infection in pulmonary endothelial cells by two hantaviruses that cause HPS, Andes virus (ANDV) and Sin Nombre virus (SNV). In vitro, we show that the surface glycoproteins of ANDV and SNV directly recognize the outermost extracellular repeat domain of PCDH1-a member of the cadherin superfamily7,8-to exploit PCDH1 for entry. In vivo, genetic ablation of PCDH1 renders Syrian golden hamsters highly resistant to a usually lethal ANDV challenge. Targeting PCDH1 could provide strategies to reduce infection and disease caused by New World hantaviruses.

3.
Cell Host Microbe ; 22(5): 688-696.e5, 2017 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-29120745

RESUMO

Arenaviruses cause fatal hemorrhagic disease in humans. Old World arenavirus glycoproteins (GPs) mainly engage α-dystroglycan as a cell-surface receptor, while New World arenaviruses hijack transferrin receptor. However, the Lujo virus (LUJV) GP does not cluster with New or Old World arenaviruses. Using a recombinant vesicular stomatitis virus containing LUJV GP as its sole attachment and fusion protein (VSV-LUJV), we demonstrate that infection is independent of known arenavirus receptor genes. A genome-wide haploid genetic screen identified the transmembrane protein neuropilin 2 (NRP2) and tetraspanin CD63 as factors for LUJV GP-mediated infection. LUJV GP binds the N-terminal domain of NRP2, while CD63 stimulates pH-activated LUJV GP-mediated membrane fusion. Overexpression of NRP2 or its N-terminal domain enhances VSV-LUJV infection, and cells lacking NRP2 are deficient in wild-type LUJV infection. These findings uncover this distinct set of host cell entry factors in LUJV infection and are attractive focus points for therapeutic intervention.


Assuntos
Lujo virus/fisiologia , Neuropilina-2/metabolismo , Tetraspanina 30/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , Proteínas de Transporte , Linhagem Celular , Interações Hospedeiro-Patógeno/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Lujo virus/genética , Lujo virus/patogenicidade , Domínios e Motivos de Interação entre Proteínas , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina , Proteínas Virais de Fusão/genética , Proteínas Virais/genética
4.
J Exp Med ; 214(9): 2563-2572, 2017 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-28724616

RESUMO

Until recently, immune responses in filovirus survivors remained poorly understood. Early studies revealed IgM and IgG responses to infection with various filoviruses, but recent outbreaks have greatly expanded our understanding of filovirus immune responses. Immune responses in survivors of Ebola virus (EBOV) and Sudan virus (SUDV) infections have provided the most insight, with T cell responses as well as detailed antibody responses having been characterized. Immune responses to Marburg virus (MARV), however, remain almost entirely uncharacterized. We report that immune responses in MARV survivors share characteristics with EBOV and SUDV infections but have some distinct differences. MARV survivors developed multivariate CD4+ T cell responses but limited CD8+ T cell responses, more in keeping with SUDV survivors than EBOV survivors. In stark contrast to SUDV survivors, rare neutralizing antibody responses in MARV survivors diminished rapidly after the outbreak. These results warrant serious consideration for any vaccine or therapeutic that seeks to be broadly protective, as different filoviruses may require different immune responses to achieve immunity.


Assuntos
Anticorpos Neutralizantes/imunologia , Doença do Vírus de Marburg/imunologia , Marburgvirus/imunologia , Células Th1/imunologia , Adolescente , Adulto , Animais , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/metabolismo , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunidade Celular/imunologia , Masculino , Doença do Vírus de Marburg/mortalidade , Pessoa de Meia-Idade , Sobreviventes , Uganda/epidemiologia , Adulto Jovem
5.
Cell ; 169(5): 891-904.e15, 2017 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-28525756

RESUMO

While neutralizing antibodies are highly effective against ebolavirus infections, current experimental ebolavirus vaccines primarily elicit species-specific antibody responses. Here, we describe an immunization-elicited macaque antibody (CA45) that clamps the internal fusion loop with the N terminus of the ebolavirus glycoproteins (GPs) and potently neutralizes Ebola, Sudan, Bundibugyo, and Reston viruses. CA45, alone or in combination with an antibody that blocks receptor binding, provided full protection against all pathogenic ebolaviruses in mice, guinea pigs, and ferrets. Analysis of memory B cells from the immunized macaque suggests that elicitation of broadly neutralizing antibodies (bNAbs) for ebolaviruses is possible but difficult, potentially due to the rarity of bNAb clones and their precursors. Unexpectedly, germline-reverted CA45, while exhibiting negligible binding to full-length GP, bound a proteolytically remodeled GP with picomolar affinity, suggesting that engineered ebolavirus vaccines could trigger rare bNAb precursors more robustly. These findings have important implications for developing pan-ebolavirus vaccine and immunotherapeutic cocktails.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Vacinas contra Ebola/imunologia , Doença pelo Vírus Ebola/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Regiões Determinantes de Complementaridade , Reações Cruzadas , Ebolavirus/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Feminino , Furões , Cobaias , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares
6.
Science ; 354(6310): 350-354, 2016 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-27608667

RESUMO

There is an urgent need for monoclonal antibody (mAb) therapies that broadly protect against Ebola virus and other filoviruses. The conserved, essential interaction between the filovirus glycoprotein, GP, and its entry receptor Niemann-Pick C1 (NPC1) provides an attractive target for such mAbs but is shielded by multiple mechanisms, including physical sequestration in late endosomes. Here, we describe a bispecific-antibody strategy to target this interaction, in which mAbs specific for NPC1 or the GP receptor-binding site are coupled to a mAb against a conserved, surface-exposed GP epitope. Bispecific antibodies, but not parent mAbs, neutralized all known ebolaviruses by coopting viral particles themselves for endosomal delivery and conferred postexposure protection against multiple ebolaviruses in mice. Such "Trojan horse" bispecific antibodies have potential as broad antifilovirus immunotherapeutics.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas de Transporte/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Glicoproteínas de Membrana/imunologia , Receptores Virais/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação/imunologia , Linhagem Celular Tumoral , Endossomos/virologia , Doença pelo Vírus Ebola/terapia , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Internalização do Vírus
7.
Clin Vaccine Immunol ; 23(8): 717-24, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27335383

RESUMO

A detailed understanding of serological immune responses to Ebola and Marburg virus infections will facilitate the development of effective diagnostic methods, therapeutics, and vaccines. We examined antibodies from Ebola or Marburg survivors 1 to 14 years after recovery from disease, by using a microarray that displayed recombinant nucleoprotein (NP), viral protein 40 (VP40), envelope glycoprotein (GP), and inactivated whole virions from six species of filoviruses. All three outbreak cohorts exhibited significant antibody responses to antigens from the original infecting species and a pattern of additional filoviruses that varied by outbreak. NP was the most cross-reactive antigen, while GP was the most specific. Antibodies from survivors of infections by Marburg marburgvirus (MARV) species were least cross-reactive, while those from survivors of infections by Sudan virus (SUDV) species exhibited the highest cross-reactivity. Based on results revealed by the protein microarray, persistent levels of antibodies to GP, NP, and VP40 were maintained for up to 14 years after infection, and survival of infection caused by one species imparted cross-reactive antibody responses to other filoviruses.


Assuntos
Anticorpos Antivirais/sangue , Reações Cruzadas , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença do Vírus de Marburg/imunologia , Marburgvirus/imunologia , Animais , Formação de Anticorpos , Antígenos Virais/imunologia , Estudos de Coortes , Surtos de Doenças , Voluntários Saudáveis , Doença pelo Vírus Ebola/epidemiologia , Humanos , Doença do Vírus de Marburg/epidemiologia , Análise em Microsséries , Análise Serial de Proteínas , Sobreviventes , Proteínas Estruturais Virais/genética
8.
Viruses ; 8(5)2016 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-27187443

RESUMO

Robust humoral and cellular immunity are critical for survival in humans during an ebolavirus infection. However, the interplay between these two arms of immunity is poorly understood. To address this, we examined residual immune responses in survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). Cytokine and chemokine expression levels in SUDV stimulated whole blood cultures were assessed by multiplex ELISA and flow cytometry. Antibody and corresponding neutralization titers were also determined. Flow cytometry and multiplex ELISA results demonstrated significantly higher levels of cytokine and chemokine responses in survivors with serological neutralizing activity. This correspondence was not detected in survivors with serum reactivity to SUDV but without neutralization activity. This previously undefined relationship between memory CD4 T cell responses and serological neutralizing capacity in SUDV survivors is key for understanding long lasting immunity in survivors of filovirus infections.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Testes de Neutralização , Sobreviventes , Uganda
9.
Sci Rep ; 6: 24897, 2016 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-27109916

RESUMO

Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities.


Assuntos
Animais Geneticamente Modificados , Anticorpos Antivirais/sangue , Anticorpos Antivirais/uso terapêutico , Bovinos , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização Passiva , Imunoglobulina G/sangue , Imunoglobulina G/uso terapêutico , Camundongos Endogâmicos BALB C , Testes de Neutralização , Ressonância de Plasmônio de Superfície , Resultado do Tratamento
10.
Proc Natl Acad Sci U S A ; 113(16): 4458-63, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27044104

RESUMO

Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/farmacologia , Febre Hemorrágica Americana/tratamento farmacológico , Febre Hemorrágica Americana/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Cobaias , Humanos , Vírus Junin , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia
11.
Elife ; 42015 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-26698106

RESUMO

Biological factors that influence the host range and spillover of Ebola virus (EBOV) and other filoviruses remain enigmatic. While filoviruses infect diverse mammalian cell lines, we report that cells from African straw-colored fruit bats (Eidolon helvum) are refractory to EBOV infection. This could be explained by a single amino acid change in the filovirus receptor, NPC1, which greatly reduces the affinity of EBOV-NPC1 interaction. We found signatures of positive selection in bat NPC1 concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls EBOV infection in Eidolon helvum cells. Our work identifies NPC1 as a genetic determinant of filovirus susceptibility in bats, and suggests that some NPC1 variations reflect host adaptations to reduce filovirus replication and virulence. A single viral mutation afforded escape from receptor control, revealing a pathway for compensatory viral evolution and a potential avenue for expansion of filovirus host range in nature.


Assuntos
Filoviridae/fisiologia , Especificidade de Hospedeiro , Glicoproteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Animais , Linhagem Celular , Quirópteros
12.
PLoS One ; 10(9): e0137786, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26422247

RESUMO

DNA vaccination of transchromosomal bovines (TcBs) with DNA vaccines expressing the codon-optimized (co) glycoprotein (GP) genes of Ebola virus (EBOV) and Sudan virus (SUDV) produce fully human polyclonal antibodies (pAbs) that recognize both viruses and demonstrate robust neutralizing activity. Each TcB was vaccinated by intramuscular electroporation (IM-EP) a total of four times and at each administration received 10 mg of the EBOV-GPco DNA vaccine and 10 mg of the SUDV-GPco DNA vaccine at two sites on the left and right sides, respectively. After two vaccinations, robust antibody responses (titers > 1000) were detected by ELISA against whole irradiated EBOV or SUDV and recombinant EBOV-GP or SUDV-GP (rGP) antigens, with higher titers observed for the rGP antigens. Strong, virus neutralizing antibody responses (titers >1000) were detected after three vaccinations when measured by vesicular stomatitis virus-based pseudovirion neutralization assay (PsVNA). Maximal neutralizing antibody responses were identified by traditional plaque reduction neutralization tests (PRNT) after four vaccinations. Neutralizing activity of human immunoglobulins (IgG) purified from TcB plasma collected after three vaccinations and injected intraperitoneally (IP) into mice at a 100 mg/kg dose was detected in the serum by PsVNA up to 14 days after administration. Passive transfer by IP injection of the purified IgG (100 mg/kg) to groups of BALB/c mice one day after IP challenge with mouse adapted (ma) EBOV resulted in 80% protection while all mice treated with non-specific pAbs succumbed. Similarly, interferon receptor 1 knockout (IFNAR(-/-)) mice receiving the purified IgG (100 mg/kg) by IP injection one day after IP challenge with wild type SUDV resulted in 89% survival. These results are the first to demonstrate that filovirus GP DNA vaccines administered to TcBs by IM-EP can elicit neutralizing antibodies that provide post-exposure protection. Additionally, these data describe production of fully human IgG in a large animal system, a system which is capable of producing large quantities of a clinical grade therapeutic product.


Assuntos
Anticorpos Antivirais/metabolismo , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Profilaxia Pós-Exposição , Vacinas de DNA/imunologia , Animais , Animais Geneticamente Modificados , Anticorpos Neutralizantes/imunologia , Bovinos/genética , Bovinos/imunologia , Cromossomos Artificiais Humanos/genética , República Democrática do Congo , Vacinas contra Ebola/imunologia , Feminino , Doença pelo Vírus Ebola/virologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Profilaxia Pós-Exposição/métodos , Receptor de Interferon alfa e beta/genética , Sudão , Vacinação/métodos
13.
Cell Rep ; 12(12): 2111-20, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26365189

RESUMO

MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/virologia , Evasão da Resposta Imune/genética , Imunização Passiva , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Sequência de Bases , Ebolavirus/genética , Ebolavirus/patogenicidade , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/mortalidade , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Macaca mulatta , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Estudos Retrospectivos , Análise de Sobrevida , Tabaco/genética , Replicação Viral
14.
MBio ; 6(3): e00565-15, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-26015498

RESUMO

UNLABELLED: Recent work demonstrated that the Niemann-Pick C1 (NPC1) protein is an essential entry receptor for filoviruses. While previous studies focused on filovirus entry requirements of NPC1 in vitro, its roles in filovirus replication and pathogenesis in vivo remain unclear. Here, we evaluated the importance of NPC1, and its partner in cholesterol transport, NPC2, by using a mouse model of Ebolavirus (EBOV) disease. We found that, whereas wild-type mice had high viral loads and succumbed to EBOV infection, Npc1(-/-) mice were entirely free of viral replication and completely protected from EBOV disease. Interestingly, Npc1(+/-) mice transiently developed high levels of viremia, but were nevertheless substantially protected from EBOV challenge. We also found Npc2(-/-) mice to be fully susceptible to EBOV infection, while Npc1(-/-) mice treated to deplete stored lysosomal cholesterol remained completely resistant to EBOV infection. These results provide mechanistic evidence that NPC1 is directly required for EBOV infection in vivo, with little or no role for NPC1/NPC2-dependent cholesterol transport. Finally, we assessed the in vivo antiviral efficacies of three compounds known to inhibit NPC1 function or NPC1-glycoprotein binding in vitro. Two compounds reduced viral titers in vivo and provided a modest, albeit not statistically significant, degree of protection. Taken together, our results show that NPC1 is critical for replication and pathogenesis in animals and is a bona fide target for development of antifilovirus therapeutics. Additionally, our findings with Npc1(+/-) mice raise the possibility that individuals heterozygous for NPC1 may have a survival advantage in the face of EBOV infection. IMPORTANCE: Researchers have been searching for an essential filovirus receptor for decades, and numerous candidate receptors have been proposed. However, none of the proposed candidate receptors has proven essential in all in vitro scenarios, nor have they proven essential when evaluated using animal models. In this report, we provide the first example of a knockout mouse that is completely refractory to EBOV infection, replication, and disease. The findings detailed here provide the first critical in vivo data illustrating the absolute requirement of NPC1 for filovirus infection in mice. Our work establishes NPC1 as a legitimate target for the development of anti-EBOV therapeutics. However, the limited success of available NPC1 inhibitors to protect mice from EBOV challenge highlights the need for new molecules or approaches to target NPC1 in vivo.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno , Proteínas/metabolismo , Replicação Viral , Animais , Colesterol/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Proteínas/genética , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/metabolismo
15.
Viruses ; 7(1): 37-51, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25569078

RESUMO

Recovery from ebolavirus infection in humans is associated with the development of both cell-mediated and humoral immune responses. According to recent studies, individuals that did not survive infection with ebolaviruses appear to have lacked a robust adaptive immune response and the expression of several early innate response markers. However, a comprehensive protective immune profile has yet to be described. Here, we examine cellular memory immune responses among survivors of two separate Ebolavirus outbreaks (EVDs) due to Sudan virus (SUDV) infection in Uganda-Gulu 2000-2001 and Kibaale 2012. Freshly collected blood samples were stimulated with inactivated SUDV, as well as with recombinant SUDV or Ebola virus (EBOV) GP (GP1-649). In addition, ELISA and plaque reduction neutralization assays were performed to determine anti-SUDV IgG titers and neutralization capacity. Cytokine expression was measured in whole blood cultures in response to SUDV and SUDV GP stimulation in both survivor pools, demonstrating recall responses that indicate immune memory. Cytokine responses between groups were similar but had distinct differences. Neutralizing, SUDV-specific IgG activity against irradiated SUDV and SUDV recombinant proteins were detected in both survivor cohorts. Furthermore, humoral and cell-mediated crossreactivity to EBOV and EBOV recombinant GP1-649 was observed in both cohorts. In conclusion, immune responses in both groups of survivors demonstrate persistent recognition of relevant antigens, albeit larger cohorts are required in order to reach greater statistical significance. The differing cytokine responses between Gulu and Kibaale outbreak survivors suggests that each outbreak may not yield identical memory responses and promotes the merits of studying the immune responses among outbreaks of the same virus. Finally, our demonstration of cross-reactive immune recognition suggests that there is potential for developing cross-protective vaccines for ebolaviruses.


Assuntos
Surtos de Doenças , Ebolavirus/imunologia , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/imunologia , Memória Imunológica , Anticorpos Antivirais/sangue , Estudos de Coortes , Reações Cruzadas , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imunidade Celular , Imunoglobulina G/sangue , Testes de Neutralização , Sudão , Sobreviventes , Uganda/epidemiologia
16.
Science ; 344(6191): 1506-10, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24970085

RESUMO

Lassa virus spreads from a rodent to humans and can lead to lethal hemorrhagic fever. Despite its broad tropism, chicken cells were reported 30 years ago to resist infection. We found that Lassa virus readily engaged its cell-surface receptor α-dystroglycan in avian cells, but virus entry in susceptible species involved a pH-dependent switch to an intracellular receptor, the lysosome-resident protein LAMP1. Iterative haploid screens revealed that the sialyltransferase ST3GAL4 was required for the interaction of the virus glycoprotein with LAMP1. A single glycosylated residue in LAMP1, present in susceptible species but absent in birds, was essential for interaction with the Lassa virus envelope protein and subsequent infection. The resistance of Lamp1-deficient mice to Lassa virus highlights the relevance of this receptor switch in vivo.


Assuntos
Vírus Lassa/fisiologia , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/virologia , Células Cultivadas , Galinhas , Distroglicanas/genética , Distroglicanas/metabolismo , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Febre Lassa/virologia , Proteína 1 de Membrana Associada ao Lisossomo/química , Lisossomos/metabolismo , Lisossomos/virologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Ligação Proteica , Sialiltransferases/metabolismo
18.
J Infect Dis ; 208(2): 299-309, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23585686

RESUMO

To better understand humoral immunity following ebolavirus infection, a serological study of the humoral immune response against the individual viral proteins of Sudan ebolavirus (Gulu) in human survivors was performed. An enzyme-linked immunosorbent assay specific for full-length recombinant viral proteins NP, VP30, VP40, and GP1-649 (GP lacking the transmembrane domain) of Sudan ebolavirus (Gulu) was used as well as a plaque reduction neutralization test. Serum samples from human survivors, which were collected up to 10 years following recovery, were screened and analyzed. Results demonstrate that samples obtained 10 years following infection contain virus-specific antibodies that can neutralize virus. Neutralization correlates well with immunoreactivity against the viral proteins NP, VP30, and GP1-649. Sera from individuals who died or those with no documented infection but immunoreactive to ebolavirus did not neutralize. This work provides insight into the duration, profile of immunoreactivity, and neutralization capacity of the humoral immune response in ebolavirus survivors.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/sangue , Linhagem Celular , Cercopithecus aethiops , Células HEK293 , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Imunidade Humoral/imunologia , Imunoglobulina A/imunologia , Testes de Neutralização/métodos , Sudão , Sobreviventes , Células Vero , Proteínas do Envelope Viral/imunologia
19.
J Virol ; 87(9): 4952-64, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23408633

RESUMO

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.


Assuntos
Ebolavirus/imunologia , Vírus da Encefalite Equina Venezuelana/genética , Doença pelo Vírus Ebola/prevenção & controle , Replicon , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Ebolavirus/genética , Vírus da Encefalite Equina Venezuelana/fisiologia , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Macaca fascicularis , Vacinação , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
20.
Proc Natl Acad Sci U S A ; 109(13): 5034-9, 2012 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-22411795

RESUMO

Antibody therapies to prevent or limit filovirus infections have received modest interest in recent years, in part because of early negative experimental evidence. We have overcome the limitations of this approach, leveraging the use of antibody from nonhuman primates (NHPs) that survived challenge to filoviruses under controlled conditions. By using concentrated, polyclonal IgG antibody from these survivors, we treated filovirus-infected NHPs with multiple doses administered over the clinical phase of disease. In the first study, Marburg virus (MARV)-infected NHPs were treated 15 to 30 min postexposure with virus-specific IgG, with additional treatments on days 4 and 8 postexposure. The postexposure IgG treatment was completely protective, with no signs of disease or detectable viremia. MARV-specific IgM antibody responses were generated, and all macaques survived rechallenge with MARV, suggesting that they generated an immune response to virus replication. In the next set of studies, NHPs were infected with MARV or Ebola virus (EBOV), and treatments were delayed 48 h, with additional treatments on days 4 and 8 postexposure. The delayed treatments protected both MARV- and EBOV-challenged NHPs. In both studies, two of the three IgG-treated NHPs had no clinical signs of illness, with the third NHP developing mild and delayed signs of disease followed by full recovery. These studies clearly demonstrate that postexposure antibody treatments can protect NHPs and open avenues for filovirus therapies for human use using established Food and Drug Administration-approved polyclonal or monoclonal antibody technologies.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Filoviridae/imunologia , Infecções por Filoviridae/prevenção & controle , Filoviridae/imunologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , Animais , Fracionamento Químico , Ebolavirus/imunologia , Infecções por Filoviridae/virologia , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Marburgvirus/imunologia , Testes de Neutralização , Especificidade da Espécie , Análise de Sobrevida
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