RESUMO
Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.
Assuntos
Anticorpos Neutralizantes/imunologia , HIV-1/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Polissacarídeos/imunologia , SARS-CoV-2/imunologia , Vírus da Imunodeficiência Símia/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , Dimerização , Epitopos/imunologia , Glicosilação , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Macaca mulatta , Polissacarídeos/química , Receptores de Antígenos de Linfócitos B/química , Vírus da Imunodeficiência Símia/genética , Vacinas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
The HIV-1-envelope (Env) trimer is covered by a glycan shield of â¼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer.
Assuntos
HIV-1/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Cristalografia por Raios X , Glicosilação , HIV-1/classificação , HIV-1/imunologia , Evasão da Resposta Imune , Modelos Moleculares , Simulação de Dinâmica Molecular , Polissacarídeos/análise , Polissacarídeos/metabolismoRESUMO
An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate. We evaluated RSV F-specific B cell responses before and after vaccination in six participants using complementary B cell sequencing methodologies and identified 555 clonal lineages. DS-Cav1-induced lineages recognized the prefusion conformation of F (pre-F) and were genetically diverse. Expressed antibodies recognized all six antigenic sites on the pre-F trimer. We identified 34 public clonotypes, and structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Thus, vaccination with DS-Cav1 generates a diverse polyclonal response targeting the antigenic sites on pre-F, supporting the development and advanced testing of pre-F-based vaccines against RSV.
Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Criança , Pré-Escolar , Estudos de Coortes , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Vacinação/métodos , Proteínas Virais de Fusão/imunologia , Adulto JovemRESUMO
Virtually the entire surface of the HIV-1-envelope trimer is recognized by neutralizing antibodies, except for a highly glycosylated region at the center of the "silent face" on the gp120 subunit. From an HIV-1-infected donor, #74, we identified antibody VRC-PG05, which neutralized 27% of HIV-1 strains. The crystal structure of the antigen-binding fragment of VRC-PG05 in complex with gp120 revealed an epitope comprised primarily of N-linked glycans from N262, N295, and N448 at the silent face center. Somatic hypermutation occurred preferentially at antibody residues that interacted with these glycans, suggesting somatic development of glycan recognition. Resistance to VRC-PG05 in donor #74 involved shifting of glycan-N448 to N446 or mutation of glycan-proximal residue E293. HIV-1 neutralization can thus be achieved at the silent face center by glycan-recognizing antibody; along with other known epitopes, the VRC-PG05 epitope completes coverage by neutralizing antibody of all major exposed regions of the prefusion closed trimer.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Polissacarídeos/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Antígenos Virais/química , Antígenos Virais/imunologia , Sítios de Ligação , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Glicopeptídeos/química , Glicopeptídeos/imunologia , Glicosilação , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Polissacarídeos/química , Ligação Proteica/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Relação Estrutura-AtividadeRESUMO
While neutralizing antibodies that target the HIV-1 fusion peptide have been elicited in mice by vaccination, antibodies reported thus far have been from only a single antibody class that could neutralize ~30% of HIV-1 strains. To explore the ability of the murine immune system to generate cross-clade neutralizing antibodies and to investigate how higher breadth and potency might be achieved, we tested 17 prime-boost regimens that utilized diverse fusion peptide-carrier conjugates and HIV-1 envelope trimers with different fusion peptides. We observed priming in mice with fusion peptide-carrier conjugates of variable peptide length to elicit higher neutralizing responses, a result we confirmed in guinea pigs. From vaccinated mice, we isolated 21 antibodies, belonging to 4 distinct classes of fusion peptide-directed antibodies capable of cross-clade neutralization. Top antibodies from each class collectively neutralized over 50% of a 208-strain panel. Structural analyses - both X-ray and cryo-EM - revealed each antibody class to recognize a distinct conformation of fusion peptide and to have a binding pocket capable of accommodating diverse fusion peptides. Murine vaccinations can thus elicit diverse neutralizing antibodies, and altering peptide length during prime can improve the elicitation of cross-clade responses targeting the fusion peptide site of HIV-1 vulnerability. IMPORTANCE The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence. We found that variation in peptide length during prime elicits enhanced neutralizing responses in mice and guinea pigs. We identified vaccine-elicited murine monoclonal antibodies from distinct classes capable of cross-clade neutralization and of diverse fusion peptide recognition. Our findings lend insight into improved immunogens and regimens for HIV-1 vaccine development.
Assuntos
Vacinas contra a AIDS , Infecções por HIV , Soropositividade para HIV , HIV-1 , Animais , Cobaias , Camundongos , Anticorpos Anti-HIV , Isotipos de Imunoglobulinas , Vacinação , Peptídeos , Anticorpos Neutralizantes , Anticorpos Amplamente Neutralizantes , Produtos do Gene env do Vírus da Imunodeficiência Humana , Infecções por HIV/prevenção & controleRESUMO
Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been identified for related pneumoviruses based on the structure-based stabilization of the fusion (F) glycoprotein trimer, with prefusion-stabilized F glycoprotein trimers eliciting significantly higher neutralizing responses than their postfusion F counterparts. However, immunization with HMPV F trimers in either prefusion or postfusion conformations has been reported to elicit equivalent neutralization responses. Here we investigate the impact of stabilizing disulfides, especially interprotomer disulfides (IP-DSs) linking protomers of the F trimer, on the elicitation of HMPV-neutralizing responses. We designed F trimer disulfides, screened for their expression, and used electron microscopy (EM) to confirm their formation, including that of an unexpected postfusion variant. In mice, IP-DS-stabilized prefusion and postfusion HMPV F elicited significantly higher neutralizing responses than non-IP-DS-stabilized HMPV Fs. In macaques, the impact of IP-DS stabilization was more measured, although IP-DS-stabilized variants of either prefusion or postfusion HMPV F induced neutralizing responses many times the average titers observed in a healthy human cohort. Serological and absorption-based analyses of macaque responses revealed elicited HMPV-neutralizing responses to be absorbed differently by IP-DS-containing and by non-IP-DS-containing postfusion Fs, suggesting IP-DS stabilization to alter not only the immunogenicity of select epitopes but their antigenicity as well. We speculate the observed increase in immunogenicity by IP-DS trimers to be related to reduced interprotomer flexibility within the HMPV F trimer.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Dissulfetos/química , Epitopos/imunologia , Glicoproteínas/imunologia , Metapneumovirus/imunologia , Mutação , Animais , Glicoproteínas/genética , Humanos , Imunização , Macaca , Metapneumovirus/genética , Camundongos , Regiões Promotoras GenéticasRESUMO
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has led to growing concerns over increased transmissibility and the ability of some variants to partially escape immunity. Sera from participants immunized on a prime-boost schedule with the mRNA-1273 COVID-19 vaccine were tested for neutralizing activity against several SARS-CoV-2 variants, including variants of concern (VOCs) and variants of interest (VOIs), compared to neutralization of the wild-type SARS-CoV-2 virus (designated D614G). Results showed minimal, statistically nonsignificant effects on neutralization titers against the B.1.1.7 (Alpha) variant (1.2-fold reduction compared with D614G); other VOCs, such as B.1.351 (Beta, including B.1.351-v1, B.1.351-v2, and B.1.351-v3), P.1 (Gamma), and B.1.617.2 (Delta), showed significantly decreased neutralization titers ranging from 2.1-fold to 8.4-fold reductions compared with D614G, although all remained susceptible to mRNA-1273-elicited serum neutralization. IMPORTANCE In light of multiple variants of SARS-CoV-2 that have been documented globally during the COVID-19 pandemic, it remains important to continually assess the ability of currently available vaccines to confer protection against newly emerging variants. Data presented herein indicate that immunization with the mRNA-1273 COVID-19 vaccine produces neutralizing antibodies against key emerging variants tested, including variants of concern and variants of interest. While the serum neutralization elicited by mRNA-1273 against most variants tested was reduced compared with that against the wild-type virus, the level of neutralization is still expected to be protective. Such data are crucial to inform ongoing and future vaccination strategies to combat COVID-19.
Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Pandemias/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacina de mRNA-1273 contra 2019-nCoV , Adulto , Anticorpos Antivirais/imunologia , Feminino , Humanos , Masculino , Mutação , Testes de Neutralização , VacinaçãoRESUMO
Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites on the HIV envelope protein (Env) trimer or on other pathogens. However, the physical characteristics of peptide immunogens can limit their pharmacokinetic and immunological properties. Here, we have designed synthetic "star" nanoparticles based on biocompatible N-[(2-hydroxypropyl)methacrylamide] (HPMA)-based polymer arms extending from a poly(amidoamine) (PAMAM) dendrimer core. In mice, these star nanoparticles trafficked to lymph nodes (LNs) by 4 hours following vaccination, where they were taken up by subcapsular macrophages and then resident dendritic cells (DCs). Immunogenicity optimization studies revealed a correlation of immunogen density with antibody titers. Furthermore, the co-delivery of Env variable loop 3 (V3) and T-helper peptides induced titers that were 2 logs higher than if the peptides were given in separate nanoparticles. Finally, we performed a nonhuman primate (NHP) study using a V3 glycopeptide minimal immunogen that was structurally optimized to be recognized by Env V3/glycan broadly neutralizing antibodies (bnAbs). When administered with a potent Toll-like receptor (TLR) 7/8 agonist adjuvant, these nanoparticles elicited high antibody binding titers to the V3 site. Similar to human V3/glycan bnAbs, certain monoclonal antibodies (mAbs) elicited by this vaccine were glycan dependent or targeted the GDIR peptide motif. To improve affinity to native Env trimer affinity, nonhuman primates (NHPs) were boosted with various SOSIP Env proteins; however, significant neutralization was not observed. Taken together, this study provides a new vaccine platform for administration of glycopeptide immunogens for focusing immune responses to specific bnAb epitopes.
Assuntos
Vacinas contra a AIDS/imunologia , HIV-1/imunologia , Nanopartículas/uso terapêutico , Animais , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Epitopos/imunologia , Feminino , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/imunologia , Soropositividade para HIV/imunologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Peptídeos , PrimatasRESUMO
Human and simian immunodeficiency viruses (HIV/SIVs) use CD4 as the primary receptor to enter target cells. Here, we show that the chimpanzee CD4 is highly polymorphic, with nine coding variants present in wild populations, and that this diversity interferes with SIV envelope (Env)-CD4 interactions. Testing the replication fitness of SIVcpz strains in CD4+ T cells from captive chimpanzees, we found that certain viruses were unable to infect cells from certain hosts. These differences were recapitulated in CD4 transfection assays, which revealed a strong association between CD4 genotypes and SIVcpz infection phenotypes. The most striking differences were observed for three substitutions (Q25R, Q40R, and P68T), with P68T generating a second N-linked glycosylation site (N66) in addition to an invariant N32 encoded by all chimpanzee CD4 alleles. In silico modeling and site-directed mutagenesis identified charged residues at the CD4-Env interface and clashes between CD4- and Env-encoded glycans as mechanisms of inhibition. CD4 polymorphisms also reduced Env-mediated cell entry of monkey SIVs, which was dependent on at least one D1 domain glycan. CD4 allele frequencies varied among wild chimpanzees, with high diversity in all but the western subspecies, which appeared to have undergone a selective sweep. One allele was associated with lower SIVcpz prevalence rates in the wild. These results indicate that substitutions in the D1 domain of the chimpanzee CD4 can prevent SIV cell entry. Although some SIVcpz strains have adapted to utilize these variants, CD4 diversity is maintained, protecting chimpanzees against infection with SIVcpz and other SIVs to which they are exposed.
Assuntos
Antígenos CD4/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Vírus da Imunodeficiência Símia/genética , Proteínas do Envelope Viral/genética , Animais , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Evolução Molecular , Variação Genética/imunologia , HIV/genética , HIV/patogenicidade , Humanos , Pan troglodytes/genética , Pan troglodytes/imunologia , Polissacarídeos/genética , Polissacarídeos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas do Envelope Viral/imunologiaRESUMO
Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.
Assuntos
Vírus da Parainfluenza 1 Humana/imunologia , Vírus da Parainfluenza 2 Humana/imunologia , Vírus da Parainfluenza 3 Humana/imunologia , Vírus da Parainfluenza 4 Humana/imunologia , Infecções por Respirovirus/imunologia , Proteínas Virais de Fusão/química , Vacinas Virais/química , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Microscopia Crioeletrônica , Feminino , Humanos , Macaca mulatta , Masculino , Camundongos , Vírus da Parainfluenza 1 Humana/química , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 2 Humana/química , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 3 Humana/química , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 4 Humana/química , Vírus da Parainfluenza 4 Humana/genética , Infecções por Vírus Respiratório Sincicial , Infecções por Respirovirus/prevenção & controle , Infecções por Respirovirus/virologia , Proteínas Virais de Fusão/administração & dosagem , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a post-fusion state. As the sole viral antigen on the HIV-1 virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5 Å resolution for an HIV-1 Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the pre-fusion conformation of gp41, indicates rearrangements needed for fusion activation, and defines parameters of immune evasion and immune recognition. Pre-fusion gp41 encircles amino- and carboxy-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry involve opening the clasp and expelling the termini. N-linked glycosylation and sequence-variable regions cover the pre-fusion closed spike; we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation.
Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/imunologia , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/imunologia , Estudos de Coortes , Cristalografia por Raios X , Variação Genética , Glicosilação , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/genética , Infecções por HIV/imunologia , Humanos , Evasão da Resposta Imune , Fusão de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Polissacarídeos/química , Polissacarídeos/imunologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Homologia Estrutural de Proteína , Internalização do VírusAssuntos
Anticorpos Neutralizantes/sangue , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Vacina de mRNA-1273 contra 2019-nCoV , Anticorpos Antivirais/sangue , COVID-19/prevenção & controle , Humanos , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Estatísticas não Paramétricas , Vacinas Sintéticas/imunologia , Vacinas de mRNARESUMO
UNLABELLED: Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained. IMPORTANCE: The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve structural and antigenic mimicry of mature Env spikes on virions. Here we show that replacement of the cleavage site between gp120 and gp41 in a lead soluble gp140 construct, BG505.SOSIP, with flexible linkers can result in molecules that do not require cleavage to fold efficiently into the mature closed state. Our results provide insights into the impact of cleavage on HIV-1 Env folding. In some contexts such as genetic immunization, optimized cleavage-independent soluble gp140 constructs may have utility over the parental BG505.SOSIP, as they would not require furin cleavage to achieve mimicry of mature Env spikes on virions.
Assuntos
HIV-1/imunologia , HIV-1/fisiologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Substituição de Aminoácidos , Anticorpos Anti-HIV , Antígenos HIV/química , Antígenos HIV/genética , Antígenos HIV/ultraestrutura , HIV-1/genética , Humanos , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mimetismo Molecular , Mutagênese Sítio-Dirigida , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
Polymorphic differences distinguishing MHC class I subtypes often permit the presentation of shared epitopes in conformationally identical formats but can affect T-cell repertoire selection, differentially impacting autoimmune susceptibilities and viral clearance in vivo. The molecular mechanisms underlying this effect are not well understood. We performed structural, thermodynamic, and functional analyses of a conserved T-cell receptor (TCR) which is frequently expanded in response to a HIV-1 epitope when presented by HLA-B*5701 but is not selected by HLA-B*5703, which differs from HLA-B*5701 by two concealed polymorphisms. Our findings illustrate that although both HLA-B*57 subtypes display the epitope in structurally conserved formats, the impact of their polymorphic differences occurs directly as a consequence of TCR ligation, primarily because of peptide adjustments required for TCR binding, which involves the interplay of polymorphic residues and water molecules. These minor differences culminate in subtype-specific differential TCR-binding kinetics and cellular function. Our data demonstrate a potential mechanism whereby the most subtle MHC class I micropolymorphisms can influence TCR use and highlight their implications for disease outcomes.
Assuntos
Genes MHC Classe I , Antígenos HLA-B/química , Antígenos HLA-B/genética , Polimorfismo Genético , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos , Células Apresentadoras de Antígenos/imunologia , Cristalografia por Raios X , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/metabolismo , HIV-1/genética , HIV-1/imunologia , Antígenos HLA-B/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , TermodinâmicaRESUMO
With the success of messenger RNA (mRNA) vaccines against coronavirus disease 2019, strategies can now focus on improving vaccine potency, breadth, and stability. We designed and evaluated domain-based mRNA vaccines encoding the wild-type spike protein receptor binding domain (RBD) or N-terminal domain (NTD) alone or in combination. An NTD-RBD-linked candidate vaccine, mRNA-1283, showed improved antigen expression, antibody responses, and stability at refrigerated temperatures (2° to 8°C) compared with the clinically available mRNA-1273, which encodes the full-length spike protein. In BALB/c mice administered mRNA-1283 as a primary series, booster, or variant-specific booster, similar or greater immune responses from viral challenge were observed against wild-type, beta, delta, or omicron (BA.1) viruses compared with mRNA-1273-immunized mice, especially at lower vaccine dosages. K18-hACE2 mice immunized with mRNA-1283 or mRNA-1273 as a primary series demonstrated similar degrees of protection from challenge with SARS-CoV-2 Delta and Omicron variants at all vaccine dosages. These results support clinical assessment of mRNA-1283, which has now entered clinical trials (NCT05137236).
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Camundongos , COVID-19/prevenção & controle , Vacina de mRNA-1273 contra 2019-nCoV , Glicoproteína da Espícula de Coronavírus/genética , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , Vacinas de mRNARESUMO
Analysis of longitudinally obtained HIV-1 env sequences from an individual with reported cross-reactive neutralizing activity revealed that the majority of viral variants obtained from serum between 4 and 7 years after seroconversion were unable to persist in peripheral blood. Here we show that these viral variants were more sensitive to autologous serum neutralization, had shorter envelopes with fewer potential N-linked glycosylation sites, and showed lower replication kinetics than successfully evolving HIV-1 variants. These data reflect the host selection pressures on phenotypic characteristics of HIV-1 and illustrate in detail the dynamic interaction between HIV-1 and its host's humoral immune responses.
Assuntos
Anticorpos Neutralizantes/sangue , Variação Genética , Anticorpos Anti-HIV/sangue , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Sequência de Aminoácidos , Reações Cruzadas , Evolução Molecular , Genes env , Infecções por HIV/imunologia , HIV-1/fisiologia , Humanos , Dados de Sequência Molecular , Análise de Sequência de Proteína , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4(+) T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 10(4) copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4(+) T-cell numbers, low viral load and limited viral divergence.
Assuntos
Antirretrovirais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Variação Genética , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Viremia/tratamento farmacológico , Substituição de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Evolução Biológica , Imunoglobulina G/sangue , Macaca fascicularis , Masculino , Glicoproteínas de Membrana/genética , Mutação/genética , Filogenia , Reação em Cadeia da Polimerase , RNA Viral , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/patogenicidade , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/virologia , Proteínas do Envelope Viral/genética , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia , Viremia/imunologiaRESUMO
With the success of mRNA vaccines against coronavirus disease 2019 (COVID-19), strategies can now focus on improving vaccine potency, breadth, and stability. We present the design and preclinical evaluation of domain-based mRNA vaccines encoding the wild-type spike-protein receptor-binding (RBD) and/or N-terminal domains (NTD). An NTD-RBD linked candidate vaccine, mRNA-1283, showed improved antigen expression, antibody responses, and stability at refrigerated temperatures (2-8°C) compared with the clinically available mRNA-1273, which encodes the full-length spike protein. In mice administered mRNA-1283 as a primary series, booster, or variant-specific booster, similar or greater immune responses and protection from viral challenge were observed against wild-type, beta, delta, or omicron (BA. 1) compared with mRNA-1273 immunized mice, especially at lower vaccine dosages. These results support clinical assessment of mRNA-1283 ( NCT05137236 ). One Sentence Summary: A domain-based mRNA vaccine, mRNA-1283, is immunogenic and protective against SARS-CoV-2 and emerging variants in mice.
RESUMO
Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Antivirais/farmacologia , Pulmão/virologia , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Infecções por Respirovirus/prevenção & controle , Proteínas Virais de Fusão/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Antivirais/imunologia , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular , Modelos Animais de Doenças , Epitopos , Interações Hospedeiro-Patógeno , Humanos , Hospedeiro Imunocomprometido , Pulmão/imunologia , Vírus da Parainfluenza 3 Humana/imunologia , Vírus da Parainfluenza 3 Humana/patogenicidade , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/virologia , Sigmodontinae , Proteínas Virais de Fusão/imunologiaRESUMO
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative infection of a global pandemic that has led to more than 2 million deaths worldwide. The Moderna mRNA-1273 vaccine has demonstrated ~94% efficacy in a Phase 3 study and has been approved under Emergency Use Authorization. The emergence of SARS-CoV-2 variants with mutations in the spike protein, most recently circulating isolates from the United Kingdom (B.1.1.7) and Republic of South Africa (B.1.351), has led to lower neutralization from convalescent serum by pseudovirus neutralization (PsVN) assays and resistance to certain monoclonal antibodies. Here, using two orthogonal VSV and lentivirus PsVN assays expressing spike variants of 20E (EU1), 20A.EU2, D614G-N439, mink cluster 5, B.1.1.7, and B.1.351 variants, we assessed the neutralizing capacity of sera from human subjects or non-human primates (NHPs) that received mRNA-1273. No significant impact on neutralization against the B.1.1.7 variant was detected in either case, however reduced neutralization was measured against the mutations present in B.1.351. Geometric mean titer (GMT) of human sera from clinical trial participants in VSV PsVN assay using D614G spike was 1/1852. VSV pseudoviruses with spike containing K417N-E484K-N501Y-D614G and full B.1.351 mutations resulted in 2.7 and 6.4-fold GMT reduction, respectively, when compared to the D614G VSV pseudovirus. Importantly, the VSV PsVN GMT of these human sera to the full B.1.351 spike variant was still 1/290, with all evaluated sera able to fully neutralize. Similarly, sera from NHPs immunized with 30 or 100µg of mRNA-1273 had VSV PsVN GMTs of ~ 1/323 or 1/404, respectively, against the full B.1.351 spike variant with a ~ 5 to 10-fold reduction compared to D614G. Individual mutations that are characteristic of the B.1.1.7 and B.1.351 variants had a similar impact on neutralization when tested in VSV or in lentivirus PsVN assays. Despite the observed decreases, the GMT of VSV PsVN titers in human vaccinee sera against the B.1.351 variant remained at ~1/300. Taken together these data demonstrate reduced but still significant neutralization against the full B.1.351 variant following mRNA-1273 vaccination.