RESUMO
Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.
Assuntos
Afinidade de Anticorpos , Linfócitos B , Centro Germinativo , Anticorpos Anti-HIV , HIV-1 , Centro Germinativo/imunologia , Animais , Camundongos , Humanos , Linfócitos B/imunologia , HIV-1/imunologia , Anticorpos Anti-HIV/imunologia , Afinidade de Anticorpos/imunologia , Anticorpos Neutralizantes/imunologia , Infecções por HIV/imunologia , Vacinas contra a AIDS/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Técnicas de Introdução de Genes , Camundongos Transgênicos , Anticorpos Amplamente Neutralizantes/imunologia , Camundongos Endogâmicos C57BLRESUMO
A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.
Assuntos
Vacinas contra a AIDS , Anticorpos Neutralizantes , Anticorpos Anti-HIV , Proteína gp41 do Envelope de HIV , Infecções por HIV , HIV-1 , Macaca mulatta , Animais , Humanos , Proteína gp41 do Envelope de HIV/imunologia , Anticorpos Anti-HIV/imunologia , Camundongos , Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , HIV-1/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Vacinação , Anticorpos Amplamente Neutralizantes/imunologia , Linfócitos B/imunologia , Nanopartículas/química , Feminino , Regiões Determinantes de Complementaridade/imunologia , Epitopos/imunologiaRESUMO
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
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
Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.
Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Vacinas Sintéticas/imunologia , Adulto , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/genética , Antígenos Virais/imunologia , Feminino , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Alinhamento de Sequência , Vacinas Sintéticas/administração & dosagemRESUMO
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
How precursor frequencies and antigen affinities impact interclonal B cell competition is a particularly relevant issue for candidate germline-targeting HIV vaccine designs because of the in vivo rarity of naive B cells that recognize broadly neutralizing epitopes. Knowing the frequencies and affinities of HIV-specific VRC01-class naive human B cells, we transferred B cells with germline VRC01 B cell receptors into congenic recipients to elucidate the roles of precursor frequency, antigen affinity, and avidity on B cell responses following immunization. All three factors were interdependently limiting for competitive success of VRC01-class B cells. In physiological high-affinity conditions using a multivalent immunogen, rare VRC01-class B cells successfully competed in germinal centers (GC), underwent extensive somatic hypermutation, and differentiated into memory B cells. The data reveal dominant influences of precursor frequency, affinity, and avidity for interclonal GC competition and indicate that germline-targeting immunogens can overcome these challenges with high-affinity multimeric designs.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , HIV-1/imunologia , Animais , Anticorpos Amplamente Neutralizantes , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Anticorpos Anti-HIV , Masculino , Camundongos , Camundongos TransgênicosRESUMO
An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-2∗02, the germline VRC01 VH segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.
Assuntos
Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos/imunologia , Antígenos CD4/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Amplamente Neutralizantes , Linhagem Celular , Feminino , Técnicas de Introdução de Genes , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polissacarídeos/químicaRESUMO
Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through nontemplated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs Vκ3-20 or Vκ1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had nonphysiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as Vκ1-33 and/or Vκ3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted the generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated Vκ-to-Jκ joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center B cell responses. Vκ3-20-based responses were enhanced by N-region addition, which generates Vκ3-20-to-Jκ junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for the generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Vacinas , Camundongos , Humanos , Animais , Anticorpos Amplamente Neutralizantes , Anticorpos Neutralizantes , HIV-1/genética , Anticorpos Anti-HIV , DNA Nucleotidilexotransferase , Regiões Determinantes de Complementaridade/genética , Infecções por HIV/prevenção & controleRESUMO
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
Animal models of human antigen-specific B cell receptors (BCRs) generally depend on "inferred germline" sequences, and thus their relationship to authentic naive human B cell BCR sequences and affinities is unclear. Here, BCR sequences from authentic naive human VRC01-class B cells from healthy human donors were selected for the generation of three BCR knockin mice. The BCRs span the physiological range of affinities found in humans, and use three different light chains (VK3-20, VK1-5, and VK1-33) found among subclasses of naive human VRC01-class B cells and HIV broadly neutralizing antibodies (bnAbs). The germline-targeting HIV immunogen eOD-GT8 60mer is currently in clinical trial as a candidate bnAb vaccine priming immunogen. To attempt to model human immune responses to the eOD-GT8 60mer, we tested each authentic naive human VRC01-class BCR mouse model under rare human physiological B cell precursor frequency conditions. B cells with high (HuGL18HL) or medium (HuGL17HL) affinity BCRs were primed, recruited to germinal centers, and they affinity matured, and formed memory B cells. Precursor frequency and affinity interdependently influenced responses. Taken together, these experiments utilizing authentic naive human VRC01-class BCRs validate a central tenet of germline-targeting vaccine design and extend the overall concept of the reverse vaccinology approach to vaccine development.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Vacinas contra a AIDS/imunologia , Sequência de Aminoácidos/genética , Animais , Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/farmacologia , Antígenos CD4/imunologia , Técnicas de Introdução de Genes/métodos , Centro Germinativo/imunologia , Antígenos HIV , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/imunologia , Vacinação/métodosRESUMO
Inducing broad spectrum neutralizing antibodies against challenging pathogens such as HIV-1 is a major vaccine design goal, but may be hindered by conformational instability within viral envelope glycoproteins (Env). Chemical cross-linking is widely used for vaccine antigen stabilization, but how this process affects structure, antigenicity and immunogenicity is poorly understood and its use remains entirely empirical. We have solved the first cryo-EM structure of a cross-linked vaccine antigen. The 4.2 Å structure of HIV-1 BG505 SOSIP soluble recombinant Env in complex with a CD4 binding site-specific broadly neutralizing antibody (bNAb) Fab fragment reveals how cross-linking affects key properties of the trimer. We observed density corresponding to highly specific glutaraldehyde (GLA) cross-links between gp120 monomers at the trimer apex and between gp120 and gp41 at the trimer interface that had strikingly little impact on overall trimer conformation, but critically enhanced trimer stability and improved Env antigenicity. Cross-links were also observed within gp120 at sites associated with the N241/N289 glycan hole that locally modified trimer antigenicity. In immunogenicity studies, the neutralizing antibody response to cross-linked trimers showed modest but significantly greater breadth against a global panel of difficult-to-neutralize Tier-2 heterologous viruses. Moreover, the specificity of autologous Tier-2 neutralization was modified away from the N241/N289 glycan hole, implying a novel specificity. Finally, we have investigated for the first time T helper cell responses to next-generation soluble trimers, and report on vaccine-relevant immunodominant responses to epitopes within BG505 that are modified by cross-linking. Elucidation of the structural correlates of a cross-linked viral glycoprotein will allow more rational use of this methodology for vaccine design, and reveals a strategy with promise for eliciting neutralizing antibodies needed for an effective HIV-1 vaccine.
Assuntos
HIV-1/química , 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/química , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Reagentes de Ligações Cruzadas , Microscopia Crioeletrônica , Anticorpos Anti-HIV/imunologia , Antígenos HIV/química , Antígenos HIV/imunologia , Antígenos HIV/ultraestrutura , Interações Hospedeiro-Patógeno/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Coelhos , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/ultraestruturaRESUMO
Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.
Assuntos
Desenho de Fármacos , Epitopos/química , Epitopos/imunologia , Estabilidade Proteica , Vacinas contra Vírus Sincicial Respiratório/química , Vacinas contra Vírus Sincicial Respiratório/imunologia , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/análise , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/imunologia , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Macaca mulatta/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Testes de Neutralização , Conformação Proteica , Vírus Sinciciais Respiratórios/química , Vírus Sinciciais Respiratórios/imunologiaRESUMO
Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Humanos , Conformação Proteica , Ressonância de Plasmônio de Superfície , Difração de Raios XRESUMO
Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix-loop-helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/química , Antivirais/farmacologia , Linhagem Celular , Cristalografia por Raios X , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Camundongos , Mutagênese , Palivizumab/farmacologia , Vacinas contra Vírus Sincicial Respiratório/química , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacosRESUMO
An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Desenho de Fármacos , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/genética , Anticorpos Anti-HIV/genética , Infecções por HIV/imunologia , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , MutaçãoRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1005815.].
RESUMO
Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.