RESUMO
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.
Assuntos
Vacinas contra a AIDS , Infecções por HIV , HIV-1 , Camundongos , Humanos , Animais , Anticorpos Anti-HIV , Anticorpos Amplamente Neutralizantes , Anticorpos Neutralizantes , RNA Mensageiro/genética , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
Vaccines generate high-affinity antibodies by recruiting antigen-specific B cells to germinal centers (GCs), but the mechanisms governing the recruitment to GCs on secondary challenges remain unclear. Here, using preclinical SARS-CoV and HIV mouse models, we demonstrated that the antibodies elicited during primary humoral responses shaped the naive B cell recruitment to GCs during secondary exposures. The antibodies from primary responses could either enhance or, conversely, restrict the GC participation of naive B cells: broad-binding, low-affinity, and low-titer antibodies enhanced recruitment, whereas, by contrast, the high titers of high-affinity, mono-epitope-specific antibodies attenuated cognate naive B cell recruitment. Thus, the directionality and intensity of that effect was determined by antibody concentration, affinity, and epitope specificity. Circulating antibodies can, therefore, be important determinants of antigen immunogenicity. Future vaccines may need to overcome-or could, alternatively, leverage-the effects of circulating primary antibodies on subsequent naive B cell recruitment.
Assuntos
Linfócitos B , Centro Germinativo , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Antígenos , Epitopos , Imunidade Humoral , CamundongosRESUMO
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.
Assuntos
Vacinas contra a AIDS , Infecções por HIV , HIV-1 , Humanos , Anticorpos Amplamente Neutralizantes , Anticorpos Anti-HIV , Produtos do Gene env do Vírus da Imunodeficiência Humana , Anticorpos Neutralizantes , Regiões Determinantes de Complementaridade/genética , Infecções por HIV/prevenção & controleRESUMO
A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos Virais/administração & dosagem , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Imunização , Imunoglobulinas/genética , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Linfócitos B/imunologia , Clonagem Molecular , Primers do DNA/química , Epitopos/imunologia , Técnicas de Introdução de Genes , Infecções por HIV/imunologia , Camundongos , Mutação , Alinhamento de SequênciaRESUMO
The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/uso terapêutico , Centro Germinativo/imunologia , Anticorpos Anti-HIV/uso terapêutico , Infecções por HIV/terapia , HIV-1/imunologia , Animais , Células Cultivadas , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Centro Germinativo/virologia , Infecções por HIV/imunologia , Humanos , Imunização , Injeções Subcutâneas , Primatas , Multimerização Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Polissacarídeos/imunologia , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Epitopos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunização/métodos , Camundongos , Camundongos Knockout , Mutação/imunologia , Alinhamento de Sequência , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Here, we describe a one-step, in vivo CRISPR/Cas9 nuclease-mediated strategy to generate knock-in mice. We produced knock-in (KI) mice wherein a 1.9-kb DNA fragment bearing a pre-arranged human B-cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease-induced double-stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double-stranded breaks are subsequently repaired via homology-directed repair by a plasmid-borne template containing the pre-arranged human immunoglobulin heavy chain. To validate our knock-in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B-cell development and performing single B-cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30-50%). In the future, we envision that such knock-in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases.
Assuntos
Linfócitos B/imunologia , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes/métodos , Cadeias Pesadas de Imunoglobulinas , Animais , Linfócitos B/citologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Camundongos , Camundongos TransgênicosRESUMO
The first protein kinase structure, solved in 1991, revealed the fold that is shared by all members of the eukaryotic protein kinase superfamily and showed how the conserved sequence motifs cluster mostly around the active site. This structure of the PKA catalytic (C) subunit showed also how a single phosphate integrated the entire molecule. Since then the EPKs have become a major drug target, second only to the G-protein coupled receptors. Although PKA provided a mechanistic understanding of catalysis that continues to serve as a prototype for the family, by comparing many active and inactive kinases we subsequently discovered a hydrophobic spine architecture that is a characteristic feature of all active kinases. The ways in which the regulatory spine is dynamically assembled is the defining feature of each protein kinase. Protein kinases have thus evolved to be molecular switches, like the G-proteins, and unlike metabolic enzymes which have evolved to be efficient catalysis. PKA also shows how the dynamic tails surround the core and serve as essential regulatory elements. The phosphorylation sites in PKA, introduced both co- and post-translationally, are very stable. The resulting C-subunit is then packaged as an inhibited holoenzyme with cAMP-binding regulatory (R) subunits so that PKA activity is regulated exclusively by cAMP, not by the dynamic turnover of an activation loop phosphate. We could not understand activation and inhibition without seeing structures of R:C complexes; however, to appreciate the structural uniqueness of each R2:C2 holoenzyme required solving structures of tetrameric holoenzymes. It is these tetrameric holoenzymes that are localized to discrete sites in the cell, typically by A Kinase Anchoring Proteins where they create discrete foci for PKA signaling. Understanding these dynamic macromolecular complexes is the challenge that we now face. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Biocatálise , Domínio Catalítico , AMP Cíclico/química , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , FosforilaçãoRESUMO
mRNA delivered using lipid nanoparticles (LNPs) has become an important subunit vaccine modality, but mechanisms of action for mRNA vaccines remain incompletely understood. Here, we synthesized a metal chelator-lipid conjugate enabling positron emission tomography (PET) tracer labeling of LNP/mRNA vaccines for quantitative visualization of vaccine trafficking in live non-human primates (NHPs). Following i.m. injection, we observed LNPs distributing through injected muscle tissue, simultaneous with rapid trafficking to draining lymph nodes (dLNs). Deltoid injection of LNPs mimicking human vaccine administration led to stochastic LNP delivery to 3 different sets of dLNs. LNP uptake in dLNs was confirmed by histology, and cellular analysis of tissues via flow cytometry identified antigen-presenting cells as the primary cell type responsible for early LNP uptake and mRNA translation. These results provide insights into the biodistribution of mRNA vaccines administered at clinically relevant doses, injection volumes, and injection sites in an important large animal model for vaccine development.
RESUMO
Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.
Assuntos
Vacinas contra a AIDS , Anticorpos Amplamente Neutralizantes , Centro Germinativo , Anticorpos Anti-HIV , HIV-1 , Imunização Secundária , Nanopartículas , Vacinas de mRNA , Animais , Humanos , Camundongos , Vacinas contra a AIDS/imunologia , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Reações Cruzadas , Técnicas de Introdução de Genes , Centro Germinativo/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , HIV-1/genética , Lipossomos , Células B de Memória/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/genética , Hipermutação Somática de Imunoglobulina , Vacinas de mRNA/imunologia , Feminino , Camundongos Endogâmicos C57BLRESUMO
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
The catalytic subunit of cAMP-dependent protein kinase (PKA) is a member of the AGC group of protein kinases. Whereas PKA has served as a structural model for the protein kinase superfamily, all previous structures of the catalytic subunit contain a phosphorylated activation loop. To understand the structural effects of activation loop phosphorylation at Thr-197 we used a PKA mutant that does not autophosphorylate at Thr-197. The enzyme crystallized in the apo-state, and the structure was solved to 3.0 Å. The N-lobe is rotated by 18° relative to the wild-type apoenzyme, which illustrates that the enzyme likely exists in a wide range of conformations in solution due to the uncoupling of the N- and C-lobes. Several regions of the protein including the activation loop are disordered in the structure, and there are alternate main chain conformations for the magnesium positioning loop and catalytic loop causing a complete loss of hydrogen bonding between these two active site structural elements. These alterations are reflected in a 20-fold decrease in the apparent phosphoryl transfer rate as measured by pre-steady-state kinetic methods.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Modelos Moleculares , Animais , Domínio Catalítico , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ligação de Hidrogênio , Camundongos , Mutação de Sentido Incorreto , Fosforilação/genética , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
The catalytic (C) subunit of cAMP-dependent protein kinase (PKA) is a serine/threonine kinase responsible for most of the effects of cAMP signaling, and PKA serves as a prototype for the entire kinase family. Despite multiple studies of PKA, the steps involved in phosphoryl transfer, the roles of the catalytically essential magnesium ions, and the processes that govern the rate-limiting step of ADP release are unresolved. Here we identified conditions that yielded slow phosphoryl transfer of the γ-phosphate from the generally nonhydrolyzable analog of ATP, adenosine-5'-(ß,γ-imido)triphosphate (AMP-PNP), onto a substrate peptide within protein crystals. By trapping both products in the crystal lattice, we now have a complete resolution profile of all the catalytic steps. One crystal structure refined to 1.55 Å resolution shows two states of the protein with 55% displaying intact AMP-PNP and an unphosphorylated substrate and 45% displaying transfer of the γ-phosphate of AMP-PNP onto the substrate peptide yielding AMP-PN and a phosphorylated substrate. Another structure refined to 2.15 Å resolution displays complete phosphoryl transfer to the substrate. These structures, in addition to trapping both products in the crystal lattice, implicate one magnesium ion, previously termed Mg2, as the more stably bound ion. Following phosphoryl transfer, Mg2 recruits a water molecule to retain an octahedral coordination geometry suggesting the strong binding character of this magnesium ion, and Mg2 remains in the active site following complete phosphoryl transfer while Mg1 is expelled. Loss of Mg1 may thus be an important part of the rate-limiting step of ADP release.
Assuntos
Adenilil Imidodifosfato/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peptídeos/metabolismo , Adenilil Imidodifosfato/química , Animais , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/química , Magnésio/química , Magnésio/metabolismo , Camundongos , Modelos Moleculares , Peptídeos/química , FosforilaçãoRESUMO
In the ongoing effort to develop a vaccine against HIV, vaccine approaches that promote strong germinal center (GC) responses may be critical to enable the selection and affinity maturation of rare B cell clones capable of evolving to produce broadly neutralizing antibodies. We previously demonstrated an approach for enhancing GC responses and overall humoral immunity elicited by alum-adjuvanted protein immunization via the use of phosphoserine (pSer) peptide-tagged immunogens that stably anchor to alum particles via ligand exchange with the alum particle surface. Here, using a clinically relevant stabilized HIV Env trimer termed MD39, we systematically evaluated the impact of several parameters relevant to pSer tag composition and trimer immunogen design to optimize this approach, including phosphate valency, amino acid sequence of the trimer C-terminus used for pSer tag conjugation, and structure of the pSer tag. We also tested the impact of co-administering a potent saponin/monophosphoryl lipid A (MPLA) nanoparticle co-adjuvant with alum-bound trimers. We identified MD39 trimer sequences bearing an optimized positively-charged C-terminal amino acid sequence, which, when conjugated to a pSer tag with four phosphates and a polypeptide spacer, bound very tightly to alum particles while retaining a native Env-like antigenicity profile. This optimized pSer-trimer design elicited robust antigen-specific GC B cell and serum IgG responses in mice. Through this optimization, we present a favorable MD39-pSer immunogen construct for clinical translation.
RESUMO
Phosphorylation of the activation loop is one of the most common mechanisms for regulating protein kinase activity. The catalytic subunit of cAMP-dependent protein kinase autophosphorylates Thr(197) in the activation loop when expressed in Escherichia coli. Although mutation of Arg(194) to Ala prevents autophosphorylation, phosphorylation of Thr(197) can still be achieved by a heterologous protein kinase, phosphoinositide-dependent protein kinase (PDK1), in vitro. In this study, we examined the structural and functional consequences of adding a single phosphate to the activation loop of cAMP-dependent protein kinase by comparing the wild type C-subunit to the R194A mutant either in the presence or the absence of activation loop phosphorylation. Phosphorylation of Thr(197) decreased the K(m) by approximately 15- and 7-fold for kemptide and ATP, respectively, increased the stability of the enzyme as measured by fluorescence and circular dichroism, and enhanced the binding between the C-subunit and IP20, a protein kinase inhibitor peptide. Additionally, deuterium exchange coupled to mass spectrometry was used to compare the structural dynamics of these proteins. All of the regions of the C-subunit analyzed underwent amide hydrogen exchange at a higher or equal rate in the unphosphorylated enzyme compared with the phosphorylated enzyme. The largest changes occurred at the C terminus of the activation segment in the p + 1 loop/APE regions and the alphaH-alphaI loop motifs and leads to the prediction of a coordinated phosphorylation-induced salt bridge between two conserved residues, Glu(208) and Arg(280).
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Mutação , Estrutura Terciária de Proteína , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Catálise , Domínio Catalítico/genética , Dicroísmo Circular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Medição da Troca de Deutério , Ativação Enzimática , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Fosforilação , Desnaturação Proteica , Dobramento de Proteína/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Treonina/metabolismo , Ureia/farmacologiaRESUMO
In vaccine design, antigens are often arrayed in a multivalent nanoparticle form, but in vivo mechanisms underlying the enhanced immunity elicited by such vaccines remain poorly understood. We compared the fates of two different heavily glycosylated HIV antigens, a gp120-derived mini-protein and a large, stabilized envelope trimer, in protein nanoparticle or "free" forms after primary immunization. Unlike monomeric antigens, nanoparticles were rapidly shuttled to the follicular dendritic cell (FDC) network and then concentrated in germinal centers in a complement-, mannose-binding lectin (MBL)-, and immunogen glycan-dependent manner. Loss of FDC localization in MBL-deficient mice or via immunogen deglycosylation significantly affected antibody responses. These findings identify an innate immune-mediated recognition pathway promoting antibody responses to particulate antigens, with broad implications for humoral immunity and vaccine design.
Assuntos
Vacinas contra a AIDS/imunologia , Formação de Anticorpos , Centro Germinativo/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Imunidade Inata , Polissacarídeos/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Testes de Fixação de Complemento , Proteínas do Sistema Complemento/imunologia , Células Dendríticas/imunologia , Feminino , Anticorpos Anti-HIV/imunologia , Infecções por HIV/prevenção & controle , Lipossomos , Lectina de Ligação a Manose/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Complexos Multiproteicos , Nanopartículas , Receptores de Complemento/imunologiaRESUMO
Vaccine induction of broadly neutralizing antibodies (bnAbs) to HIV remains a major challenge. Germline-targeting immunogens hold promise for initiating the induction of certain bnAb classes; yet for most bnAbs, a strong dependence on antibody heavy chain complementarity-determining region 3 (HCDR3) is a major barrier. Exploiting ultradeep human antibody sequencing data, we identified a diverse set of potential antibody precursors for a bnAb with dominant HCDR3 contacts. We then developed HIV envelope trimer-based immunogens that primed responses from rare bnAb-precursor B cells in a mouse model and bound a range of potential bnAb-precursor human naïve B cells in ex vivo screens. Our repertoire-guided germline-targeting approach provides a framework for priming the induction of many HIV bnAbs and could be applied to most HCDR3-dominant antibodies from other pathogens.
Assuntos
Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Evolução Molecular Direcionada/métodos , Anticorpos Anti-HIV/imunologia , Imunogenicidade da Vacina , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/química , Regiões Determinantes de Complementaridade/imunologia , Modelos Animais de Doenças , Células HEK293 , Anticorpos Anti-HIV/química , Humanos , Camundongos , Camundongos Knockout , Células Precursoras de Linfócitos B/imunologiaRESUMO
Elicitation of broadly neutralizing antibodies (bnAbs) is a primary HIV vaccine goal. Native-like trimers mimicking virion-associated spikes present nearly all bnAb epitopes and are therefore promising vaccine antigens. However, first generation native-like trimers expose epitopes for non-neutralizing antibodies (non-nAbs), which may hinder bnAb induction. We here employ computational and structure-guided design to develop improved native-like trimers that reduce exposure of non-nAb epitopes in the V3-loop and trimer base, minimize both CD4 reactivity and CD4-induced non-nAb epitope exposure, and increase thermal stability while maintaining bnAb antigenicity. In rabbit immunizations with native-like trimers of the 327c isolate, improved trimers suppress elicitation of V3-directed and tier-1 neutralizing antibodies and induce robust autologous tier-2 neutralization, unlike a first-generation trimer. The improved native-like trimers from diverse HIV isolates, and the design methods, have promise to assist in the development of a HIV vaccine.
Assuntos
Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Anticorpos Antivirais/imunologia , Antígenos CD4/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Animais , Anticorpos Neutralizantes/imunologia , Antígenos CD4/genética , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Infecções por HIV/imunologia , HIV-1/química , HIV-1/genética , Humanos , Imunização , Multimerização Proteica , Coelhos , Produtos do Gene env do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
Protein kinases have evolved in eukaryotes to be highly dynamic molecular switches that regulate a plethora of biological processes. Two motifs, a dynamic activation segment and a GHI helical subdomain, distinguish the eukaryotic protein kinases (EPKs) from the more primitive eukaryotic-like kinases. The EPKs are themselves highly regulated, typically by phosphorylation, and this allows them to be rapidly turned on and off. The EPKs have a novel hydrophobic architecture that is typically regulated by the dynamic assembly of two hydrophobic spines that is usually mediated by the phosphorylation of an activation loop phosphate. Cyclic AMP-dependent protein kinase (protein kinase A (PKA)) is used as a prototype to exemplify these features of the PKA superfamily. Specificity in PKA signalling is achieved in large part by packaging the enzyme as inactive tetrameric holoenzymes with regulatory subunits that then are localized to macromolecular complexes in close proximity to dedicated substrates by targeting scaffold proteins. In this way, the cell creates discrete foci that most likely represent the physiological environment for cyclic AMP-mediated signalling.
Assuntos
Eucariotos/enzimologia , Evolução Molecular , Fosforilação , Proteínas Quinases/química , Regulação Alostérica , Domínio Catalítico , Ativação Enzimática , Estabilidade Enzimática , Eucariotos/química , Holoenzimas/química , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Transdução de Sinais , Especificidade por SubstratoRESUMO
The catalytic (C) subunit of cAMP-dependent protein kinase [protein kinase A (PKA)] is a major target of cAMP signaling, and its regulation is of fundamental importance to biological processes. One mode of regulation is N-myristylation, which has eluded structural and functional characterization so far because most crystal structures are of the non-myristylated enzyme, are phosphorylated on Ser10, and generally lack electron density for the first 13 residues. We crystallized myristylated wild-type (WT) PKA and a K7C mutant as binary (bound to a substrate peptide) and ternary [bound to a substrate peptide and adenosine-5'-(ß,γ-imido)triphosphate] complexes. There was clear electron density for the entire N-terminus in the binary complexes, both refined to 2.0 Å, and K7C ternary complex, refined to 1.35 Å. The N-termini in these three structures display a novel conformation with a previously unseen helix from residues 1 to 7. The K7C mutant appears to have a more stable N-terminus, and this correlated with a significant decrease in the B-factors for the N-terminus in the myr-K7C complexes compared to the WT binary complex. The N-terminus of the myristylated WT ternary complex, refined to 2.0 Å, was disordered as in previous structures. In addition to a more ordered N-terminus, the myristylated K7C mutant exhibited a 53% increase in k(cat). The effect of nucleotide binding on the structure of the N-terminus in the WT protein and the kinetic changes in the K7C protein suggest that myristylation or occupancy of the myristyl binding pocket may serve as a site for allosteric regulation in the C-subunit.