RESUMEN
Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as human volunteers challenged with ETEC, diarrheal severity is significantly increased severity in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of human volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected human volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design.
RESUMEN
Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.
Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Humanos , Anticuerpos ampliamente neutralizantes , Anticuerpos Anti-VIH , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Anticuerpos Neutralizantes , Regiones Determinantes de Complementariedad/genética , Infecciones por VIH/prevención & controlRESUMEN
Eliciting broadly neutralizing antibodies (bnAbs) is the core of HIV vaccine design. bnAbs specific to the V2-apex region of the HIV envelope acquire breadth and potency with modest somatic hypermutation, making them attractive vaccination targets. To evaluate Apex germline-targeting (ApexGT) vaccine candidates, we engineered knockin (KI) mouse models expressing the germline B cell receptor (BCR) of the bnAb PCT64. We found that high affinity of the ApexGT immunogen for PCT64-germline BCRs was necessary to specifically activate KI B cells at human physiological frequencies, recruit them to germinal centers, and select for mature bnAb mutations. Relative to protein, mRNA-encoded membrane-bound ApexGT immunization significantly increased activation and recruitment of PCT64 precursors to germinal centers and lowered their affinity threshold. We have thus developed additional models for HIV vaccine research, validated ApexGT immunogens for priming V2-apex bnAb precursors, and identified mRNA-LNP as a suitable approach to substantially improve the B cell response.
Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Ratones , Humanos , Animales , Anticuerpos Anti-VIH , Anticuerpos ampliamente neutralizantes , Anticuerpos Neutralizantes , ARN Mensajero/genética , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
SIVmac239 infection of macaques is a favored model of human HIV infection. However, the SIVmac239 envelope (Env) trimer structure, glycan occupancy, and the targets and ability of neutralizing antibodies (nAbs) to protect against SIVmac239 remain unknown. Here, we report the isolation of SIVmac239 nAbs that recognize a glycan hole and the V1/V4 loop. A high-resolution structure of a SIVmac239 Env trimer-nAb complex shows many similarities to HIV and SIVcpz Envs, but with distinct V4 features and an extended V1 loop. Moreover, SIVmac239 Env has a higher glycan shield density than HIV Env that may contribute to poor or delayed nAb responses in SIVmac239-infected macaques. Passive transfer of a nAb protects macaques from repeated intravenous SIVmac239 challenge at serum titers comparable to those described for protection of humans against HIV infection. Our results provide structural insights for vaccine design and shed light on antibody-mediated protection in the SIV model.
Asunto(s)
Infecciones por VIH , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Infecciones por VIH/prevención & control , Humanos , Macaca mulatta , PolisacáridosRESUMEN
Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Formación de Anticuerpos/inmunología , Anticuerpos Anti-VIH/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/ultraestructura , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/ultraestructura , Microscopía por Crioelectrón/métodos , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Glicosilación , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/ultraestructura , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , VIH-1/metabolismo , Humanos , Macaca mulatta , Modelos Moleculares , Conformación Proteica , Productos del Gen env del Virus de la Inmunodeficiencia Humana/ultraestructuraRESUMEN
Dense surface glycosylation on the HIV-1 envelope (Env) protein acts as a shield from the adaptive immune system. However, the molecular complexity and flexibility of glycans make experimental studies a challenge. Here we have integrated high-throughput atomistic modeling of fully glycosylated HIV-1 Env with graph theory to capture immunologically important features of the shield topology. This is the first complete all-atom model of HIV-1 Env SOSIP glycan shield that includes both oligomannose and complex glycans, providing physiologically relevant insights of the glycan shield. This integrated approach including quantitative comparison with cryo-electron microscopy data provides hitherto unexplored details of the native shield architecture and its difference from the high-mannose glycoform. We have also derived a measure to quantify the shielding effect over the antigenic protein surface that defines regions of relative vulnerability and resilience of the shield and can be harnessed for rational immunogen design.
RESUMEN
The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the "glycan shield," is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure-function analysis. Here, we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same cell line used for vaccine production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner, we found that highly connected glycan clusters are resistant to digestion and help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.
Asunto(s)
VIH-1/inmunología , Polisacáridos/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Anticuerpos Neutralizantes/inmunología , Formación de Anticuerpos , Simulación por Computador , Microscopía por Crioelectrón/métodos , Epítopos/química , Glicosilación , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , Seropositividad para VIH , VIH-1/metabolismo , Humanos , Evasión Inmune/inmunología , Espectrometría de Masas/métodos , Modelos Moleculares , Productos del Gen env del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of HIV Env trimer immunogens; however, the optimal implementation to different immunization strategies remains to be determined.
Asunto(s)
Anticuerpos Anti-VIH/inmunología , Antígenos VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Nanopartículas/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Epítopos/inmunología , Femenino , Infecciones por VIH/virología , Humanos , Inmunización , Nanopartículas/administración & dosificación , Conejos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Potent broadly neutralizing antibodies (bnAbs) to HIV have been very challenging to elicit by vaccination in wild-type animals. Here, by x-ray crystallography, cryo-electron microscopy, and site-directed mutagenesis, we structurally and functionally elucidate the mode of binding of a potent bnAb (NC-Cow1) elicited in cows by immunization with the HIV envelope (Env) trimer BG505 SOSIP.664. The exceptionally long (60 residues) third complementarity-determining region of the heavy chain (CDR H3) of NC-Cow1 forms a mini domain (knob) on an extended stalk that navigates through the dense glycan shield on Env to target a small footprint on the gp120 CD4 receptor binding site with no contact of the other CDRs to the rest of the Env trimer. These findings illustrate, in molecular detail, how an unusual vaccine-induced cow bnAb to HIV Env can neutralize with high potency and breadth.
Asunto(s)
Infecciones por VIH , VIH-1 , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Anticuerpos ampliamente neutralizantes , Bovinos , Microscopía por Crioelectrón , Femenino , Anticuerpos Anti-VIH/metabolismo , Infecciones por VIH/prevención & controlRESUMEN
Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development focuses on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein. Coronavirus S proteins are extensively glycosylated, encoding around 66-87 N-linked glycosylation sites per trimeric spike. Here, we reveal a specific area of high glycan density on MERS S that results in the formation of oligomannose-type glycan clusters, which were absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Overall, our data reveal how organisation of glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the protein surface.
Asunto(s)
Glicoproteínas/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio , Polisacáridos , Glicoproteína de la Espiga del Coronavirus/inmunología , Proteínas Virales de Fusión/inmunología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Microscopía por Crioelectrón , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Glicoproteínas/química , Glicoproteínas/ultraestructura , Glicosilación , Células HEK293 , VIH-1/inmunología , VIH-1/metabolismo , Humanos , Evasión Inmune/fisiología , Virus Lassa/inmunología , Virus Lassa/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Orthomyxoviridae/inmunología , Orthomyxoviridae/metabolismo , Polisacáridos/química , Polisacáridos/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/ultraestructura , Proteínas Virales/química , Proteínas Virales/inmunología , Proteínas Virales/ultraestructuraRESUMEN
Structural and functional studies of HIV envelope glycoprotein (Env) as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstitute a full-length Env clone into a nanodisc, complex it with a membrane-proximal external region (MPER) targeting antibody 10E8, and structurally define the full quaternary epitope of 10E8 consisting of lipid, MPER, and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapt the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with a peptidisc scaffold.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/genética , VIH-1/genética , Membrana Dobles de Lípidos/metabolismo , HumanosRESUMEN
The development of native-like HIV-1 envelope (Env) trimer antigens has enabled the induction of neutralizing antibody (NAb) responses against neutralization-resistant HIV-1 strains in animal models. However, NAb responses are relatively weak and narrow in specificity. Displaying antigens in a multivalent fashion on nanoparticles (NPs) is an established strategy to increase their immunogenicity. Here we present the design and characterization of two-component protein NPs displaying 20 stabilized SOSIP trimers from various HIV-1 strains. The two-component nature permits the incorporation of exclusively well-folded, native-like Env trimers into NPs that self-assemble in vitro with high efficiency. Immunization studies show that the NPs are particularly efficacious as priming immunogens, improve the quality of the Ab response over a conventional one-component nanoparticle system, and are most effective when SOSIP trimers with an apex-proximate neutralizing epitope are displayed. Their ability to enhance and shape the immunogenicity of SOSIP trimers make these NPs a promising immunogen platform.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/inmunología , Animales , Antígenos Virales/biosíntesis , Línea Celular , Epítopos/inmunología , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Nanopartículas , Multimerización de Proteína/inmunología , Estructura Terciaria de Proteína , ConejosRESUMEN
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.
Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos , Glicoproteínas/metabolismo , Anticuerpos Anti-VIH/metabolismo , Pruebas de Neutralización , Virus de la Inmunodeficiencia de los Simios/inmunología , Proteínas del Envoltorio Viral/metabolismo , Línea Celular , Humanos , Unión Proteica , Virus de la Inmunodeficiencia de los Simios/crecimiento & desarrolloRESUMEN
As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.
Asunto(s)
VIH-1/inmunología , Polisacáridos/inmunología , Polisacáridos/metabolismo , Anticuerpos Neutralizantes/inmunología , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/metabolismo , VIH-1/patogenicidad , Humanos , Multimerización de ProteínaRESUMEN
Broadly neutralizing antibodies (bnAbs) targeting the HIV envelope glycoprotein (Env) typically take years to develop. Longitudinal analyses of both neutralizing antibody lineages and viruses at serial time points during infection provide a basis for understanding the co-evolutionary contest between HIV and the humoral immune system. Here, we describe the structural characterization of an apex-targeting antibody lineage and autologous clade A viral Env from a donor in the Protocol C cohort. Comparison of Ab-Env complexes at early and late time points reveals that, within the antibody lineage, the CDRH3 loop rigidifies, the bnAb angle of approach steepens, and surface charges are mutated to accommodate glycan changes. Additionally, we observed differences in site-specific glycosylation between soluble and full-length Env constructs, which may be important for tuning optimal immunogenicity in soluble Env trimers. These studies therefore provide important guideposts for design of immunogens that prime and mature nAb responses to the Env V2-apex.
Asunto(s)
Vacunas contra el SIDA/metabolismo , Anticuerpos Neutralizantes/metabolismo , Evolución Molecular , Anticuerpos Anti-VIH/metabolismo , VIH-1/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Sitios de Unión de Anticuerpos , Microscopía por Crioelectrón , Epítopos/química , Epítopos/inmunología , Glicosilación , Células HEK293 , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Humanos , Simulación del Acoplamiento Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design.
Asunto(s)
Anticuerpos Neutralizantes/fisiología , Linaje de la Célula , Anticuerpos Anti-VIH/fisiología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/química , Regiones Determinantes de Complementariedad , Anticuerpos Anti-VIH/química , HumanosRESUMEN
SUMMARY: The Electron Microscopy Hole Punch (EMHP) is a streamlined suite of tools for quick assessment, sorting and hole masking of electron micrographs. With recent advances in single-particle electron cryo-microscopy (cryo-EM) data processing allowing for the rapid determination of protein structures using a smaller computational footprint, we saw the need for a fast and simple tool for data pre-processing that could run independent of existing high-performance computing (HPC) infrastructures. EMHP provides a data preprocessing platform in a small package that requires minimal python dependencies to function. AVAILABILITY AND IMPLEMENTATION: https://www.bitbucket.org/chazbot/emhp Apache 2.0 License. CONTACT: bowman@scripps.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón , Procesamiento de Imagen Asistido por Computador/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Proteínas/químicaRESUMEN
No immunogen to date has reliably elicited broadly neutralizing antibodies to HIV in humans or animal models. Advances in the design of immunogens that antigenically mimic the HIV envelope glycoprotein (Env), such as the soluble cleaved trimer BG505 SOSIP, have improved the elicitation of potent isolate-specific antibody responses in rabbits and macaques, but so far failed to induce broadly neutralizing antibodies. One possible reason for this failure is that the relevant antibody repertoires are poorly suited to target the conserved epitope regions on Env, which are somewhat occluded relative to the exposed variable epitopes. Here, to test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach more than 70 amino acids in length. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody isolated from this cow harboured an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC50 of 0.028 µg ml-1. Breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/aislamiento & purificación , Bovinos/inmunología , VIH/inmunología , Inmunización , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Células HEK293 , Proteínas gp160 de Envoltorio del VIH/inmunología , HumanosRESUMEN
We compare forces resisting DNA packaging and forces driving DNA ejection in bacteriophage phi29 with theoretical predictions. Ejection of DNA from prohead-motor complexes is triggered by heating complexes after in vitro packaging and force is inferred from the suppression of ejection by applied osmotic pressure. Ejection force from 0% to 80% filling is found to be in quantitative agreement with predictions of a continuum mechanics model that assumes a repulsive DNA-DNA interaction potential based on DNA condensation studies and predicts an inverse-spool conformation. Force resisting DNA packaging from â¼80% to 100% filling inferred from optical tweezers studies is also consistent with the predictions of this model. The striking agreement with these two different measurements suggests that the overall energetics of DNA packaging is well described by the model. However, since electron microscopy studies of phi29 do not reveal a spool conformation, our findings suggest that the spool model overestimates the role of bending rigidity and underestimates the role of intrastrand repulsion. Below â¼80% filling the inferred forces resisting packaging are unexpectedly lower than the inferred ejection forces, suggesting that in this filling range the forces are less accurately determined or strongly temperature dependent.
Asunto(s)
Fagos de Bacillus/fisiología , Empaquetamiento del ADN , ADN Viral , Integración Viral , Fenómenos Biomecánicos , Empaquetamiento del ADN/fisiología , ADN Viral/fisiología , Elasticidad , Tamaño del Genoma , Microscopía Electrónica , Modelos Biológicos , Conformación de Ácido Nucleico , Pinzas Ópticas , Presión Osmótica , Polietilenglicoles , Ensamble de Virus/fisiología , Integración Viral/fisiologíaRESUMEN
We report evidence for an unconventional type of allosteric regulation of a biomotor. We show that the genome-packaging motor of phage Ï29 is regulated by a sensor that detects the density and conformation of the DNA packaged inside the viral capsid, and slows the motor by a mechanism distinct from the effect of a direct load force on the motor. Specifically, we show that motor-ATP interactions are regulated by a signal that is propagated allosterically from inside the viral shell to the motor mounted on the outside. This signal continuously regulates the motor speed and pausing in response to changes in either density or conformation of the packaged DNA, and slows the motor before the buildup of large forces resisting DNA confinement. Analysis of motor slipping reveals that the force resisting packaging remains low (<1 pN) until â¼ 70% and then rises sharply to â¼ 23 pN at high filling, which is a several-fold lower value than was previously estimated under the assumption that force alone slows the motor. These findings are consistent with recent studies of the stepping kinetics of the motor. The allosteric regulatory mechanism we report allows double-stranded DNA viruses to achieve rapid, high-density packing of their genomes by limiting the buildup of nonequilibrium load forces on the motor.