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1.
BMC Infect Dis ; 19(1): 940, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31699037

RESUMO

BACKGROUND: Bacillus anthracis causes a highly lethal infectious disease primarily due to toxin-mediated injury. Antibiotics are no longer effective to treat the accumulation of anthrax toxin, thereby new strategies of antibody treatment are essential. Two anti- anthrax protective antigen (PA) antibodies, hmPA6 and PA21, have been reported by our lab previously. METHODS: The mechanisms of the two antibodies were elucidated by Electrophoresis, Competitive Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test, and in vitro, in vivo (F344 rats) treatment test. The epitopes of the two antibodies were proved by Western blot and Enzyme-linked immune sorbent assay with different domains of PA. RESULTS: In this study, we compared affinity and neutralization of these two antibodies. PA21 was better in protecting cells and rats, whereas hmPA6 had higher affinity. Furthermore, the neutralization mechanisms of the two antibodies and their recognition domains of PA were studied. The results showed that hmPA6 recognized domain IV, thus PA could not bind to cell receptors. Conversely, PA21 recognized domain II, thereby limiting heptamer oligomerization of PA63 in cells. CONCLUSIONS: Our studies elucidated the mechanisms and epitopes of hmPA6 and PA21. The present investigation can advance future use of the two antibodies in anthrax treatment or prophylaxis, and potentially as a combination treatment as the antibodies target different epitopes.


Assuntos
Anticorpos Antibacterianos/metabolismo , Anticorpos Neutralizantes/metabolismo , Bacillus anthracis/imunologia , Animais , Antraz/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eletroforese , Epitopos/análise , Epitopos/imunologia , Imunoensaio , Ratos , Ratos Endogâmicos F344
2.
Immunity ; 51(4): 724-734.e4, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31586542

RESUMO

HIV- and SIV-envelope (Env) trimers are both extensively glycosylated, and antibodies identified to date have been unable to fully neutralize SIVmac239. Here, we report the isolation, structure, and glycan interactions of antibody ITS90.03, a monoclonal antibody that completely neutralized the highly neutralization-resistant isolate, SIVmac239. The co-crystal structure of a fully glycosylated SIVmac239-gp120 core in complex with rhesus CD4 and the antigen-binding fragment of ITS90.03 at 2.5-Å resolution revealed that ITS90 recognized an epitope comprised of 45% glycan. SIV-gp120 core, rhesus CD4, and their complex could each be aligned structurally to their human counterparts. The structure revealed that glycans masked most of the SIV Env protein surface, with ITS90 targeting a glycan hole, which is occupied in ∼83% of SIV strains by glycan N238. Overall, the SIV glycan shield appears to functionally resemble its HIV counterpart in coverage of spike, shielding from antibody, and modulation of receptor accessibility.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Infecções por HIV/imunologia , HIV/fisiologia , Polissacarídeos/química , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Antígenos CD4/metabolismo , Células Cultivadas , Cristalização , Cristalografia por Raios X , Modelos Animais de Doenças , Glicosilação , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Macaca mulatta , Glicoproteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismo
3.
Nat Commun ; 10(1): 3859, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455769

RESUMO

Induction of long-lived antibody responses during infection or vaccination is often essential for subsequent protection, but the relative contributions of T follicular helper (Tfh) cells and T helper 1 (Th1) cells for induction of antigen specific antibody responses to viruses are unclear. Here, we establish an acute Zika virus (ZIKV) infection model in immunocompetent mice, and show that ZIKV infection elicits robust Th1-like Tfh cell and protective antibody responses. While these Th1-like Tfh cells share phenotypic and transcriptomic profiles with both Tfh and Th1 cells, they also have unique surface markers and gene expression characteristics, and are dependent on T-bet for their development. Th1-like Tfh cells, but not Th1 cells, are essential for class switching of ZIKV-specific IgG2c antibodies and maintenance of long-term neutralizing antibody responses. Our study suggests that specific modulation of the Th1-like Tfh cell response during infection or vaccination may augment the induction of antiviral antibody response to ZIKV and other viruses.


Assuntos
Switching de Imunoglobulina/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais/imunologia , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Células Vero , Infecção por Zika virus/virologia
4.
Immunity ; 51(1): 141-154.e6, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315032

RESUMO

The VH1-2 restricted VRC01-class of antibodies targeting the HIV envelope CD4 binding site are a major focus of HIV vaccine strategies. However, a detailed analysis of VRC01-class antibody development has been limited by the rare nature of these responses during natural infection and the lack of longitudinal sampling of such responses. To inform vaccine strategies, we mapped the development of a VRC01-class antibody lineage (PCIN63) in the subtype C infected IAVI Protocol C neutralizer PC063. PCIN63 monoclonal antibodies had the hallmark VRC01-class features and demonstrated neutralization breadth similar to the prototype VRC01 antibody, but were 2- to 3-fold less mutated. Maturation occurred rapidly within ∼24 months of emergence of the lineage and somatic hypermutations accumulated at key contact residues. This longitudinal study of broadly neutralizing VRC01-class antibody lineage reveals early binding to the N276-glycan during affinity maturation, which may have implications for vaccine design.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/fisiologia , Vacinas contra a AIDS/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Afinidade de Anticorpos , Linfócitos B/imunologia , Antígenos CD4/metabolismo , Regiões Determinantes de Complementaridade/genética , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Polissacarídeos/metabolismo , Ligação Proteica
5.
Nat Commun ; 10(1): 3068, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296843

RESUMO

Most neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) target the receptor-binding domain (RBD) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). The epitopes and mechanisms of mAbs targeting non-RBD regions have not been well characterized yet. Here we report the monoclonal antibody 7D10 that binds to the N-terminal domain (NTD) of the spike glycoprotein and inhibits the cell entry of MERS-CoV with high potency. Structure determination and mutagenesis experiments reveal the epitope and critical residues on the NTD for 7D10 binding and neutralization. Further experiments indicate that the neutralization by 7D10 is not solely dependent on the inhibition of DPP4 binding, but also acts after viral cell attachment, inhibiting the pre-fusion to post-fusion conformational change of the spike. These properties give 7D10 a wide neutralization breadth and help explain its synergistic effects with several RBD-targeting antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/sangue , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Linhagem Celular Tumoral , Infecções por Coronavirus/sangue , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Dipeptidil Peptidase 4/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Testes de Neutralização , Ligação Proteica/imunologia , Domínios Proteicos/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Células Vero , Internalização do Vírus
6.
BMC Res Notes ; 12(1): 331, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186065

RESUMO

OBJECTIVE: The bone morphogenetic protein (BMP) signaling pathway comprises the largest subdivision of the transforming growth factor (TGFß) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. Dysregulated BMP signaling underlies human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms and regulation of BMP signaling may yield translational opportunities. Unfortunately, limited tools are available to evaluate this pathway, and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. Specific inhibitors for type 2 receptors are poorly represented. Thus, we sought to identify and validate an antibody that neutralizes the ligand-binding function of BMP receptor type 2 (BMPR2) extracellular domain (ECD). RESULTS: Using a modified, cell-free immunoprecipitation assay, we examined the neutralizing ability of the mouse monoclonal antibody 3F6 and found a dose-dependent inhibition of BMPR2-ECD ligand-binding. Consistent with this, 3F6 blocks endogenous BMPR2 function in the BMP-responsive cell line HEK293T. The specificity of 3F6 action was confirmed by demonstrating that this antibody has no effect on BMP-responsiveness in HEK293T cells in which BMPR2 expression is knocked-down. Our results provide important proof-of-concept data for future studies interrogating BMPR2 function.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células HEK293 , Humanos , Camundongos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
7.
Cell Host Microbe ; 25(6): 873-883.e5, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31194940

RESUMO

The fusion peptide (FP) of HIV-1 envelope glycoprotein (Env) is essential for mediating viral entry. Detection of broadly neutralizing antibodies (bnAbs) that interact with the FP has revealed it as a site of vulnerability. We delineate X-ray and cryo-electron microscopy (cryo-EM) structures of bnAb ACS202, from an HIV-infected elite neutralizer, with an FP and with a soluble Env trimer (AMC011 SOSIP.v4.2) derived from the same patient. We show that ACS202 CDRH3 forms a "ß strand" interaction with the exposed hydrophobic FP and recognizes a continuous region of gp120, including a conserved N-linked glycan at N88. A cryo-EM structure of another previously identified bnAb VRC34.01 with AMC011 SOSIP.v4.2 shows that it also penetrates through glycans to target the FP. We further demonstrate that the FP can twist and present different conformations for recognition by bnAbs, which enables approach to Env from diverse angles. The variable recognition of FP by bnAbs thus provides insights for vaccine design.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Anticorpos Neutralizantes/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
8.
PLoS Pathog ; 15(5): e1007759, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116791

RESUMO

Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV.


Assuntos
Hepacivirus/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Anticorpos Neutralizantes/metabolismo , Células HEK293 , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Conformação Proteica , Multimerização Proteica , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/genética , Internalização do Vírus
9.
PLoS Biol ; 17(4): e3000229, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31039149

RESUMO

Hepatitis A virus (HAV), an enigmatic and ancient pathogen, is a major causative agent of acute viral hepatitis worldwide. Although there are effective vaccines, antivirals against HAV infection are still required, especially during fulminant hepatitis outbreaks. A more in-depth understanding of the antigenic characteristics of HAV and the mechanisms of neutralization could aid in the development of rationally designed antiviral drugs targeting HAV. In this paper, 4 new antibodies-F4, F6, F7, and F9-are reported that potently neutralize HAV at 50% neutralizing concentration values (neut50) ranging from 0.1 nM to 0.85 nM. High-resolution cryo-electron microscopy (cryo-EM) structures of HAV bound to F4, F6, F7, and F9, together with results of our previous studies on R10 fragment of antigen binding (Fab)-HAV complex, shed light on the locations and nature of the epitopes recognized by the 5 neutralizing monoclonal antibodies (NAbs). All the epitopes locate within the same patch and are highly conserved. The key structure-activity correlates based on the antigenic sites have been established. Based on the structural data of the single conserved antigenic site and key structure-activity correlates, one promising drug candidate named golvatinib was identified by in silico docking studies. Cell-based antiviral assays confirmed that golvatinib is capable of blocking HAV infection effectively with a 50% inhibitory concentration (IC50) of approximately 1 µM. These results suggest that the single conserved antigenic site from complete HAV capsid is a good antiviral target and that golvatinib could function as a lead compound for anti-HAV drug development.


Assuntos
Anticorpos Neutralizantes/ultraestrutura , Desenho de Drogas , Vírus da Hepatite A/imunologia , Aminopiridinas/metabolismo , Aminopiridinas/farmacologia , Anticorpos Monoclonais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais , Antígenos Virais , Capsídeo/metabolismo , Simulação por Computador , Epitopos , Antígenos da Hepatite A/metabolismo , Antígenos da Hepatite A/ultraestrutura , Vírus da Hepatite A/patogenicidade , Vírus da Hepatite A/ultraestrutura , Humanos , Piperazinas/metabolismo , Piperazinas/farmacologia , Ligação Proteica
10.
Nat Commun ; 10(1): 2105, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068578

RESUMO

The respiratory syncytial virus (RSV) F glycoprotein is a class I fusion protein that mediates viral entry and is a major target of neutralizing antibodies. Structures of prefusion forms of RSV F, as well as other class I fusion proteins, have revealed compact trimeric arrangements, yet whether these trimeric forms can transiently open remains unknown. Here, we perform structural and biochemical studies on a recently isolated antibody, CR9501, and demonstrate that it enhances the opening of prefusion-stabilized RSV F trimers. The 3.3 Å crystal structure of monomeric RSV F bound to CR9501, combined with analysis of over 25 previously determined RSV F structures, reveals a breathing motion of the prefusion conformation. We also demonstrate that full-length RSV F trimers transiently open and dissociate on the cell surface. Collectively, these findings have implications for the function of class I fusion proteins, as well as antibody prophylaxis and vaccine development for RSV.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas Virais de Fusão/metabolismo , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Linfócitos B/virologia , Simulação por Computador , Cristalografia por Raios X , Desenvolvimento de Medicamentos , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Multimerização Proteica/fisiologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia
11.
Nat Commun ; 10(1): 2190, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097697

RESUMO

HIV-infected infants develop broadly neutralizing plasma responses with more rapid kinetics than adults, suggesting the ontogeny of infant responses could better inform a path to achievable vaccine targets. Here we reconstruct the developmental lineage of BF520.1, an infant-derived HIV-specific broadly neutralizing antibody (bnAb), using computational methods developed specifically for this purpose. We find that the BF520.1 inferred naive precursor binds HIV Env. We also show that heterologous cross-clade neutralizing activity evolved in the infant within six months of infection and that, ultimately, only 2% SHM is needed to achieve the full breadth of the mature antibody. Mutagenesis and structural analyses reveal that, for this infant bnAb, substitutions in the kappa chain were critical for activity, particularly in CDRL1. Overall, the developmental pathway of this infant antibody includes features distinct from adult antibodies, including several that may be amenable to better vaccine responses.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Vacinas contra a AIDS/imunologia , Fatores Etários , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Biologia Computacional/métodos , Reações Cruzadas/imunologia , Desenho de Drogas , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/isolamento & purificação , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/metabolismo , Lactente , Leucócitos Mononucleares , Mutagênese , Análise de Sequência de DNA , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
12.
MAbs ; 11(4): 653-665, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929560

RESUMO

The development of alternative therapeutic strategies to tumor necrosis factor (TNF)-blocking antibodies for the treatment of inflammatory diseases has generated increasing interest. In particular, selective inhibition of TNF receptor 1 (TNFR1) promises a more precise intervention, tackling only the pro-inflammatory responses mediated by TNF while leaving regenerative and pro-survival signals transduced by TNFR2 untouched. We recently generated a monovalent anti-TNFR1 antibody fragment (Fab 13.7) as an efficient inhibitor of TNFR1. To improve the pharmacokinetic properties of Fab 13.7, the variable domains of the heavy and light chains were fused to the N-termini of newly generated heterodimerizing Fc chains. This novel Fc heterodimerization technology, designated "Fc-one/kappa" (Fc1κ) is based on interspersed constant Ig domains substituting the CH3 domains of a γ1 Fc. The interspersed immunoglobulin (Ig) domains originate from the per se heterodimerizing constant CH1 and CLκ domains and contain sequence stretches of an IgG1 CH3 domain, destined to enable interaction with the neonatal Fc receptor, and thus promote extended serum half-life. The resulting monovalent Fv-Fc1κ fusion protein (Atrosimab) retained strong binding to TNFR1 as determined by enzyme-linked immunosorbent assay and quartz crystal microbalance, and potently inhibited TNF-induced activation of TNFR1. Atrosimab lacks agonistic activity for TNFR1 on its own and in the presence of anti-human IgG antibodies and displays clearly improved pharmacokinetic properties.


Assuntos
Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Fragmentos Fc das Imunoglobulinas/genética , Imunoterapia/métodos , Inflamação/terapia , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Dimerização , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Proteínas Recombinantes de Fusão/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores
13.
Cell ; 177(3): 524-540, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31002794

RESUMO

B cells and the antibodies they produce have a deeply penetrating influence on human physiology. Here, we review current understanding of how B cell responses are initiated; the different paths to generate short- and long-lived plasma cells, germinal center cells, and memory cells; and how each path impacts antibody diversity, selectivity, and affinity. We discuss how basic research is informing efforts to generate vaccines that induce broadly neutralizing antibodies against viral pathogens, revealing the special features associated with allergen-reactive IgE responses and uncovering the antibody-independent mechanisms by which B cells contribute to health and disease.


Assuntos
Linfócitos B/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Antígenos/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , Memória Imunológica , Plasmócitos/imunologia , Plasmócitos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas/imunologia
14.
Nat Commun ; 10(1): 1788, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996276

RESUMO

Three Ebolavirus genus viruses cause lethal disease and lack targeted therapeutics: Ebola virus, Sudan virus and Bundibugyo virus. Monoclonal antibody (mAb) cocktails against the surface glycoprotein (GP) present a potential therapeutic strategy. Here we report two crystal structures of the antibody BDBV223, alone and complexed with its GP2 stalk epitope, an interesting site for therapeutic/vaccine design due to its high sequence conservation among ebolaviruses. BDBV223, identified in a human survivor of Bundibugyo virus disease, neutralizes both Bundibugyo virus and Ebola virus, but not Sudan virus. Importantly, the structure suggests that BDBV223 binding interferes with both the trimeric bundle assembly of GP and the viral membrane by stabilizing a conformation in which the monomers are separated by GP lifting or bending. Targeted mutagenesis of BDBV223 to enhance SUDV GP recognition indicates that additional determinants of antibody binding likely lie outside the visualized interactions, and perhaps involve quaternary assembly or membrane-interacting regions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , Reações Cruzadas/imunologia , Cristalografia por Raios X , Ebolavirus/imunologia , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Hibridomas , Mutagênese , Sobreviventes , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
15.
Arch Virol ; 164(6): 1619-1628, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30953201

RESUMO

Control of classical swine fever (CSF) in developing countries is achieved by immunization with attenuated vaccines, such as the lapinized C-strain vaccine that has been widely used in China. However, C-strain has relatively low growth rate in cell cultures, thus affecting productivity of the vaccine for the industry. In this study, eight amino acid residues were mutated on the C-strain backbone, resulting in a cell-adapted strain Cmut8. The mutant strain exhibited rapid growth with titer of about 100 fold higher than its parental C-strain. The mutation sites located at structural proteins Erns and E2 contributed more to cell adaptation than those located in non-structural proteins. Sera collected from pigs inoculated with Cmut8 and C-strain at the same dose showed similar antibody levels and neutralization titers. Pigs inoculated with different doses of Cmut8 (low, medium and high) and with C-strain offered full protection against challenge with a virulent strain, shown as absence of fever and other symptoms, marginal low levels of viral load, and no obvious gross pathological changes in major organs. Unvaccinated control pigs challenged with the virulent strain showed high fever from day 2 post-challenge and apparent clinical symptoms with two deaths. Viral load were markedly elevated in these control pigs after challenge. The pigs inoculated with high dose of Cmut8 did not show fever or other typical CSF symptoms, and no apparent pathological changes were observed in major organs. Besides, the Cmut8 strain did not induce typical fever response in rabbits. These results demonstrate that the cell-adapted Cmut8 strain remains non-pathogenic to the weaned pigs, provides full protection and could be a good candidate vaccine strain for improved yield at lower cost.


Assuntos
Anticorpos Neutralizantes/metabolismo , Vírus da Febre Suína Clássica/patogenicidade , Peste Suína Clássica/virologia , Mutação , Proteínas Estruturais Virais/genética , Adaptação Fisiológica , Animais , Anticorpos Antivirais/metabolismo , Linhagem Celular , Peste Suína Clássica/imunologia , Peste Suína Clássica/mortalidade , Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/crescimento & desenvolvimento , Vírus da Febre Suína Clássica/imunologia , Coelhos , Suínos , Vacinação , Carga Viral , Proteínas Estruturais Virais/imunologia
16.
Emerg Microbes Infect ; 8(1): 272-281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866781

RESUMO

Because of its high infectivity in humans and the lack of effective vaccines, Nipah virus is classified as a category C agent and handling has to be performed under biosafety level 4 conditions in non-endemic countries, which has hindered the development of vaccines. Based on a highly efficient pseudovirus production system using a modified HIV backbone vector, a pseudovirus-based mouse model has been developed for evaluating the efficacy of Nipah vaccines in biosafety level 2 facilities. For the first time, the correlates of protection have been identified in a mouse model. The limited levels of neutralizing antibodies against immunogens fusion protein (F), glycoprotein (G), and combination of F and G (FG) were found to be 148, 275, and 115, respectively, in passive immunization. Relatively lower limited levels of protection of 52, and 170 were observed for immunogens F, and G, respectively, in an active immunization model. Although the minimal levels for protection of neutralizing antibody in passive immunization were slightly higher than those in active immunization, neutralizing antibody played a key role in protection against Nipah virus infection. The immunogens F and G provided similar protection, and the combination of these immunogens did not provide better outcomes. Either immunogen F or G would provide sufficient protection for Nipah vaccine. The Nipah pseudovirus mouse model, which does not involve highly pathogenic virus, has the potential to greatly facilitate the standardization and implementation of an assay to propel the development of NiV vaccines.


Assuntos
Infecções por Henipavirus/prevenção & controle , Vírus Nipah/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Animais , Anticorpos Neutralizantes/metabolismo , Contenção de Riscos Biológicos , Cães , Infecções por Henipavirus/imunologia , Humanos , Células Madin Darby de Rim Canino , Camundongos , Vacinas Virais/imunologia
17.
Biologicals ; 59: 56-61, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30898479

RESUMO

The Rapid Fluorescent Focus Inhibition Test (RFFIT) is a standard assay used to detect and assess the titers of rabies virus neutralizing antibodies (RVNA) in blood sera. To simplify the multistep RFFIT procedure by eliminating the immunostaining step, we generated a new recombinant RV expressing a green fluorescent protein (rRV-GFP) and assess its suitability for quantifying RVNA. We rescued the rRV-GFP virus from plasmid DNA carrying a full-length genome of the CVS-N2c strain of RV in which the eGFP gene was inserted between the glycoprotein and RNA-polymerase genes. The recombinant virus was genetically stable and grew efficiently in appropriate cells expressing sufficient GFP fluorescence to detect directly 20 h post infection (hpi). We evaluated the feasibility of using rRV-GFP in RFFIT by comparing RVNA titers in 27 serum samples measured by conventional RFFIT and RFFIT-GFP. A linear regression analysis of the data demonstrated a good agreement between these two methods (r = 0.9776) including results with samples having RVNA titers close to the minimally acceptable vaccine potency threshold (0.5 IU/ml). Study results showed that the rRV-GFP virus could replace the CVS-11 challenge virus currently used in the conventional RFFIT and enabling more rapid, simpler, and less expensive detection and quantitation of RVNA.


Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Animais , Anticorpos Neutralizantes/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cobaias , Humanos , Medições Luminescentes/métodos , Camundongos , Testes de Neutralização , Coelhos , Raiva/prevenção & controle , Raiva/virologia , Vacinas Antirrábicas/administração & dosagem , Vírus da Raiva/genética , Vírus da Raiva/metabolismo , Recombinação Genética
18.
PLoS One ; 14(3): e0210477, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30845175

RESUMO

Enterovirus (EV) 71 is the main pathogen associated with hand-foot-mouth disease (HFMD) and can lead to the disease with severe mortality in children. Since 2009, in the Republic of Korea, an outbreak of EV71 C4a infection with neurologic involvement emerged, where in HFMD involvement was identified and central nervous system complications were reported. In this study, EV71 C4a virus-like particles (VLPs) produced by recombinant technology were generated in a baculovirus expression system. To improve the production yield, EV71 VLP was constructed using the dual promoter system baculovirus P1 and 3CD (baculo-P1-3CD), which harbored both the structural protein-encoding P1 region under the control of the polyhedron promoter and the 3CD protease gene under the regulation of the CMV-IE, lef3, gp41, or chitinase promoters to augment the level of gene transcription. Efficient VLP expression was demonstrated through optimization of incubation time and insect cell type. In addition, to evaluate the potential of VLP as a vaccine candidate, we tested the neutralizing antibodies and total anti-EV71 IgG from the purified EV71 C4a VLP serum. The recombinant EV71 VLP exhibited the morphology of self-assembled VLP, as determined by electron microscopy. Use of baculo-P1-3CD-gp41 led to a high yield (11.3mg/L < 40kDa) of VLPs in High-FiveTM cells at 3 days post-infection. Furthermore, the potential of VLP as a vaccine was evaluated through the neutralizing ability elicited by the purified EV71 VLP after immunization of BALB/c mice, which was shown to induce potent and long-lasting humoral immune responses as evidenced by the cross-neutralization titer. Our results could be used to expedite the developmental process for vaccines under clinical trials and to ensure manufacturing consistency for licensing requirements.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Enterovirus Humano A/genética , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Baculoviridae , Enterovirus Humano A/imunologia , Feminino , Engenharia Genética , Camundongos , Camundongos Endogâmicos BALB C , Células Sf9 , Vacinação , Vacinas de Partículas Semelhantes a Vírus/imunologia , Células Vero
19.
Cell ; 176(6): 1420-1431.e17, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849373

RESUMO

Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses ∼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.


Assuntos
Anticorpos Neutralizantes/imunologia , Vírus Sinciciais Respiratórios/imunologia , Vacinação/métodos , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Caveolina 1 , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/uso terapêutico , Cultura Primária de Células , Vírus Sinciciais Respiratórios/patogenicidade , Vacinas/imunologia , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/fisiologia
20.
Biomed Microdevices ; 21(1): 14, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30725230

RESUMO

Minimally invasive point-of-care diagnostic devices are of great interest for rapid detection of biomarkers in diverse settings. Although blood is the most common source of biomarkers, interstitial fluid (ISF) is an alternate body fluid that does not clot or contain red blood cells that often complicate analysis. However, ISF is difficult to collect. In this study, we assessed the utility of a microneedle patch to sample microliter volumes of ISF in a simple and minimally invasive manner. We demonstrated the use of ISF collected in this way for therapeutic drug monitoring by showing similar vancomycin pharmacokinetic profiles in ISF and serum from rats. We also measured polio-specific neutralizing antibodies and anti-polio IgG in ISF similar to serum in rats immunized with polio vaccine. These studies demonstrate the potential utility of ISF collected by microneedle patch in therapeutic drug monitoring and immunodiagnostic applications.


Assuntos
Derme/metabolismo , Monitoramento de Medicamentos , Líquido Extracelular/metabolismo , Agulhas , Vancomicina , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Biomarcadores/metabolismo , Monitoramento de Medicamentos/instrumentação , Monitoramento de Medicamentos/métodos , Feminino , Imunoglobulina G/metabolismo , Injeções Intradérmicas/instrumentação , Injeções Intradérmicas/métodos , Poliovirus/metabolismo , Vacinas contra Poliovirus/farmacologia , Ratos , Ratos Wistar , Vancomicina/farmacocinética , Vancomicina/farmacologia
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