RESUMO
The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.
Assuntos
Vacinas contra a AIDS , HIV-1 , Animais , Humanos , Anticorpos Amplamente Neutralizantes , Antígenos CD4 , Moléculas de Adesão Celular , HIV-1/fisiologia , Macaca , Vacinas contra a AIDS/imunologiaRESUMO
A critical roadblock to HIV vaccine development is the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans. In people living with HIV-1, bnAbs take years to develop. The HVTN 133 clinical trial studied a peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs (NCT03934541). Here, we report MPER peptide-liposome induction of polyclonal HIV-1 B cell lineages of mature bnAbs and their precursors, the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains with lineage initiation after the second immunization. Neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids. This study demonstrates proof of concept for rapid vaccine induction of human B cell lineages with heterologous neutralizing activity and selection of antibody improbable mutations and outlines a path for successful HIV-1 vaccine development.
Assuntos
Vacinas contra a AIDS , Anticorpos Neutralizantes , Linfócitos B , Anticorpos Anti-HIV , HIV-1 , Humanos , Vacinas contra a AIDS/imunologia , HIV-1/imunologia , Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Linhagem da Célula , Lipossomos , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Mutação , Proteína gp41 do Envelope de HIV/imunologiaRESUMO
How different classes of the B cell antigen receptor (BCR) sense viral antigens used in vaccination protocols is poorly understood. Here, we study antigen binding and sensing of human Ramos B cells expressing a BCR of either the IgM or IgG1 class with specificity for the CD4-binding-site of the envelope (Env) protein of the HIV-1. Both BCRs carry an identical antigen binding site derived from the broad neutralizing antibody (bnAb) CH31. We find a five times higher expression of the IgG1-BCR in comparison to the IgM-BCR on the surface of transfected Ramos B cells. The two BCR classes also differ from each other in their interaction with cognate HIV Env antigens in that the IgG1-BCR and IgM-BCR bind preferentially to polyvalent and monovalent antigens, respectively. By generating an IgM/IgG1 chimeric BCR, we found that the class-specific BCR expression and antigen-sensing behavior can be transferred with the CH1γ domain from the IgG1-BCR to the IgM-BCR. Thus, the class of CH1 domain has an impact on BCR assembly and expression as well as on antigen sensing.
Assuntos
HIV-1 , Imunoglobulina G , Imunoglobulina M , Receptores de Antígenos de Linfócitos B , Humanos , Imunoglobulina M/imunologia , Imunoglobulina G/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , HIV-1/imunologia , HIV-1/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Anticorpos Anti-HIV/imunologia , Domínios Proteicos , Anticorpos Neutralizantes/imunologiaRESUMO
Antibodies perform both neutralizing and non-neutralizing effector functions that protect against certain pathogen-induced diseases. A human antibody directed at the SARS-CoV-2 Spike N-terminal domain (NTD), DH1052, was recently shown to be non-neutralizing, yet it protected mice and cynomolgus macaques from severe disease. The mechanisms of NTD non-neutralizing antibody-mediated protection are unknown. Here we show that Fc effector functions mediate NTD non-neutralizing antibody (non-nAb) protection against SARS-CoV-2 MA10 viral challenge in mice. Though non-nAb prophylactic infusion did not suppress infectious viral titers in the lung as potently as neutralizing antibody (nAb) infusion, disease markers including gross lung discoloration were similar in nAb and non-nAb groups. Fc functional knockout substitutions abolished non-nAb protection and increased viral titers in the nAb group. Fc enhancement increased non-nAb protection relative to WT, supporting a positive association between Fc functionality and degree of protection from SARS-CoV-2 infection. For therapeutic administration of antibodies, non-nAb effector functions contributed to virus suppression and lessening of lung discoloration, but the presence of neutralization was required for optimal protection from disease. This study demonstrates that non-nAbs can utilize Fc-mediated mechanisms to lower viral load and prevent lung damage due to coronavirus infection.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Fragmentos Fc das Imunoglobulinas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , SARS-CoV-2/imunologia , Camundongos , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Anticorpos Antivirais/imunologia , Anticorpos Neutralizantes/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Humanos , Feminino , Domínios Proteicos/imunologia , Carga Viral , Pulmão/virologia , Pulmão/imunologia , Pulmão/patologiaRESUMO
Small-molecule viral entry inhibitors, such as BMS-626529 (BMS-529), allosterically block CD4 binding to HIV-1 envelope (Env) and inhibit CD4-induced structural changes in Env trimers. Here, we show that the binding of BMS-529 to clade C soluble chimeric gp140 SOSIP (ch.SOSIP) and membrane-bound trimers with intact transmembrane domain (gp150) prevented trimer conformational transitions and enhanced their immunogenicity. When complexed to BMS-529, ch.SOSIP trimers retained their binding to broadly neutralizing antibodies (bNAbs) and to their unmutated common ancestor (UCA) antibodies, while exposure of CD4-induced (CD4i) non-bNAb epitopes was inhibited. BMS-529-complexed gp150 trimers in detergent micelles, which were isolated from CHO cells, bound to bNAbs, including UCA and intermediates of the CD4 binding site (bs) CH103 bNAb lineage, and showed limited exposure of CD4i epitopes and a glycosylation pattern with a preponderance of high-mannose glycans. In rabbits, BMS-529-complexed V3 glycan-targeting ch.SOSIP immunogen induced in the majority of immunized animals higher neutralization titers against both autologous and select high mannose-bearing heterologous tier 2 pseudoviruses than those immunized with the noncomplexed ch.SOSIP. In rhesus macaques, BMS-529 complexed to CD4 bs-targeting ch.SOSIP immunogen induced stronger neutralization against tier 2 pseudoviruses bearing high-mannose glycans than noncomplexed ch.SOSIP trimer immunogen. When immunized with gp150 complexed to BMS-529, rhesus macaques showed neutralization against tier 2 pseudoviruses with targeted glycan deletion and high-mannose glycan enrichment. These results demonstrated that stabilization of Env trimer conformation with BMS-529 improved the immunogenicity of select chimeric SOSIP trimers and elicited tier 2 neutralizing antibodies of higher potency than noncomplexed trimers.IMPORTANCE Soluble forms of HIV-1 envelope trimers exhibit conformational heterogeneity and undergo CD4-induced (CD4i) exposure of epitopes of non-neutralizing antibodies that can potentially hinder induction of broad neutralizing antibody responses. These limitations have been mitigated through recent structure-guided approaches and include trimer-stabilizing mutations that resist trimer conformational transition and exposure of CD4i epitopes. The use of small-molecule viral inhibitors that allosterically block CD4 binding represents an alternative strategy for stabilizing Env trimer in the pre-CD4-triggered state of both soluble and membrane-bound trimers. In this study, we report that the viral entry inhibitor BMS-626529 restricts trimer conformational transition and improves the immunogenicity of select Env trimer immunogens.
Assuntos
Fármacos Anti-HIV/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Infecções por HIV/tratamento farmacológico , Imunoconjugados/farmacologia , Piperazinas/farmacologia , Triazóis/farmacologia , Animais , Fármacos Anti-HIV/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Antígenos CD4/antagonistas & inibidores , Antígenos CD4/genética , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células CHO , Cricetulus , Glicosilação , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Humanos , Imunoconjugados/química , Macaca mulatta , Piperazinas/química , Multimerização Proteica , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Triazóis/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
In the RV144 gp120 HIV vaccine trial, decreased transmission risk was correlated with Abs that reacted with a linear epitope at a lysine residue at position 169 (K169) in the HIV-1 envelope (Env) V2 region. The K169 V2 response was restricted to Abs bearing Vλ rearrangements that expressed aspartic acid/glutamic acid in CDR L2. The AE.A244 gp120 in AIDSVAX B/E also bound to the unmutated ancestor of a V2-glycan broadly neutralizing Ab, but this Ab type was not induced in the RV144 trial. In this study, we sought to determine whether immunodominance of the V2 linear epitope could be overcome in the absence of human Vλ rearrangements. We immunized IgH- and Igκ-humanized mice with the AE.A244 gp120 Env. In these mice, the V2 Ab response was focused on a linear epitope that did not include K169. V2 Abs were isolated that used the same human VH gene segment as an RV144 V2 Ab but paired with a mouse λ L chain. Structural characterization of one of these V2 Abs revealed how the linear V2 epitope could be engaged, despite the lack of aspartic acid/glutamic acid encoded in the mouse repertoire. Thus, despite the absence of the human Vλ locus in these humanized mice, the dominance of Vλ pairing with human VH for HIV-1 Env V2 recognition resulted in human VH pairing with mouse λ L chains instead of allowing otherwise subdominant V2-glycan broadly neutralizing Abs to develop.
Assuntos
Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Motivos de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Epitopos , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias lambda de Imunoglobulina/imunologia , CamundongosRESUMO
G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the ß2-adrenoceptor (ß2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Regulação Alostérica/efeitos dos fármacos , Aptâmeros de Nucleotídeos/química , Benzoxazinas/química , Benzoxazinas/farmacologia , Humanos , Modelos Moleculares , Conformação Proteica , Receptores Adrenérgicos beta 2/químicaRESUMO
Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Doença Crônica , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/patologiaRESUMO
Achieving an AIDS-free generation will require elimination of postnatal transmission of HIV-1 while maintaining the nutritional and immunologic benefits of breastfeeding for infants in developing regions. Maternal/infant antiretroviral prophylaxis can reduce postnatal HIV-1 transmission, yet toxicities and the development of drug-resistant viral strains may limit the effectiveness of this strategy. Interestingly, in the absence of antiretroviral prophylaxis, greater than 90% of infants exposed to HIV-1 via breastfeeding remain uninfected, despite daily mucosal exposure to the virus for up to 2 y. Moreover, milk of uninfected women inherently neutralizes HIV-1 and prevents virus transmission in animal models, yet the factor(s) responsible for this anti-HIV activity is not well-defined. In this report, we identify a primary HIV-1-neutralizing protein in breast milk, Tenascin-C (TNC). TNC is an extracellular matrix protein important in fetal development and wound healing, yet its antimicrobial properties have not previously been established. Purified TNC captured and neutralized multiclade chronic and transmitted/founder HIV-1 variants, and depletion of TNC abolished the HIV-1-neutralizing activity of milk. TNC bound the HIV-1 Envelope protein at a site that is induced upon engagement of its primary receptor, CD4, and is blocked by V3 loop- (19B and F39F) and chemokine coreceptor binding site-directed (17B) monoclonal antibodies. Our results demonstrate the ability of an innate mucosal host protein found in milk to neutralize HIV-1 via binding to the chemokine coreceptor site, potentially explaining why the majority of HIV-1-exposed breastfed infants are protected against mucosal HIV-1 transmission.
Assuntos
Síndrome da Imunodeficiência Adquirida/transmissão , HIV-1/efeitos dos fármacos , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Leite Humano/química , Tenascina/farmacologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Western Blotting , Linhagem Celular , Cromatografia por Troca Iônica , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunoprecipitação , Concentração Inibidora 50 , Espectrometria de Massas , Tenascina/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. Broadly neutralizing antibodies (BnAbs) are not induced by current vaccines, but are found in plasma in â¼20% of HIV-1-infected individuals after several years of infection. One strategy for induction of unfavored antibody responses is to produce homogeneous immunogens that selectively express BnAb epitopes but minimally express dominant strain-specific epitopes. Here we report that synthetic, homogeneously glycosylated peptides that bind avidly to variable loop 1/2 (V1V2) BnAbs PG9 and CH01 bind minimally to strain-specific neutralizing V2 antibodies that are targeted to the same envelope polypeptide site. Both oligomannose derivatization and conformational stabilization by disulfide-linked dimer formation of synthetic V1V2 peptides were required for strong binding of V1V2 BnAbs. An HIV-1 vaccine should target BnAb unmutated common ancestor (UCA) B-cell receptors of naïve B cells, but to date no HIV-1 envelope constructs have been found that bind to the UCA of V1V2 BnAb PG9. We demonstrate herein that V1V2 glycopeptide dimers bearing Man5GlcNAc2 glycan units bind with apparent nanomolar affinities to UCAs of V1V2 BnAbs PG9 and CH01 and with micromolar affinity to the UCA of a V2 strain-specific antibody. The higher-affinity binding of these V1V2 glycopeptides to BnAbs and their UCAs renders these glycopeptide constructs particularly attractive immunogens for targeting subdominant HIV-1 envelope V1V2-neutralizing antibody-producing B cells.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Glicopeptídeos/imunologia , HIV-1/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos B/genética , Ressonância de Plasmônio de SuperfícieRESUMO
The rare, broadly neutralizing antibodies, 4E10 and 2F5, that target the HIV-1 membrane proximal external region also associate with HIV-1 membrane lipids as part of a required first-step in HIV-1 neutralization. HIV-1 virions have high concentration of cholesterol and sphingomyelin, which are able to organize into liquid-ordered domains (i.e., lipid rafts), and could influence the interaction of neutralizing antibodies with epitopes proximal to the membrane. The objective of this research is to understand how these lipid domains contribute to 2F5/4E10 membrane interactions and to antigen presentation in liposomal form of HIV-1 vaccines. To this end we have engineered biomimetic supported lipid bilayers and are able to use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody-membrane interactions. Our results demonstrate that 2F5/4E10 do not interact with highly ordered gel and liquid-ordered domains and exclusively bind to a liquid-disordered lipid phase. This suggests that vaccine liposomes that contain key viral membrane components, such as high cholesterol content, may not be advantageous for 2F5/4E10 vaccine strategies. Rather, vaccine liposomes that primarily contain a liquid-disordered phase may be more likely to elicit production of lipid reactive, 2F5- and 4E10-like antibodies.
Assuntos
Anticorpos Monoclonais Murinos , Apresentação de Antígeno/efeitos dos fármacos , Materiais Biomiméticos , Anticorpos Anti-HIV , HIV-1/imunologia , Microdomínios da Membrana , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/farmacologia , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Humanos , Lipossomos/química , Lipossomos/farmacologia , Microdomínios da Membrana/química , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , CamundongosRESUMO
UNLABELLED: Mucosal epithelial cell surface galactosylceramide (Galcer) has been postulated to be a receptor for HIV-1 envelope (Env) interactions with mucosal epithelial cells. Disruption of the HIV-1 Env interaction with such alternate receptors could be one strategy to prevent HIV-1 entry through the mucosal barrier. To study antibody modulation of HIV-1 Env-Galcer interactions, we used Galcer-containing liposomes to assess whether natural- and vaccine-induced monoclonal antibodies can block HIV-1 Env binding to Galcer. HIV-1 Env gp140 proteins bound to Galcer liposomes with Kds (dissociation constants) in the nanomolar range. Several HIV-1 ALVAC/AIDSVAX vaccinee-derived monoclonal antibodies (MAbs) specific for the gp120 first constant (C1) region blocked Galcer binding of a transmitted/founder HIV-1 Env gp140. Among the C1-specific MAbs that showed Galcer blocking, the antibody-dependent cellular cytotoxicity-mediating CH38 IgG and its natural IgA isotype were the most potent blocking antibodies. C1-specific IgG monoclonal antibodies that blocked Env binding to Galcer induced upregulation of the gp120 CD4-inducible (CD4i) epitope bound by MAb 17B, demonstrating that a conformational change in gp120 may be required for Galcer blocking. However, the MAb 17B itself did not block Env-Galcer binding, suggesting that the C1 antibody-induced gp120 conformational changes resulted in alteration in a Galcer binding site distant from the CD4i 17B MAb binding site. IMPORTANCE: Galactosyl ceramide, a glycosphingolipid, has been postulated to be a receptor for the HIV-1 envelope glycoprotein (Env) interaction with mucosal epithelial cells. Here, we have mimicked this interaction by using an artificial membrane containing synthetic Galcer and recombinant HIV-1 Env proteins to identify antibodies that would block the HIV-1 Env-Galcer interaction. Our study revealed that a class of vaccine-induced human antibodies potently blocks HIV-1 Env-Galcer binding by perturbing the HIV-1 Env conformation.
Assuntos
Vacinas contra a AIDS/imunologia , Antígenos CD4/imunologia , Epitopos/imunologia , Galactosilceramidas/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Sítios de Ligação/imunologia , Anticorpos Anti-HIV/imunologia , Humanos , Imunoglobulina G/imunologia , Lipossomos/imunologia , Ligação Proteica/imunologiaRESUMO
An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.
Assuntos
Vacinas contra a AIDS/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Deleção de Sequência , Vacinas contra a AIDS/genética , Animais , Afinidade de Anticorpos , Epitopos/imunologia , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/genética , Humanos , Macaca mulattaRESUMO
The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although remarkable progress has been made in understanding the structures of various Env conformations, microsecond timescale dynamics have not been studied experimentally. Here, we used time-resolved, temperature-jump small-angle x-ray scattering to monitor structural rearrangements in an HIV-1 Env SOSIP ectodomain construct with microsecond precision. In two distinct Env variants, we detected a transition that correlated with known Env structure rearrangements with a time constant in the hundreds of microseconds range. A previously unknown structural transition was also observed, which occurred with a time constant below 10 µs, and involved an order-to-disorder transition in the trimer apex. Using this information, we engineered an Env SOSIP construct that locks the trimer in the prefusion closed state by connecting adjacent protomers via disulfides. Our findings show that the microsecond timescale structural dynamics play an essential role in controlling the Env conformation with impacts on vaccine design.
Assuntos
HIV-1 , Produtos do Gene env do Vírus da Imunodeficiência Humana , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Anticorpos Anti-HIV , Conformação Molecular , Multimerização Proteica , Conformação ProteicaRESUMO
Critical to the search for an effective HIV-1 vaccine is the development of immunogens capable of inducing broadly neutralizing antibodies (BnAbs). A key first step in this process is to design immunogens that can be recognized by known BnAbs. The monoclonal antibody PG9 is a BnAb that neutralizes diverse strains of HIV-1 by targeting a conserved carbohydrate-protein epitope in the variable 1 and 2 (V1V2) region of the viral envelope. Important for recognition are two closely spaced N-glycans at Asn(160) and Asn(156). Glycopeptides containing this synthetically challenging bis-N-glycosylated motif were prepared by convergent assembly, and were shown to be antigenic for PG9. Synthetic glycopeptides such as these may be useful for the development of HIV-1 vaccines based on the envelope V1V2 BnAb epitope.
Assuntos
Vacinas contra a AIDS/imunologia , Antígenos/imunologia , Glicopeptídeos/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/química , Antígenos/química , Configuração de Carboidratos , Cristalografia por Raios X , Glicopeptídeos/química , Proteína gp120 do Envelope de HIV/química , Modelos Moleculares , Dados de Sequência MolecularRESUMO
Antibody affinity maturation enables adaptive immune responses to a wide range of pathogens. In some individuals broadly neutralizing antibodies develop to recognize rapidly mutating pathogens with extensive sequence diversity. Vaccine design for pathogens such as HIV-1 and influenza has therefore focused on recapitulating the natural affinity maturation process. Here, we determine structures of antibodies in complex with HIV-1 Envelope for all observed members and ancestral states of the broadly neutralizing HIV-1 V3-glycan targeting DH270 antibody clonal B cell lineage. These structures track the development of neutralization breadth from the unmutated common ancestor and define affinity maturation at high spatial resolution. By elucidating contacts mediated by key mutations at different stages of antibody development we identified sites on the epitope-paratope interface that are the focus of affinity optimization. Thus, our results identify bottlenecks on the path to natural affinity maturation and reveal solutions for these that will inform immunogen design aimed at eliciting a broadly neutralizing immune response by vaccination.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Infecções por HIV/prevenção & controle , HIV-1/genética , Anticorpos Neutralizantes , Anticorpos Anti-HIV , PolissacarídeosRESUMO
Antibodies perform both neutralizing and non-neutralizing effector functions that protect against certain pathogen-induced diseases. A human antibody directed at the SARS-CoV-2 Spike N-terminal domain (NTD), DH1052, was recently shown to be non-neutralizing yet it protected mice and cynomolgus macaques from severe disease. The mechanisms of this non-neutralizing antibody-mediated protection are unknown. Here we show that Fc effector functions mediate non-neutralizing antibody (non-nAb) protection against SARS-CoV-2 MA10 viral challenge in mice. Though non-nAb infusion did not suppress infectious viral titers in the lung as potently as NTD neutralizing antibody (nAb) infusion, disease markers including gross lung discoloration were similar in nAb and non-nAb groups. Fc functional knockout substitutions abolished non-nAb protection and increased viral titers in the nAb group. Finally, Fc enhancement increased non-nAb protection relative to WT, supporting a positive association between Fc functionality and degree of protection in SARS-CoV-2 infection. This study demonstrates that non-nAbs can utilize Fc-mediated mechanisms to lower viral load and prevent lung damage due to coronavirus infection.
RESUMO
Vaccine development targeting rapidly evolving pathogens such as HIV-1 requires induction of broadly neutralizing antibodies (bnAbs) with conserved paratopes and mutations, and, in some cases, the same Ig-heavy chains. The current trial-and-error search for immunogen modifications that improve selection for specific bnAb mutations is imprecise. To precisely engineer bnAb boosting immunogens, we used molecular dynamics simulations to examine encounter states that form when antibodies collide with the HIV-1 Envelope (Env). By mapping how bnAbs use encounter states to find their bound states, we identified Env mutations that were predicted to select for specific antibody mutations in two HIV-1 bnAb B cell lineages. The Env mutations encoded antibody affinity gains and selected for desired antibody mutations in vivo. These results demonstrate proof-of-concept that Env immunogens can be designed to directly select for specific antibody mutations at residue-level precision by vaccination, thus demonstrating the feasibility of sequential bnAb-inducing HIV-1 vaccine design.
RESUMO
HIV-1 gp41 envelope antibodies, which are frequently induced in HIV-1-infected individuals, are predominantly nonneutralizing. The rare and difficult-to-induce neutralizing antibodies (2F5 and 4E10) that target gp41 membrane-proximal epitopes (MPER) are polyspecific and require lipid binding for HIV-1 neutralization. These results raise the questions of how prevalent polyreactivity is among gp41 antibodies and how the binding properties of gp41-nonneutralizing antibodies differ from those of antibodies that are broadly neutralizing. In this study, we have characterized a panel of human gp41 antibodies with binding specificities within the immunodominant cluster I (gp41 amino acids [aa] 579 to 613) or cluster II (gp41 aa 644 to 667) for reactivity to autoantigens, to the gp140 protein, and with MPER peptide-lipid conjugates. We report that while none of the gp41 cluster I antibodies studied were polyspecific, all three gp41 cluster II antibodies bound either to lipids or autoantigens, thus showing the propensity of cluster II antibodies to manifest polyreactivity. All cluster II gp41 monoclonal antibodies (MAbs), including those that were lipid reactive, failed to bind to gp41 MPER peptide-lipid complexes. Cluster II antibodies bound strongly with nanomolar binding affinity (dissociation constant [K(d)]) to oligomeric gp140 proteins, and thus, they recognize conformational epitopes on gp41 that are distinct from those of neutralizing gp41 antibodies. These results demonstrate that lipid-reactive gp41 cluster II antibodies are nonneutralizing due to their inability to bind to the relevant neutralizing epitopes on gp41.
Assuntos
Anticorpos Monoclonais/imunologia , Autoantígenos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Fosfolipídeos/imunologia , Humanos , Cinética , Ligação ProteicaRESUMO
Genetic factors, as well as antigenic stimuli, can influence antibody repertoire formation. Moreover, the affinity of antigen for unmutated naïve B cell receptors determines the threshold for activation of germinal center antibody responses. The gp41 2F5 broadly neutralizing antibody (bNAb) uses the V(H)2-5 gene, which has 10 distinct alleles that use either a heavy-chain complementarity-determining region 2 (HCDR2) aspartic acid (D(H54)) or an HCDR2 asparagine (N(H54)) residue. The 2F5 HCDR2 D(H54) residue has been shown to form a salt bridge with gp41 (665)K; the V(H)2-5 germ line allele variant containing N(H54) cannot do so and thus should bind less avidly to gp41. Thus, the induction of 2F5 bNAb is dependent on both genetic and structural factors that could affect antigen affinity of unmutated naïve B cell receptors. Here, we studied allelic variants of the V(H)2-5 inferred germ line forms of the HIV-1 gp41 bNAb 2F5 for their antigen binding affinities to gp41 linear peptide and conformational protein antigens. Both V(H)2-5 2F5 inferred germ line variants bound to gp41 peptides and protein, including the fusion intermediate protein mimic, although more weakly than the mature 2F5 antibody. As predicted, the affinity of the N(H54) variant for fusion-intermediate conformation was an order of magnitude lower than that of the D(H54) V(H)2-5 germ line antibody, demonstrating that allelic variants of 2F5 germ line antibodies differentially bind to gp41. Thus, these data demonstrate a genetically determined trait that may affect host responses to HIV-1 envelope epitopes recognized by broadly neutralizing antibodies and has implications for unmutated ancestor-based immunogen design.