RESUMO
Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.
Assuntos
Afinidade de Anticorpos , Linfócitos B , Movimento Celular , Células Clonais , Centro Germinativo , Anticorpos Anti-HIV , Imunização , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Células Clonais/citologia , Células Clonais/imunologia , Epitopos de Linfócito B/imunologia , Perfilação da Expressão Gênica , Centro Germinativo/citologia , Centro Germinativo/imunologia , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Imunização Secundária , Macaca mulatta/imunologia , Macaca mulatta/virologia , Células B de Memória/citologia , Células B de Memória/imunologia , Análise de Célula Única , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Fatores de Tempo , Produtos do Gene env do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.
Assuntos
Vacinas contra a AIDS , Anticorpos Neutralizantes , Anticorpos Anti-HIV , Animais , Humanos , Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Camundongos , Vacinação , Imunização Secundária , HIV-1/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Anticorpos Amplamente Neutralizantes/imunologiaRESUMO
Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.
Assuntos
Vacinas contra a AIDS , Anticorpos Amplamente Neutralizantes , Regiões Determinantes de Complementaridade , Centro Germinativo , Anticorpos Anti-HIV , Animais , Humanos , Vacinas contra a AIDS/imunologia , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Regiões Determinantes de Complementaridade/imunologia , Microscopia Crioeletrônica , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Centro Germinativo/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Macaca mulatta , Células B de Memória/imunologiaRESUMO
Vaccine priming immunogens that activate germline precursors for broadly neutralizing antibodies (bnAbs) have promise for development of precision vaccines against major human pathogens. In a clinical trial of the eOD-GT8 60mer germline-targeting immunogen, higher frequencies of vaccine-induced VRC01-class bnAb-precursor B cells were observed in the high dose compared to the low dose group. Through immunoglobulin heavy chain variable (IGHV) genotyping, statistical modeling, quantification of IGHV1-2 allele usage and B cell frequencies in the naive repertoire for each trial participant, and antibody affinity analyses, we found that the difference between dose groups in VRC01-class response frequency was best explained by IGHV1-2 genotype rather than dose and was most likely due to differences in IGHV1-2 B cell frequencies for different genotypes. The results demonstrate the need to define population-level immunoglobulin allelic variations when designing germline-targeting immunogens and evaluating them in clinical trials.
RESUMO
The downregulation of translation through eIF2α phosphorylation is a cellular response to diverse stresses, including viral infection, and is mediated by the GCN2 kinase, protein kinase R (PKR), protein kinase-like endoplasmic reticulum kinase (PERK), and heme-regulated inhibitor kinase (HRI). Although PKR plays a major role in defense against viruses, other eIF2α kinases also may respond to viral infection and contribute to the shutdown of protein synthesis. Here we describe the recessive, loss-of-function mutation atchoum (atc) in Eif2ak4, encoding GCN2, which increased susceptibility to infection by the double-stranded DNA viruses mouse cytomegalovirus (MCMV) and human adenovirus. This mutation was identified by screening macrophages isolated from mice carrying N-ethyl-N-nitrosourea (ENU)-induced mutations. Cells from Eif2ak4(atc/atc) mice failed to phosphorylate eIF2α in response to MCMV. Importantly, homozygous Eif2ak4(atc) mice showed a modest increase in susceptibility to MCMV infection, demonstrating that translational arrest dependent on GCN2 contributes to the antiviral response in vivo.
Assuntos
Infecções por Vírus de DNA/genética , Predisposição Genética para Doença , Mutação , Proteínas Serina-Treonina Quinases/genética , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Vaccine priming immunogens that activate germline precursors for broadly neutralizing antibodies (bnAbs) have promise for development of precision vaccines against major human pathogens. In a clinical trial of the eOD-GT8 60mer germline-targeting immunogen, higher frequencies of vaccine-induced VRC01-class bnAb-precursor B cells were observed in the high dose compared to the low dose group. Through immunoglobulin heavy chain variable (IGHV) genotyping, statistical modeling, quantification of IGHV1-2 allele usage and B cell frequencies in the naive repertoire for each trial participant, and antibody affinity analyses, we found that the difference between dose groups in VRC01-class response frequency was best explained by IGHV1-2 genotype rather than dose and was most likely due to differences in IGHV1-2 B cell frequencies for different genotypes. The results demonstrate the need to define population-level immunoglobulin allelic variations when designing germline-targeting immunogens and evaluating them in clinical trials. One-Sentence Summary: Human genetic variation can modulate the strength of vaccine-induced broadly neutralizing antibody precursor B cell responses.
RESUMO
Maturation of adenoviruses is distinguished by proteolytic processing of several interior minor capsid proteins and core proteins by the adenoviral protease and subsequent reorganization of adenovirus core. We report the results derived from the icosahedrally averaged cryo-EM structure of a cell entry defective form of adenovirus, designated ts1, at a resolution of 3.7 Å as well as of the localized reconstructions of unique hexons and penton base. The virion structure revealed the structures and organization of precursors of minor capsid proteins, pIIIa, pVI and pVIII, which are closely associated with the hexons on the capsid interior. In addition to a well-ordered helical domain (a.a. 310-397) of pIIIa, highlights of the structure include the precursors of VIII display significantly different structures near the cleavage sites. Moreover, we traced residues 4-96 of the membrane lytic protein (pVI) that includes an amphipathic helix occluded deep in the hexon cavity suggesting the possibility of co-assembly of hexons with the precursors of VI. In addition, we observe a second copy of pVI ordered up to residue L40 in the peripentonal hexons and a few fragments of density corresponding to 2nd and 3rd copies of pVI in other hexons. However, we see no evidence of precursors of VII binding in the hexon cavity. These findings suggest the possibility that differently bound pVI molecules undergo processing at the N-terminal cleavage sites at varying efficiencies, subsequently creating competition between the cleaved and uncleaved forms of VI, followed by reorganization, processing, and release of VI molecules from the hexon cavities.
Assuntos
Adenovírus Humanos/fisiologia , Proteínas do Capsídeo/química , Capsídeo/química , Precursores de Proteínas/genética , Internalização do Vírus , Humanos , Modelos Moleculares , Conformação Proteica , Vírion/metabolismo , Montagem de VírusRESUMO
Elicitation of HIV broadly neutralizing antibodies (bnAbs) is challenging because unmutated bnAb precursors are rare and seldom bind HIV envelope glycoprotein (Env) trimers. One strategy to initiate bnAb responses is to use germline-targeting (GT) immunogens with high affinity to bnAb-class precursor B cells and then shepherd affinity maturation with booster immunogens that successively look more like native Env. In a mouse model where the frequency of VRC01-precursor (VRC01gHL) B cells mimics that of humans, we show that following a GT HIV Env trimer protein prime, VRC01-class B cells in the germinal center (GC) acquire high-affinity VRC01-class B cell somatic hypermutations (SHMs). Many GC-derived VRC01gHL antibodies robustly bind N276 glycan-deficient Env trimers and neutralize several N276 glycan-deficient tier 2 HIV strains. These results are encouraging for GT Env trimer vaccine designs and demonstrate accumulation of substantial SHMs, including deletions, uncommon point mutations, and functional bnAb features, after a single immunization.
Assuntos
Vacinas contra a AIDS , Infecções por HIV , HIV-1 , Animais , Anticorpos Neutralizantes , Antígenos Virais , Anticorpos Amplamente Neutralizantes , Anticorpos Anti-HIV , Imunização , Camundongos , Polissacarídeos/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
Broadly neutralizing antibodies (bnAbs) can protect against HIV infection but have not been induced by human vaccination. A key barrier to bnAb induction is vaccine priming of rare bnAb-precursor B cells. In a randomized, double-blind, placebo-controlled phase 1 clinical trial, the HIV vaccine-priming candidate eOD-GT8 60mer adjuvanted with AS01B had a favorable safety profile and induced VRC01-class bnAb precursors in 97% of vaccine recipients with median frequencies reaching 0.1% among immunoglobulin G B cells in blood. bnAb precursors shared properties with bnAbs and gained somatic hypermutation and affinity with the boost. The results establish clinical proof of concept for germline-targeting vaccine priming, support development of boosting regimens to induce bnAbs, and encourage application of the germline-targeting strategy to other targets in HIV and other pathogens.
Assuntos
Vacinas contra a AIDS , Anticorpos Amplamente Neutralizantes , Células Germinativas , Anticorpos Anti-HIV , Infecções por HIV , Cadeias Pesadas de Imunoglobulinas , Cadeias Leves de Imunoglobulina , Humanos , Adjuvantes Imunológicos , Vacinas contra a AIDS/imunologia , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Amplamente Neutralizantes/imunologia , Infecções por HIV/prevenção & controle , Vacinação , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Células Germinativas/imunologia , Linfócitos B/imunologia , Mutação , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Masculino , Feminino , AdultoRESUMO
A structure of adenovirus type 12 (HAdV12) complexed with a soluble form of integrin alphavbeta5 was determined by cryo-electron microscopy (cryoEM) image reconstruction. Subnanometer resolution (8 A) was achieved for the icosahedral capsid with moderate resolution (27 A) for integrin density above each penton base. Modeling with alphavbeta3 and alpha(IIb)beta3 crystal structures indicates that a maximum of four integrins fit over the pentameric penton base. The close spacing (approximately 60 A) of the RGD protrusions on penton base precludes integrin binding in the same orientation to neighboring RGD sites. Flexible penton-base RGD loops and incoherent averaging of bound integrin molecules explain the moderate resolution observed for the integrin density. A model with four integrins bound to a penton base suggests that integrin might extend one RGD-loop in the direction that could induce a conformational change in the penton base involving clockwise untwisting of the pentamer. A global conformational change in penton base could be one step on the way to the release of Ad vertex proteins during cell entry. Comparison of the cryoEM structure with bent and extended models for the integrin ectodomain reveals that integrin adopts an extended conformation when bound to the Ad penton base, a multivalent viral ligand. These findings shed further light on the structural basis of integrin binding to biologically relevant ligands, as well as on the molecular events leading to HAdV cell entry.
Assuntos
Adenoviridae/ultraestrutura , Proteínas do Capsídeo/ultraestrutura , Receptores de Vitronectina/ultraestrutura , Animais , Capsídeo/ultraestrutura , Proteínas do Capsídeo/química , Linhagem Celular , Microscopia Crioeletrônica , Conformação Proteica , Receptores de Vitronectina/química , Ligação ViralRESUMO
We report the revised crystal structure of a pseudo-typed human adenovirus at 3.8-Å resolution that is consistent with the atomic models of minor proteins determined by cryo-electron microscopy. The diffraction data from multiple crystals were rescaled and merged to increase the data completeness. The densities for the minor proteins were initially identified in the phase-refined omit maps that were further improved by the phases from docked poly-alanine models to build atomic structures. While the trimeric fiber molecules are disordered due to flexibility and imposition of 5-fold symmetry, the remaining major capsid proteins hexon and penton base are clearly ordered, with the exception of hypervariable region 1 of hexons, the RGD containing loop, and the N-termini of the penton base. The exterior minor protein IX together with the interior minor proteins IIIa and VIII stabilizes the adenovirus virion. A segment of N-terminal pro-peptide of VI is found in the interior cavities of peripentonal hexons, and the rest of VI is disordered. While the triskelion substructures formed by the N-termini of IX conform to excellent quasi 3-fold symmetry, the tetrameric coiled-coils formed by the C-termini and organized in parallel and anti-parallel arrangement do not exhibit any quasi-symmetry. This observation also conveys the pitfalls of using the quasi-equivalence as validation criteria for the structural analysis of extended (non-modular) capsid proteins such as IX. Together, these results remedy certain discrepancies in the previous X-ray model in agreement with the cryo-electron microscopy models.
Assuntos
Adenovírus Humanos/química , Adenovírus Humanos/ultraestrutura , Proteínas do Capsídeo/química , Substâncias Macromoleculares/química , Modelos Moleculares , Conformação Proteica , Capsídeo/química , Capsídeo/ultraestrutura , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Substâncias Macromoleculares/ultraestruturaRESUMO
Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.
Assuntos
Infecções por Adenoviridae/etiologia , Adenovírus Humanos/patogenicidade , Antígenos CD/fisiologia , Ceratoconjuntivite/etiologia , Glicoproteínas de Membrana/fisiologia , Receptores Virais/fisiologia , Infecções por Adenoviridae/virologia , Adenovírus Humanos/classificação , Adenovírus Humanos/genética , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/genética , Sequência de Bases , Células CHO , Linhagem Celular , Cricetinae , DNA Viral/genética , Células HeLa , Humanos , Ceratoconjuntivite/virologia , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Receptores Virais/química , Receptores Virais/genéticaRESUMO
The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.