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1.
Nat Microbiol ; 4(5): 734-747, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30886356

RESUMO

Antibodies serve as critical barriers to viral infection. Humoral immunity to a virus is achieved through the dual role of antibodies in communicating the presence of invading pathogens in infected cells to effector cells, and in interfering with processes essential to the viral life cycle (chiefly entry into the host cell). For individuals that successfully control infection, virus-elicited antibodies can provide lifelong surveillance and protection from future insults. One approach to understand the nature of a successful immune response has been to utilize structural biology to uncover the molecular details of antibodies derived from vaccines or natural infection and how they interact with their cognate microbial antigens. The ability to isolate antigen-specific B-cells and rapidly solve structures of functional, monoclonal antibodies in complex with viral glycoprotein surface antigens has greatly expanded our knowledge of the sites of vulnerability on viruses. In this Review, we compare the adaptive humoral immune responses to human immunodeficiency virus (HIV), influenza and filoviruses, with a particular focus on neutralizing antibodies. The pathogenesis of each of these viruses is quite different, providing an opportunity for comparison of immune responses: HIV causes a persistent, chronic infection; influenza, an acute infection with multiple exposures during a lifetime and annual vaccination; filoviruses, a virulent, acute infection. Neutralizing antibodies that develop under these different constraints are therefore sentinels that can provide insight into the underlying humoral immune responses, as well as important lessons to guide future development of vaccines and immunotherapeutics.


Assuntos
Anticorpos Antivirais/imunologia , Viroses/imunologia , Vírus/imunologia , Animais , Formação de Anticorpos , Humanos , Viroses/virologia , Vírus/genética
2.
Cell Rep ; 24(10): 2723-2732.e4, 2018 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-30184505

RESUMO

Monoclonal antibodies (mAbs) with pan-ebolavirus cross-reactivity are highly desirable, but development of such mAbs is limited by a lack of a molecular understanding of cross-reactive epitopes. The antibody ADI-15878 was previously identified from a human survivor of Ebola virus Makona variant (EBOV/Mak) infection. This mAb demonstrated potent neutralizing activity against all known ebolaviruses and provided protection in rodent and ferret models against three ebolavirus species. Here, we describe the unliganded crystal structure of ADI-15878 as well as the cryo-EM structures of ADI-15878 in complex with the EBOV/Mak and Bundibugyo virus (BDBV) glycoproteins (GPs). ADI-15878 binds through an induced-fit mechanism by targeting highly conserved residues in the internal fusion loop (IFL), bridging across GP protomers via the heptad repeat 1 (HR1) region. Our structures provide a more complete description of the ebolavirus immunogenic landscape, as well as a molecular basis for how rare but potent antibodies target conserved filoviral fusion machinery.

3.
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.

4.
J Infect Dis ; 218(suppl_5): S612-S626, 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-29860496

RESUMO

Background: For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases. Methods: In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses. Results: Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent. Conclusions: This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.

5.
Nat Microbiol ; 3(6): 670-677, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29736037

RESUMO

Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMapp, which has been shown to be effective in nonhuman primate models of infection 1 and has been used under compassionate-treatment protocols in humans 2 . ZMapp is a mixture of three chimerized murine monoclonal antibodies (mAbs)3-6 that target EBOV-specific epitopes on the surface glycoprotein7,8. However, ZMapp mAbs do not neutralize other species from the genus Ebolavirus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here, we describe three naturally occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane-proximal external region (MPER) of the glycoprotein. The identification of a conserved neutralizing antigenic site in the glycoprotein suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2-MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.

6.
Cell ; 169(5): 878-890.e15, 2017 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-28525755

RESUMO

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, we mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Our findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Vacinas contra Ebola/imunologia , Doença pelo Vírus Ebola/imunologia , Sobreviventes , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Cercopithecus aethiops , Reações Cruzadas , Ebolavirus/classificação , Ebolavirus/imunologia , Feminino , Furões , Doença pelo Vírus Ebola/virologia , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Alinhamento de Sequência , Células Vero
7.
Nat Microbiol ; 1(9): 16128, 2016 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-27562261

RESUMO

The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little is known regarding its structure or functional role. A minor product, resulting from transcriptional editing, is the transmembrane-anchored, trimeric viral surface glycoprotein (GP). GP mediates attachment to and entry into host cells, and is the intended target of antibody therapeutics. Because large portions of sequence are shared between GP and sGP, it has been hypothesized that sGP may potentially subvert the immune response or may contribute to pathogenicity. In this study, we present cryo-electron microscopy structures of GP and sGP in complex with GP-specific and GP/sGP cross-reactive antibodies undergoing human clinical trials. The structure of the sGP dimer presented here, in complex with both an sGP-specific antibody and a GP/sGP cross-reactive antibody, permits us to unambiguously assign the oligomeric arrangement of sGP and compare its structure and epitope presentation to those of GP. We also provide biophysical evaluation of naturally occurring GP/sGP mutations that fall within the footprints identified by our high-resolution structures. Taken together, our data provide a detailed and more complete picture of the accessible Ebolavirus glycoprotein landscape and a structural basis to evaluate patient and vaccine antibody responses towards differently structured products of the GP gene.


Assuntos
Anticorpos Monoclonais/química , Ebolavirus/imunologia , Glicoproteínas/química , Doença pelo Vírus Ebola/imunologia , Glicoproteínas de Membrana/química , Modelos Estruturais , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Reações Cruzadas , Microscopia Crioeletrônica , Ebolavirus/patogenicidade , Epitopos/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Mutação , Multimerização Proteica , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/imunologia
8.
Cell Rep ; 15(7): 1514-1526, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27160900

RESUMO

Previous efforts to identify cross-neutralizing antibodies to the receptor-binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS; cross-neutralizes Ebola (EBOV), Sudan (SUDV), and, to a lesser extent, Bundibugyo viruses; and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™ is remarkably effective against EBOV (Zaire) but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross-protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Ebolavirus/fisiologia , Epitopos/imunologia , Glicoproteínas/metabolismo , Doença pelo Vírus Ebola/imunologia , Receptores Virais/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/ultraestrutura , Anticorpos Neutralizantes , Anticorpos Antivirais/química , Sítios de Ligação , Modelos Animais de Doenças , Feminino , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/ultraestrutura , Cobaias , Células HEK293 , Humanos , Cinética , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação/genética , Coloração Negativa , Testes de Neutralização , Resultado do Tratamento
9.
Science ; 351(6277): 1078-83, 2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-26912366

RESUMO

Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/uso terapêutico , Formação de Anticorpos , Complexo Antígeno-Anticorpo/química , República Democrática do Congo/epidemiologia , Surtos de Doenças , Vacinas contra Ebola/imunologia , Vacinas contra Ebola/uso terapêutico , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/terapia , Humanos , Imunização Passiva , Camundongos , Sobreviventes , Doadores de Tecidos , Proteínas do Envelope Viral/química , Vírion/imunologia
10.
Cell ; 164(3): 392-405, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26806128

RESUMO

Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses, including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Sobreviventes , Animais , Reações Cruzadas , Modelos Animais de Doenças , Mapeamento de Epitopos , Cobaias , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Modelos Moleculares , Mutagênese , Uganda
12.
PLoS Pathog ; 11(6): e1005016, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26115029

RESUMO

The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel "wing" feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Doença pelo Vírus Ebola/imunologia , Imunização , Doença do Vírus de Marburg/prevenção & controle , Marburgvirus/imunologia , Animais , Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Ebolavirus/imunologia , Feminino , Masculino , Doença do Vírus de Marburg/imunologia , Camundongos Endogâmicos BALB C
13.
Cell ; 160(5): 893-903, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25723164

RESUMO

The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Complexo Antígeno-Anticorpo/ultraestrutura , Doença do Vírus de Marburg/imunologia , Marburgvirus/química , Proteínas do Envelope Viral/química , Adulto , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Linfócitos B/imunologia , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Marburgvirus/genética , Marburgvirus/imunologia , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Proteínas do Envelope Viral/metabolismo
14.
Proc Natl Acad Sci U S A ; 111(48): 17182-7, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25404321

RESUMO

Ebola virus (EBOV) and related filoviruses cause severe hemorrhagic fever, with up to 90% lethality, and no treatments are approved for human use. Multiple recent outbreaks of EBOV and the likelihood of future human exposure highlight the need for pre- and postexposure treatments. Monoclonal antibody (mAb) cocktails are particularly attractive candidates due to their proven postexposure efficacy in nonhuman primate models of EBOV infection. Two candidate cocktails, MB-003 and ZMAb, have been extensively evaluated in both in vitro and in vivo studies. Recently, these two therapeutics have been combined into a new cocktail named ZMapp, which showed increased efficacy and has been given compassionately to some human patients. Epitope information and mechanism of action are currently unknown for most of the component mAbs. Here we provide single-particle EM reconstructions of every mAb in the ZMapp cocktail, as well as additional antibodies from MB-003 and ZMAb. Our results illuminate key and recurring sites of vulnerability on the EBOV glycoprotein and provide a structural rationale for the efficacy of ZMapp. Interestingly, two of its components recognize overlapping epitopes and compete with each other for binding. Going forward, this work now provides a basis for strategic selection of next-generation antibody cocktails against Ebola and related viruses and a model for predicting the impact of ZMapp on potential escape mutations in ongoing or future Ebola outbreaks.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Epitopos/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Linhagem Celular , Ebolavirus/metabolismo , Epitopos/química , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Fragmentos Fab das Imunoglobulinas/imunologia , Microscopia Eletrônica , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas Virais/imunologia
15.
J Virol ; 88(17): 10177-88, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24965454

RESUMO

UNLABELLED: The neutralizing anti-HIV-1 antibody 2G12 is of particular interest due to the sterilizing protection it provides from viral challenge in animal models. 2G12 is a unique, domain-exchanged antibody that binds exclusively to conserved N-linked glycans that form the high-mannose patch on the gp120 outer domain centered on a glycan at position N332. Several glycans in and around the 2G12 epitope have been shown to interact with other potent, broadly neutralizing antibodies; therefore, this region constitutes a supersite of vulnerability on gp120. While crystal structures of 2G12 and 2G12 bound to high-mannose glycans have been solved, no structural information that describes the interaction of 2G12 with gp120 or the Env trimer is available. Here, we present a negative-stain single-particle electron microscopy reconstruction of 2G12 Fab2 in complex with a soluble, trimeric Env at ∼17-Šresolution that reveals the antibody's interaction with its native and fully glycosylated epitope. We also mapped relevant glycans in this epitope by fitting high-resolution crystal structures and by performing neutralization assays of glycan knockouts. In addition, a reconstruction at ∼26 Šof the ternary complex formed by 2G12 Fab2, soluble CD4, and Env indicates that 2G12 may block membrane fusion by induced steric hindrance upon primary receptor binding, thereby abrogating Env's interaction with coreceptor(s). These structures provide a basis for understanding 2G12 binding and neutralization, and our low-resolution model and glycan assignments provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope. IMPORTANCE: HIV-1 is a human virus that results in the deaths of millions of people around the world each year. While there are several effective therapeutics available to prolong life, a vaccine is the best long-term solution for curbing this global epidemic. Here, we present structural data that reveal the viral binding site of one of the first HIV-1-neutralizing antibodies isolated, 2G12, and provide a rationale for its effectiveness. These structures provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope, which will aid in vaccine design and antibody-based therapies.


Assuntos
Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Substâncias Macromoleculares/ultraestrutura , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Coloração e Rotulagem/métodos
16.
Nat Struct Mol Biol ; 20(7): 796-803, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23708606

RESUMO

A substantial proportion of the broadly neutralizing antibodies (bnAbs) identified in certain HIV-infected donors recognize glycan-dependent epitopes on HIV-1 gp120. Here we elucidate how the bnAb PGT 135 binds its Asn332 glycan-dependent epitope from its 3.1-Å crystal structure with gp120, CD4 and Fab 17b. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield and access the gp120 protein surface. EM reveals that PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. Combined structural studies of PGT 135, PGT 128 and 2G12 show that this Asn332-dependent antigenic region is highly accessible and much more extensive than initially appreciated, which allows for multiple binding modes and varied angles of approach; thereby it represents a supersite of vulnerability for antibody neutralization.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Alcaloides/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Reações Antígeno-Anticorpo , Sítios de Ligação de Anticorpos , Biopolímeros , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Sequência de Carboidratos , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Glicosilação/efeitos dos fármacos , Células HEK293 , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/ultraestrutura , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Polissacarídeos/fisiologia , Conformação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Relação Estrutura-Atividade , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
17.
Proc Natl Acad Sci U S A ; 110(11): 4351-6, 2013 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-23426631

RESUMO

PG9 is the founder member of an expanding family of glycan-dependent human antibodies that preferentially bind the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralize the virus. Here, we show that a soluble SOSIP.664 gp140 trimer constructed from the Clade A BG505 sequence binds PG9 with high affinity (∼11 nM), enabling structural and biophysical characterizations of the PG9:Env trimer complex. The BG505 SOSIP.664 gp140 trimer is remarkably stable as assessed by electron microscopy (EM) and differential scanning calorimetry. EM, small angle X-ray scattering, size exclusion chromatography with inline multiangle light scattering and isothermal titration calorimetry all indicate that only a single PG9 fragment antigen-binding (Fab) binds to the Env trimer. An ∼18 ŠEM reconstruction demonstrates that PG9 recognizes the trimer asymmetrically at its apex via contact with two of the three gp120 protomers, possibly contributing to its reported preference for a quaternary epitope. Molecular modeling and isothermal titration calorimetry binding experiments with an engineered PG9 mutant suggest that, in addition to the N156 and N160 glycan interactions observed in crystal structures of PG9 with a scaffolded V1/V2 domain, PG9 makes secondary interactions with an N160 glycan from an adjacent gp120 protomer in the antibody-trimer complex. Together, these structural and biophysical findings should facilitate the design of HIV-1 immunogens that possess all elements of the quaternary PG9 epitope required to induce broadly neutralizing antibodies against this region.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Epitopos/química , Anticorpos Anti-HIV/química , HIV-1/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Linhagem Celular , Humanos , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Difração de Raios X
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