RESUMO
Circulating influenza viruses evade neutralization in their human hosts by acquiring escape mutations at epitopes of prevalent antibodies. A goal for next-generation influenza vaccines is to reduce escape likelihood by selectively eliciting antibodies recognizing conserved surfaces on the viral hemagglutinin (HA). The receptor-binding site (RBS) on the HA "head" and a region near the fusion peptide on the HA "stem" are two such sites. We describe here a human antibody clonal lineage, designated CL6649, members of which bind a third conserved site ("lateral patch") on the side of the H1-subtype, HA head. A crystal structure of HA with bound Fab6649 shows the conserved antibody footprint. The site was invariant in isolates from 1977 (seasonal) to 2012 (pdm2009); antibodies in CL6649 recognize HAs from the entire period. In 2013, human H1 viruses acquired mutations in this epitope that were retained in subsequent seasons, prompting modification of the H1 vaccine component in 2017. The mutations inhibit Fab6649 binding. We infer from the rapid spread of these mutations in circulating H1 influenza viruses that the previously subdominant, conserved lateral patch had become immunodominant for individuals with B-cell memory imprinted by earlier H1 exposure. We suggest that introduction of the pdm2009 H1 virus, to which most of the broadly prevalent, neutralizing antibodies did not bind, conferred a selective advantage in the immune systems of infected hosts to recall of memory B cells that recognized the lateral patch, the principal exposed epitope that did not change when pdm2009 displaced previous seasonal H1 viruses.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Memória Imunológica , Vacinas contra Influenza/imunologia , Epitopos/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genéticaRESUMO
Avian influenza viruses, such as A(H5N1) and A(H7N9), are primary public health concerns due to their pandemic potential. Influenza vaccines represent the most effective response to this threat especially with timely provision. The current pandemic response timelines require a substantial period for strain-specific reference antigen and sera preparation for use with single-radial immunodiffusion (SRID), the accepted vaccine potency assay. To address this time lag, the isotope dilution mass spectrometry (IDMS) method was developed to quantify the absolute hemagglutinin (HA, the main influenza antigen) amount in the vaccine without the need for purified, inactivated, and calibrated virus reference antigens. However, an additional challenge in determining potency is to differentiate between vaccine antigens in their most potent form from other less potent, stressed antigen forms. The limited trypsin digestion (LTD) method has been developed and does not require strain-specific full-length reference antigens or antibodies; instead, stressed HA is selectively degraded, leaving the more potent form to be measured. LTD, followed by precipitation and IDMS, allows for efficient differentiation between potent and significantly less potent HA for vaccine release and potency testing across the vaccine's shelf life. In this study, we tested the LTD-IDMS assay on A(H5N1) vaccine material that had been stressed by low pH, heat, and multiple freeze-thaw cycles. The results showed that the LTD-IDMS method effectively quantified the potent HA in A(H5N1) vaccine material with results comparable to SRID. As such, it shows great promise to complement and potentially replace SRID in a pandemic when strain-specific reagents may not be readily available.
Assuntos
Hemaglutininas/metabolismo , Virus da Influenza A Subtipo H5N1/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/imunologia , Espectrometria de Massas/métodos , HumanosRESUMO
Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.
Assuntos
Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Glicoproteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Anticorpos Neutralizantes/imunologia , Sítios de Ligação/genética , Western Blotting , Cromatografia de Afinidade , Sequência Conservada/genética , Citomegalovirus/metabolismo , Dissulfetos/metabolismo , Citometria de Fluxo , Humanos , Processamento de Imagem Assistida por Computador , Espectrometria de Massas , Glicoproteínas de Membrana/química , Microscopia Eletrônica , Complexos Multiproteicos/química , Mutagênese , Mutagênese Sítio-Dirigida , Mutação/genética , Ligação Proteica , Proteínas do Envelope Viral/químicaRESUMO
Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV.
Assuntos
Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Citomegalovirus/imunologia , Epitopos de Linfócito B/imunologia , Proteínas Virais de Fusão/imunologia , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Linhagem Celular , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Humanos , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em Tandem , Transfecção , Internalização do VírusRESUMO
Affinity maturation refines a naive B-cell response by selecting mutations in antibody variable domains that enhance antigen binding. We describe a B-cell lineage expressing broadly neutralizing influenza virus antibodies derived from a subject immunized with the 2007 trivalent vaccine. The lineage comprises three mature antibodies, the unmutated common ancestor, and a common intermediate. Their heavy-chain complementarity determining region inserts into the conserved receptor-binding pocket of influenza HA. We show by analysis of structures, binding kinetics and long time-scale molecular dynamics simulations that antibody evolution in this lineage has rigidified the initially flexible heavy-chain complementarity determining region by two nearly independent pathways and that this preconfiguration accounts for most of the affinity gain. The results advance our understanding of strategies for developing more broadly effective influenza vaccines.
Assuntos
Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Linfócitos B/imunologia , Sítios de Ligação de Anticorpos/genética , Vacinas contra Influenza/imunologia , Modelos Moleculares , Orthomyxoviridae/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Cristalografia por Raios X , Evolução Molecular , Fragmentos Fab das Imunoglobulinas/química , Região Variável de Imunoglobulina/genética , Simulação de Dinâmica Molecular , Dados de Sequência MolecularRESUMO
Non-enveloped viruses of different types have evolved distinct mechanisms for penetrating a cellular membrane during infection. Rotavirus penetration appears to occur by a process resembling enveloped-virus fusion: membrane distortion linked to conformational changes in a viral protein. Evidence for such a mechanism comes from crystallographic analyses of fragments of VP4, the rotavirus-penetration protein, and infectivity analyses of structure-based VP4 mutants. We describe here the structure of an infectious rotavirus particle determined by electron cryomicroscopy (cryoEM) and single-particle analysis at about 4.3 Å resolution. The cryoEM image reconstruction permits a nearly complete trace of the VP4 polypeptide chain, including the positions of most side chains. It shows how the two subfragments of VP4 (VP8(*) and VP5(*)) retain their association after proteolytic cleavage, reveals multiple structural roles for the ß-barrel domain of VP5(*), and specifies interactions of VP4 with other capsid proteins. The virion model allows us to integrate structural and functional information into a coherent mechanism for rotavirus entry.
Assuntos
Proteínas do Capsídeo/genética , Modelos Moleculares , Rotavirus/ultraestrutura , Vírion/ultraestrutura , Internalização do Vírus , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Cristalografia , Estrutura Secundária de Proteína , Rotavirus/metabolismoRESUMO
Respiratory syncytial virus (RSV), the main cause of infant bronchiolitis, remains a major unmet vaccine need despite more than 40 years of vaccine research. Vaccine candidates based on a chief RSV neutralization antigen, the fusion (F) glycoprotein, have foundered due to problems with stability, purity, reproducibility, and potency. Crystal structures of related parainfluenza F glycoproteins have revealed a large conformational change between the prefusion and postfusion states, suggesting that postfusion F antigens might not efficiently elicit neutralizing antibodies. We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals. The 3.2-Å X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigen's efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.
Assuntos
Anticorpos Antivirais/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Sítios de Ligação de Anticorpos , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Imunização , Lactente , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Palivizumab , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/metabolismo , Homologia de Sequência de Aminoácidos , Sigmodontinae , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/ultraestruturaRESUMO
Background: Self-amplifying mRNA vaccines have the potential to increase the magnitude and duration of protection against COVID-19 by boosting neutralizing antibody titers and cellular responses. Methods: In this study, we used the immunogenicity data from a phase 3 randomized trial comparing the immunogenicity of ARCT-154, a self-amplifying mRNA COVID-19 vaccine, with BNT162b2 mRNA COVID-19 vaccine to estimate the relative vaccine efficacy (rVE) of the two vaccines over time in younger (<60 years) and older (≥60 years) adults. Results: By day 181 post-vaccination, the rVE against symptomatic and severe Wuhan-Hu-1 disease was 9.2-11.0% and 1.2-1.5%, respectively, across age groups whereas the rVE against symptomatic and severe Omicron BA.4/5 disease was 26.8-48.0% and 5.2-9.3%, respectively, across age groups. Sensitivity analysis showed that varying the threshold titer for 50% protection against severe disease up to 10% of convalescent sera revealed incremental benefits of ARCT-154 over BNT162b2, with an rVE of up to 28.0% against Omicron BA.4/5 in adults aged ≥60 year. Conclusions: Overall, the results of this study indicate that ARCT-154 elicits broader and more durable immunogenicity against SARS-CoV-2, translating to enhanced disease protection, particularly for older adults against Omicron BA.4/5.
RESUMO
Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8â Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel ß-sheet flanked by four α-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe-4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005), J. Biol. Chem. 280, 26645-26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe-4S] cluster, prior to cluster assembly.
Assuntos
Proteínas Ferro-Enxofre/química , Modelos Químicos , Complexos Multienzimáticos/química , Pyrococcus furiosus/enzimologia , Sequência de Aminoácidos , Vias Biossintéticas/genética , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Duplicação Gênica/genética , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Multimerização Proteica/genética , Estrutura Terciária de Proteína/genética , Pyrococcus horikoshii/enzimologia , Pyrococcus horikoshii/genética , Especificidade por SubstratoRESUMO
Binding of herpes simplex virus (HSV) glycoprotein D (gD) to a cell surface receptor is required to trigger membrane fusion during entry into host cells. Nectin-1 is a cell adhesion molecule and the main HSV receptor in neurons and epithelial cells. We report the structure of gD bound to nectin-1 determined by x-ray crystallography to 4.0 Å resolution. The structure reveals that the nectin-1 binding site on gD differs from the binding site of the HVEM receptor. A surface on the first Ig-domain of nectin-1, which mediates homophilic interactions of Ig-like cell adhesion molecules, buries an area composed by residues from both the gD N- and C-terminal extensions. Phenylalanine 129, at the tip of the loop connecting ß-strands F and G of nectin-1, protrudes into a groove on gD, which is otherwise occupied by C-terminal residues in the unliganded gD and by N-terminal residues in the gD/HVEM complex. Notably, mutation of Phe129 to alanine prevents nectin-1 binding to gD and HSV entry. Together these data are consistent with previous studies showing that gD disrupts the normal nectin-1 homophilic interactions. Furthermore, the structure of the complex supports a model in which gD-receptor binding triggers HSV entry through receptor-mediated displacement of the gD C-terminal region.
Assuntos
Moléculas de Adesão Celular/química , Herpesvirus Humano 1/química , Receptores Virais/química , Proteínas do Envelope Viral/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Herpesvirus Humano 1/fisiologia , Humanos , Nectinas , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Virais/genética , Receptores Virais/metabolismo , Relação Estrutura-Atividade , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Internalização do VírusRESUMO
The protein PCSK9 (proprotein convertase subtilisin/kexin type 9) is a key regulator of low-density lipoprotein receptor (LDLR) levels and cardiovascular health. We have determined the crystal structure of LDLR bound to PCSK9 at neutral pH. The structure shows LDLR in a new extended conformation. The PCSK9 C-terminal domain is solvent exposed, enabling cofactor binding, whereas the catalytic domain and prodomain interact with LDLR epidermal growth factor(A) and ß-propeller domains, respectively. Thus, PCSK9 seems to hold LDLR in an extended conformation and to interfere with conformational rearrangements required for LDLR recycling.
Assuntos
Pró-Proteína Convertases/química , Receptores de LDL/química , Receptores de LDL/metabolismo , Serina Endopeptidases/química , Regulação para Baixo , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteólise , Serina Endopeptidases/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
[This corrects the article DOI: 10.1016/j.omtm.2022.09.013.].
RESUMO
Currently licensed influenza vaccines focus immune responses on viral hemagglutinin (HA), while the other major surface glycoprotein neuraminidase (NA) is not tightly controlled in inactivated vaccine formulations despite evidence that anti-NA antibodies reduce clinical disease. We utilized a bicistronic self-amplifying mRNA (sa-mRNA) platform encoding both HA and NA from four seasonal influenza strains, creating a quadrivalent influenza vaccine. sa-mRNA vaccines encoding an NA component induced the production of NA-inhibiting antibodies and CD4+ T-cell responses in both monovalent and quadrivalent formulations. Including NA in the vaccine enabled cross-neutralization against antigenically drifted strains and provided greater protection than HA alone upon A(H3N2) challenge in ferrets. These results demonstrate that next-generation bicistronic sa-mRNA vaccines expressing HA and NA induce potent antibodies against both viral coat proteins, as well as vaccine-specific cell-mediated immunity. When formulated as a quadrivalent seasonal influenza vaccine, the sa-mRNA platform provides an opportunity to increase the breadth of protection through cross-neutralizing anti-NA antibodies.
RESUMO
Rotaviruses, major causes of childhood gastroenteritis, are nonenveloped, icosahedral particles with double-strand RNA genomes. By the use of electron cryomicroscopy and single-particle reconstruction, we have visualized a rotavirus particle comprising the inner capsid coated with the trimeric outer-layer protein, VP7, at a resolution (4 A) comparable with that of X-ray crystallography. We have traced the VP7 polypeptide chain, including parts not seen in its X-ray crystal structure. The 3 well-ordered, 30-residue, N-terminal "arms" of each VP7 trimer grip the underlying trimer of VP6, an inner-capsid protein. Structural differences between free and particle-bound VP7 and between free and VP7-coated inner capsids may regulate mRNA transcription and release. The Ca(2+)-stabilized VP7 intratrimer contact region, which presents important neutralizing epitopes, is unaltered upon capsid binding.
Assuntos
Microscopia Crioeletrônica/métodos , Rotavirus/ultraestrutura , Vírion/ultraestrutura , Antígenos Virais/química , Antígenos Virais/metabolismo , Antígenos Virais/ultraestrutura , Cálcio/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Cristalografia por Raios X , Modelos Biológicos , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Rotavirus/metabolismo , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo , Proteínas do Core Viral/ultraestrutura , Vírion/metabolismoRESUMO
Vaccines are the primary intervention against influenza. Currently licensed inactivated vaccines focus immunity on viral hemagglutinin (HA). Self-amplifying mRNA (sa-mRNA) vaccines offer an opportunity to generate immunity to multiple viral proteins, including additional neuraminidase (NA). This evaluation of a bicistronic approach for sa-mRNA vaccine development compared subgenomic promoter and internal ribosome entry site strategies and found consistent and balanced expression of both HA and NA proteins in transfected cells. In mice, sa-mRNA bicistronic A/H5N1 vaccines raised potent anti-HA and anti-NA neutralizing antibody responses and HA- or NA-specific CD4+ and CD8+ T cell responses. The addition of NA also boosted the cross-neutralizing response to heterologous A/H1N1. Similar immunogenicity results were obtained for bicistronic seasonal A/H3N2 and B/Yamagata vaccines. In ferrets, sa-mRNA bicistronic A/H1N1 vaccine fully protected lung from infection by homologous virus and showed significant reduction of viral load in upper respiratory tract, warranting further evaluation of sa-mRNA bicistronic vaccine in humans.
RESUMO
The spike (S) protein of SARS-CoV-2 plays a crucial role in cell entry, and the nucleocapsid (N) protein is highly conserved among human coronavirus homologs. For potentially broad effectiveness against both original virus and emerging variants, we developed Alphavirus-based self-amplifying mRNA (sa-mRNA) SARS-CoV-2 vaccines: an sa-mRNA S encoding a full-length S protein stabilized in a prefusion conformation and an sa-mRNA S-N co-expressing S and N proteins for the original virus. We show that these sa-mRNA SARS-CoV-2 vaccines raised potent neutralizing antibody responses in mice against not only the original virus but also the Alpha, Beta, Gamma, and Delta variants. sa-mRNA S vaccines against the Alpha and Beta variants also raised robust cross-reactive neutralizing antibody responses against their homologous viruses and heterologous variants. sa-mRNA S and sa-mRNA S-N vaccines elicited Th1-dominant, antigen-specific CD4+ T cell responses to S and N proteins and robust and broad CD8+ T cell responses to S protein. Hamsters immunized with either vaccine were fully protected from lung infection and showed significant reduction of viral load in upper respiratory tract. Our findings demonstrate that sa-mRNA SARS-CoV-2 vaccines are potent in animal models with potential to be highly effective against SARS-CoV-2 infection in humans.
RESUMO
The native envelope (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) is trimeric, and thus trimeric Env vaccine immunogens are currently being explored in preclinical immunogenicity studies. Key challenges have included the production and purification of biochemically homogeneous and stable trimers and the evaluation of these immunogens utilizing standardized virus panels for neutralization assays. Here we report the binding and neutralizing antibody (NAb) responses elicited by clade A (92UG037.8) and clade C (CZA97.012) Env gp140 trimer immunogens in guinea pigs. These trimers have been selected and engineered for optimal biochemical stability and have defined antigenic properties. Purified gp140 trimers with Ribi adjuvant elicited potent, cross-clade NAb responses against tier 1 viruses as well as detectable but low-titer NAb responses against select tier 2 viruses from clades A, B, and C. In particular, the clade C trimer elicited NAbs that neutralized 27%, 20%, and 47% of tier 2 viruses from clades A, B, and C, respectively. Heterologous DNA prime, protein boost as well as DNA prime, recombinant adenovirus boost regimens expressing these antigens, however, did not result in an increased magnitude or breadth of NAb responses in this system. These data demonstrate the immunogenicity of stable, homogeneous clade A and clade C gp140 trimers and exemplify the utility of standardized tier 1 and tier 2 virus panels for assessing the NAb responses of candidate HIV-1 Env immunogens.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Anti-HIV/sangue , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Cobaias , Imunização , Dados de Sequência Molecular , Produtos do Gene env do Vírus da Imunodeficiência Humana/químicaRESUMO
Electron cryomicroscopy (cryo-EM) yields images of macromolecular assemblies and their components, from which 3D structures can be determined, by using an image processing method commonly known as "single-particle reconstruction." During the past two decades, this technique has become an important tool for 3D structure determination, but it generally has not been possible to determine atomic models. In principle, individual molecular images contain high-resolution information contaminated by a much higher level of noise. In practice, it has been unclear whether current averaging methods are adequate to extract this information from the background. We present here a reconstruction, obtained by using recently developed image processing methods, of the rotavirus inner capsid particle ("double-layer particle" or DLP) at a resolution suitable for interpretation by an atomic model. The result establishes single-particle reconstruction as a high-resolution technique. We show by direct comparison that the cryo-EM reconstruction of viral protein 6 (VP6) of the rotavirus DLP is similar in clarity to a 3.8-A resolution map obtained from x-ray crystallography. At this resolution, most of the amino acid side chains produce recognizable density. The icosahedral symmetry of the particle was an important factor in achieving this resolution in the cryo-EM analysis, but as the size of recordable datasets increases, single-particle reconstruction also is likely to yield structures at comparable resolution from samples of much lower symmetry. This potential has broad implications for structural cell biology.
Assuntos
Microscopia Crioeletrônica/métodos , Proteínas Virais/química , Cristalografia por Raios X , Conformação ProteicaRESUMO
Vaccination is the most effective approach to reduce the substantial morbidity and mortality caused by influenza infection. Vaccine efficacy is highly sensitive to antigenic changes causing differences between circulating and vaccine viruses. Adjuvants such as MF59 increase antibody-mediated cross-reactive immunity and therefore may provide broader seasonal protection. A recent clinical trial showed that an MF59-adjuvanted vaccine was more efficacious than a nonadjuvanted comparator in subjects < 2 years of age, although not in those ≥ 2 years, during influenza seasons in which the predominant circulating virus was an A/H3N2 strain that was antigenically different from the vaccine virus. This finding suggested that the increased efficacy of the adjuvanted vaccine in younger subjects may be mediated by strain cross-reactive antibodies. A subset of the trial population, representing subjects with distinct age and/or immunological history, was tested for antibody responses to the vaccine A/H3N2 strain as well as A/H3N2 drifted strains antigenically matching the viruses circulating during the trial seasons. The neutralizing tests showed that, compared with nonadjuvanted vaccine, the adjuvanted vaccine improved not only the neutralizing antibody response to the vaccine strain but also the cross-reactive antibody response to the drifted strains in subjects with lower preexisting antibody titers, regardless of their age or vaccine history. The results demonstrated an immunological benefit and suggested a potential efficacy benefit by adjuvanted vaccine in subjects with lower preexisting antibody responses.
Assuntos
Vacinas contra Influenza , Influenza Humana , Anticorpos Antivirais , Formação de Anticorpos , Criança , Pré-Escolar , Humanos , Vírus da Influenza A Subtipo H3N2 , Influenza Humana/prevenção & controle , Polissorbatos , EsqualenoRESUMO
Children with sickle cell disease (SCD) suffer life-threatening transient aplastic crisis (TAC) when infected with parvovirus B19. In utero, infection of healthy fetuses may result in anemia, hydrops, and death. Unfortunately, although promising vaccine candidates exist, no product has yet been licensed. One barrier to vaccine development has been the lack of a cost-effective, standardized parvovirus B19 neutralization assay. To fill this void, we evaluated the unique region of VP1 (VP1u), which contains prominent targets of neutralizing antibodies. We discovered an antigenic cross-reactivity between VP1 and VP2 that, at first, thwarted the development of a surrogate neutralization assay. We overcame the cross-reactivity by designing a mutated VP1u (VP1uAT) fragment. A new VP1uAT ELISA yielded results well correlated with neutralization (Spearman's correlation coefficient = 0.581; p = 0.001), superior to results from a standard clinical diagnostic ELISA or an ELISA with virus-like particles. Virus-specific antibodies from children with TAC, measured by the VP1uAT and neutralization assays, but not other assays, gradually increased from days 0 to 120 post-hospitalization. We propose that this novel and technically simple VP1uAT ELISA might now serve as a surrogate for the neutralization assay to support rapid development of a parvovirus B19 vaccine.