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1.
Proc Natl Acad Sci U S A ; 116(30): 15194-15199, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31296560

RESUMO

Several next-generation (universal) influenza vaccines and broadly neutralizing antibodies (bNAbs) are in clinical development. Some of these mediate inhibitions of virus replication at the postentry stage or use Fc-dependent mechanisms. Nonneutralizing antibodies have the potential to mediate enhancement of viral infection or disease. In the current study, two monoclonal antibodies (MAbs) 72/8 and 69/1, enhanced respiratory disease (ERD) in mice following H3N2 virus challenge by demonstrating increased lung pathology and changes in lung cytokine/chemokine levels. MAb 78/2 caused changes in the lung viral loads in a dose-dependent manner. Both MAbs increased HA sensitivity to trypsin cleavage at a higher pH range, suggesting MAb-induced conformational changes. pHrodo-labeled virus particles' entry and residence time in the endocytic compartment were tracked during infection of Madin-Darby canine kidney (MDCK) cells. Both MAbs reduced H3N2 virus residence time in the endocytic pathway, suggesting faster virus fusion kinetics. Structurally, 78/2 and 69/1 Fabs bound the globular head or base of the head domain of influenza hemagglutinin (HA), respectively, and induced destabilization of the HA stem domain. Together, this study describes Mab-induced destabilization of the influenza HA stem domain, faster kinetics of influenza virus fusion, and ERD in vivo. The in vivo animal model and in vitro assays described could augment preclinical safety evaluation of antibodies and next-generation influenza vaccines that generate antibodies which do not block influenza virus-receptor interaction.

2.
MBio ; 10(4)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266872

RESUMO

As a consequence of their independent evolutionary origins in apes and Old World monkeys, human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency viruses of the SIVsmm/mac lineage express phylogenetically and antigenically distinct envelope glycoproteins. Thus, HIV-1 Env-specific antibodies do not typically cross-react with the Env proteins of SIVsmm/mac isolates. Here we show that PGT145, a broadly neutralizing antibody to a quaternary epitope at the V2 apex of HIV-1 Env, directs the lysis of SIVsmm/mac-infected cells by antibody-dependent cellular cytotoxicity (ADCC) but does not neutralize SIVsmm/mac infectivity. Amino acid substitutions in the V2 loop of SIVmac239 corresponding to the epitope for PGT145 in HIV-1 Env modulate sensitivity to this antibody. Whereas a substitution in a conserved N-linked glycosylation site (N171Q) eliminates sensitivity to ADCC, a lysine-to-serine substitution in this region (K180S) increases ADCC and renders the virus susceptible to neutralization. These differences in function correlate with an increase in the affinity of PGT145 binding to Env on the surface of virus-infected cells and to soluble Env trimers. To our knowledge, this represents the first instance of an HIV-1 Env-specific antibody that cross-reacts with SIVsmm/mac Env and illustrates how differences in antibody binding affinity for Env can differentiate sensitivity to ADCC from neutralization.IMPORTANCE Here we show that PGT145, a potent broadly neutralizing antibody to HIV-1, directs the lysis of SIV-infected cells by antibody-dependent cellular cytotoxicity but does not neutralize SIV infectivity. This represents the first instance of cross-reactivity of an HIV-1 Env-specific antibody with SIVsmm/mac Env and reveals that antibody binding affinity can differentiate sensitivity to ADCC from neutralization.

3.
PLoS Pathog ; 15(7): e1007920, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31306470

RESUMO

The HIV-1 envelope glycoprotein (Env) trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env trimer mimetics, such as SOSIP trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length unmodified Env trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP trimers. Antigenically, the full-length and SOSIP trimers were comparable, but in contrast to the full-length trimer, the SOSIP trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP trimer. Furthermore, the glycan composition of full-length and SOSIP trimers was similar overall, but the SOSIP trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env trimers as immunogens.

4.
Nat Struct Mol Biol ; 26(6): 518-525, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31160780

RESUMO

Otopetrins (Otop1-Otop3) comprise one of two known eukaryotic proton-selective channel families. Otop1 is required for otoconia formation and a candidate mammalian sour taste receptor. Here we report cryo-EM structures of zebrafish Otop1 and chicken Otop3 in lipid nanodiscs. The structures reveal a dimeric architecture, with each subunit forming 12 transmembrane helices divided into structurally similar amino (N) and carboxy (C) domains. Cholesterol-like molecules occupy various sites in Otop1 and Otop3 and occlude a central tunnel. In molecular dynamics simulations, hydrophilic vestibules formed by the N and C domains and in the intrasubunit interface between N and C domains form conduits for water entry into the membrane core, suggesting three potential proton conduction pathways. By mutagenesis, we tested the roles of charged residues in each putative permeation pathway. Our results provide a structural basis for understanding selective proton permeation and gating of this conserved family of proton channels.

5.
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.

6.
Nat Commun ; 10(1): 2342, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138817

RESUMO

Recent history is punctuated by the emergence of highly pathogenic coronaviruses such as SARS- and MERS-CoV into human circulation. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Here, we present the 3.1 Å resolution structure of the SARS-CoV nsp12 polymerase bound to its essential co-factors, nsp7 and nsp8, using single particle cryo-electron microscopy. nsp12 possesses an architecture common to all viral polymerases as well as a large N-terminal extension containing a kinase-like fold and is bound by two nsp8 co-factors. This structure illuminates the assembly of the coronavirus core RNA-synthesis machinery, provides key insights into nsp12 polymerase catalysis and fidelity and acts as a template for the design of novel antiviral therapeutics.


Assuntos
Coenzimas/ultraestrutura , RNA Polimerases Dirigidas por DNA/ultraestrutura , Vírus da SARS/ultraestrutura , Proteínas não Estruturais Virais/ultraestrutura , Microscopia Crioeletrônica , Genoma Viral , Vírus da SARS/metabolismo
7.
Nat Commun ; 10(1): 2355, 2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142746

RESUMO

Stabilized HIV-1 envelope glycoproteins (Env) that resemble the native Env are utilized in vaccination strategies aimed at inducing broadly neutralizing antibodies (bNAbs). To limit the exposure of rare isolate-specific antigenic residues/determinants we generated a SOSIP trimer based on a consensus sequence of all HIV-1 group M isolates (ConM). The ConM trimer displays the epitopes of most known bNAbs and several germline bNAb precursors. The crystal structure of the ConM trimer at 3.9 Å resolution resembles that of the native Env trimer and its antigenic surface displays few rare residues. The ConM trimer elicits strong NAb responses against the autologous virus in rabbits and macaques that are significantly enhanced when it is presented on ferritin nanoparticles. The dominant NAb specificity is directed against an epitope at or close to the trimer apex. Immunogens based on consensus sequences might have utility in engineering vaccines against HIV-1 and other viruses.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Sequência Consenso , Macaca , Multimerização Proteica , Coelhos
8.
Cell Rep ; 27(8): 2426-2441.e6, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31116986

RESUMO

Epitope-targeted HIV vaccine design seeks to focus antibody responses to broadly neutralizing antibody (bnAb) sites by sequential immunization. A chimpanzee simian immunodeficiency virus (SIV) envelope (Env) shares a single bnAb site, the variable loop 2 (V2)-apex, with HIV, suggesting its possible utility in an HIV immunization strategy. Here, we generate a chimpanzee SIV Env trimer, MT145K, which displays selective binding to HIV V2-apex bnAbs and precursor versions, but no binding to other HIV specificities. We determine the structure of the MT145K trimer by cryo-EM and show that its architecture is remarkably similar to HIV Env. Immunization of an HIV V2-apex bnAb precursor Ab-expressing knockin mouse with the chimpanzee MT145K trimer induces HIV V2-specific neutralizing responses. Subsequent boosting with an HIV trimer cocktail induces responses that exhibit some virus cross-neutralization. Overall, the chimpanzee MT145K trimer behaves as expected from design both in vitro and in vivo and is an attractive potential component of a sequential immunization regimen to induce V2-apex bnAbs.

9.
Cell ; 177(5): 1153-1171.e28, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31080066

RESUMO

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.

10.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 340-347, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045563

RESUMO

Ebola virus is an emerging virus that is capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome are carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 Šresolution single-particle cryo-electron microscopy structure of the nucleoprotein-RNA helical filament presented here resembles previous structures determined at lower resolution, while providing improved molecular details of protein-protein and protein-RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid.


Assuntos
Ebolavirus/química , Nucleocapsídeo/química , Nucleoproteínas/química , RNA Viral/química , Proteínas Virais/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleocapsídeo/ultraestrutura , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Proteínas Virais/metabolismo
11.
Cell ; 177(5): 1136-1152.e18, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100268

RESUMO

Here, we describe the discovery of a naturally occurring human antibody (Ab), FluA-20, that recognizes a new site of vulnerability on the hemagglutinin (HA) head domain and reacts with most influenza A viruses. Structural characterization of FluA-20 with H1 and H3 head domains revealed a novel epitope in the HA trimer interface, suggesting previously unrecognized dynamic features of the trimeric HA protein. The critical HA residues recognized by FluA-20 remain conserved across most subtypes of influenza A viruses, which explains the Ab's extraordinary breadth. The Ab rapidly disrupted the integrity of HA protein trimers, inhibited cell-to-cell spread of virus in culture, and protected mice against challenge with viruses of H1N1, H3N2, H5N1, or H7N9 subtypes when used as prophylaxis or therapy. The FluA-20 Ab has uncovered an exceedingly conserved protective determinant in the influenza HA head domain trimer interface that is an unexpected new target for anti-influenza therapeutics and vaccines.

12.
Cell Rep ; 27(2): 586-598.e6, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970260

RESUMO

Despite recent progress in engineering native trimeric HIV-1 envelope glycoprotein (Env) mimics as vaccine candidates, Env trimers often induce vaccine-matched neutralizing antibody (NAb) responses. Understanding the specificities of autologous NAb responses and the underlying molecular mechanisms restricting the neutralization breadth is therefore informative to improve vaccine efficacy. Here, we delineate the response specificity by single B cell sorting and serum analysis of guinea pigs immunized with BG505 SOSIP.664 Env trimers. Our results reveal a prominent immune target containing both conserved and strain-specific residues in the C3/V4 region of Env in trimer-vaccinated animals. The defined NAb response shares a high degree of similarity with the early NAb response developed by a naturally infected infant from whom the HIV virus strain BG505 was isolated and later developed a broadly NAb response. Our study describes strain-specific responses and their possible evolution pathways, thereby highlighting the potential to broaden NAb responses by immunogen re-design.

13.
Molecules ; 24(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013646

RESUMO

Small-molecule HIV-1 entry inhibitors are an extremely attractive therapeutic modality. We have previously demonstrated that the entry inhibitor class can be optimized by using computational means to identify and extend the chemotypes available. Here we demonstrate unique and differential effects of previously published antiviral compounds on the gross structure of the HIV-1 Env complex, with an azabicyclohexane scaffolded inhibitor having a positive effect on glycoprotein thermostability. We demonstrate that modification of the methyltriazole-azaindole headgroup of these entry inhibitors directly effects the potency of the compounds, and substitution of the methyltriazole with an amine-oxadiazole increases the affinity of the compound 1000-fold over parental by improving the on-rate kinetic parameter. These findings support the continuing exploration of compounds that shift the conformational equilibrium of HIV-1 Env as a novel strategy to improve future inhibitor and vaccine design efforts.


Assuntos
Fármacos Anti-HIV , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Internalização do Vírus/efeitos dos fármacos , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Humanos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade
14.
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
15.
PLoS Biol ; 17(2): e3000139, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30716060

RESUMO

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The "breathing" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.

16.
Nat Commun ; 10(1): 763, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770829

RESUMO

The N-terminal fusion peptide (FP) of the human immunodeficiency virus (HIV)-1 envelope glycoprotein (Env) gp41 subunit plays a critical role in cell entry. However, capturing the structural flexibility in the unbound FP is challenging in the native Env trimer. Here, FP conformational isomerism is observed in two crystal structures of a soluble clade B transmitted/founder virus B41 SOSIP.664 Env with broadly neutralizing antibodies (bNAbs) PGT124 and 35O22 to aid in crystallization and that are not specific for binding to the FP. Large rearrangements in the FP and fusion peptide proximal region occur around M530, which remains anchored in the tryptophan clasp (gp41 W623, W628, W631) in the B41 Env prefusion state. Further, we redesigned the FP at position 518 to reinstate the bNAb VRC34.01 epitope. These findings provide further structural evidence for the dynamic nature of the FP and how a bNAb epitope can be restored during vaccine design.


Assuntos
Anticorpos Neutralizantes/imunologia , Proteínas Virais/química , Proteínas Virais/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
17.
J Biol Chem ; 294(14): 5616-5631, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30728245

RESUMO

A successful HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that target the envelope glycoprotein (Env) spike on the virus. Native-like recombinant Env trimers of the SOSIP design now serve as a platform for achieving this challenging goal. However, SOSIP trimers usually do not bind efficiently to the inferred germline precursors of bNAbs (gl-bNAbs). We hypothesized that the inherent flexibilities of the V1 and V2 variable loops in the Env trimer contribute to the poor recognition of gl-bNAb epitopes at the trimer apex that extensively involve V2 residues. To reduce local V2 flexibility and improve the binding of V2-dependent bNAbs and gl-bNAbs, we designed BG505 SOSIP.664 trimer variants containing newly created disulfide bonds intended to stabilize the V2 loop in an optimally antigenic configuration. The first variant, I184C/E190C, contained a new disulfide bond within the V2 loop, whereas the second variant, E153C/R178C, had a new disulfide bond that cross-linked V2 and V1. The resulting engineered native-like trimer variants were both more reactive with and were neutralized by V2 bNAbs and gl-bNAbs, a finding that may be valuable in the design of germline targeting and boosting trimer immunogens to create an antigenic conformation optimal for HIV vaccine development.

18.
Immunity ; 50(2): 283-285, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30784575

RESUMO

HIV envelope glycoprotein (Env) exhibits extreme antigenic variation that can be countered by an amazing class of immunoglobulins known as broadly neutralizing antibodies. Dingens et al. (2019) use saturating mutagenesis of Env to play out all of the potential bnAb escape strategies and in doing so define the functional epitopes of these important vaccine and immunotherapeutic targets.


Assuntos
Anticorpos Neutralizantes , HIV-1/imunologia , Epitopos , Anticorpos Anti-HIV , Produtos do Gene env do Vírus da Imunodeficiência Humana
19.
J Pharm Sci ; 108(7): 2264-2277, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30776383

RESUMO

The induction of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an effective vaccine against HIV-1. A soluble, trimeric, germline (gI) bNAb-targeting variant of the HIV-1 envelope glycoprotein (termed BG505 SOSIP.v4.1-GT1.1 gp140, abbreviated to GT1.1) has recently been developed. Here, we have compared this new immunogen with the parental trimer from which it was derived, BG505 SOSIP.664 gp140. We used a comprehensive suite of biochemical and biophysical methods to determine physicochemical similarities and differences between the 2 trimers, and thereby assessed whether additional formulation development efforts were needed for the GT1.1 vaccine candidate. The overall higher order structure and oligomeric states of the 2 vaccine antigens were quite similar, as were their thermal, chemical, and colloidal stability profiles, as evaluated during accelerated stability studies. Overall, we conclude that the primary sequence changes made to create the gl bNAb-targeting GT1.1 trimer did not detrimentally affect its physicochemical properties or stability profiles from a pharmaceutical perspective. This developability assessment of the BG505 GT1.1 vaccine antigen supports using the formulation and storage conditions previously identified for the parental SOSIP.664 trimer and enables the development of GT1.1 for phase I clinical studies.

20.
J Mol Biol ; 431(4): 842-856, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30597163

RESUMO

Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA-receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA-sfGFP proteins was studied using glycan arrays and tissue staining. The HA-sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA-sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.

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