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1.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33688035

RESUMO

Modified vaccinia virus Ankara (MVA) is a replication-restricted smallpox vaccine, and numerous clinical studies of recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases, including COVID-19, are in progress. Here, we characterize rMVAs expressing the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Modifications of full-length S individually or in combination included two proline substitutions, mutations of the furin recognition site, and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) is flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected cells and was recognized by anti-RBD antibody and soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced antibodies, which neutralized a pseudovirus in vitro and, upon passive transfer, protected hACE2 transgenic mice from lethal infection with SARS-CoV-2, as well as S-specific CD3+CD8+IFNγ+ T cells. Antibody boosting occurred following a second rMVA or adjuvanted purified RBD protein. Immunity conferred by a single vaccination of hACE2 mice prevented morbidity and weight loss upon intranasal infection with SARS-CoV-2 3 wk or 7 wk later. One or two rMVA vaccinations also prevented detection of infectious SARS-CoV-2 and subgenomic viral mRNAs in the lungs and greatly reduced induction of cytokine and chemokine mRNAs. A low amount of virus was found in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of low levels of subgenomic mRNAs in turbinates indicated that replication was aborted in immunized animals.


Assuntos
/imunologia , Vetores Genéticos/genética , Vacinas de DNA/imunologia , Vírus Vaccinia/genética , /genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , /genética , Modelos Animais de Doenças , Expressão Gênica , Humanos , Imunização , Imunização Passiva , Imunoglobulina G/imunologia , Camundongos , Camundongos Transgênicos , Glicoproteína da Espícula de Coronavírus/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética
2.
Nat Commun ; 12(1): 1102, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597521

RESUMO

The four-dengue virus (DENV) serotypes infect several hundred million people annually. For the greatest safety and efficacy, tetravalent DENV vaccines are designed to stimulate balanced protective immunity to all four serotypes. However, this has been difficult to achieve. Clinical trials with a leading vaccine demonstrated that unbalanced replication and immunodominance of one vaccine component over others can lead to low efficacy and vaccine enhanced severe disease. The Laboratory of Infectious Diseases at the National Institutes of Health has developed a live attenuated tetravalent DENV vaccine (TV003), which is currently being tested in phase 3 clinical trials. Here we report, our study to determine if TV003 stimulate balanced and serotype-specific (TS) neutralizing antibody (nAb) responses to each serotype. Serum samples from twenty-one dengue-naive individuals participated under study protocol CIR287 (ClinicalTrials.gov NCT02021968) are analyzed 6 months after vaccination. Most subjects (76%) develop TS nAbs to 3 or 4 DENV serotypes, indicating immunity is induced by each vaccine component. Vaccine-induced TS nAbs map to epitopes known to be targets of nAbs in people infected with wild type DENVs. Following challenge with a partially attenuated strain of DENV2, all 21 subjects are protected from the efficacy endpoints. However, some vaccinated individuals develop post challenge nAb boost, while others mount post-challenge antibody responses that are consistent with sterilizing immunity. TV003 vaccine induced DENV2 TS nAbs are associated with sterilizing immunity. Our results indicate that nAbs to TS epitopes on each serotype may be a better correlate than total levels of nAbs currently used for guiding DENV vaccine development.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Dengue/prevenção & controle , Dengue/virologia , Vacinas contra Dengue/administração & dosagem , Vírus da Dengue/classificação , Epitopos/imunologia , Humanos , Sorotipagem , Especificidade da Espécie , Resultado do Tratamento , Vacinação/métodos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia
3.
Int J Nanomedicine ; 16: 715-724, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33542626

RESUMO

Objective: The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is now rapidly spreading globally. Serological tests are an important method to assist in the diagnosis of COVID-19, used for epidemiological investigations. In this study, we aimed to investigate the impact of different types of vacuum collection tubes on the detection of SARS-CoV-2 IgM and IgG antibodies, using the colloidal gold immunochromatographic assay (GICA). Patients and Methods: A total of 112 patients with COVID-19 and 200 healthy control subjects with no infection were enrolled in this study. Their serum and plasma were collected into four different types of vacuum blood collection tubes. SARS-CoV-2 IgM and IgG specific antibodies in the plasma and serum were then detected by GICA and chemiluminescence assay (CA), respectively. In addition, the particle sizes of different colloidal gold solutions in the presence of different anticoagulants and coagulants were evaluated by both laser diffraction (Malvern) and confocal laser microscope, respectively. Results: Our results revealed that anticoagulated plasma with EDTA-K2 improved the positive detection rate of SARS-CoV-2 IgM antibodies. Furthermore, our results shown that the detection results by GICA and CA were highly consistent, especially, the results of EDTA-K2 anticoagulated plasma detected by GICA was more consistent with CA results. We confirmed that EDTA-K2 could improve the detection sensitivity of SARS-CoV-2 IgG antibodies by chelating excessive colloidal gold compared with sodium citrate or lithium heparin, these methodologies did not appear to cause false positives. Colloidal gold particles could be chelated and aggregated by EDTA-K2, but not by sodium citrate, lithium heparin and coagulants. Conclusion: GICA is widely used to detect antibodies for the advantages of convenient, fast, low cost, suitable for screening large sample and require minimal equipment. In this study, we found that EDTA-K2 amplified the positive antibody signal by chelating colloidal gold and improved the detection sensitivity of SARS-CoV-2 IgM and IgG antibodies when using the GICA. Therefore, we suggested that EDTA-K2 anticoagulated plasma was more suitable for the detection of SARS-CoV-2 antibodies.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Quelantes/química , Ácido Edético/química , Coloide de Ouro/química , Imunoensaio/métodos , Imunoglobulina G/isolamento & purificação , Imunoglobulina M/isolamento & purificação , /imunologia , Adulto , Anticorpos Antivirais/sangue , Especificidade de Anticorpos/imunologia , /imunologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Peso Molecular , Tamanho da Partícula , Polímeros/química , Sensibilidade e Especificidade
4.
Methods Mol Biol ; 2183: 537-547, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959266

RESUMO

Direct ELISA allows for the measurement of antibody levels to a particular antigen. Serum or plasma from the vaccinated subject are incubated on high-binding capacity microplates precoated with the antigen of interest and detected utilizing an enzyme-linked secondary antibody. Herein, using influenza hemagglutinin as model antigen, we describe the quantification of antigen-specific IgG titers in mouse serum to measure vaccine-induced humoral responses.


Assuntos
Especificidade de Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Imunoglobulina G/imunologia , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Camundongos , Vacinas/imunologia
5.
Methods Mol Biol ; 2183: 9-18, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959237

RESUMO

The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here allowed for a pathogen-specific hmAb cloning efficiency of >80%.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos/imunologia , Bactérias/imunologia , Imunoensaio/métodos , Sondas Moleculares , Anticorpos Antibacterianos/imunologia , Afinidade de Anticorpos , Formação de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunoglobulina G/imunologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Streptococcus pneumoniae/imunologia
6.
Methods Mol Biol ; 2183: 19-28, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959238

RESUMO

Chlamydia trachomatis is one of the most prevalent sexually transmitted infectious agents in the world and the leading cause of infectious blindness. The role of antibodies in the prevention and clearance of infection is still not fully understood, but the analysis of the immunoglobulin response to novel vaccine candidates is an important part of many of these studies. In this chapter, we describe a novel method to identify and isolate Chlamydia-specific memory B cells by fluorescence-activated cell sorting (FACS) using fluorescently labeled whole bacteria from cryopreserved human PBMC samples. This method allows for live single cells to be sorted for cell culture, in vitro assays, single-cell RNA sequencing, and cloning of paired heavy and light chains for recombinant monoclonal antibody production.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Chlamydia/imunologia , Sondas Moleculares , Anticorpos Antibacterianos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Vacinas Bacterianas/imunologia , Criopreservação , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Memória Imunológica , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo
7.
Viruses ; 12(12)2020 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-33371460

RESUMO

African swine fever (ASF) has become the major threat for the global swine industry. Furthermore, the epidemiological situation of African swine fever virus (ASFV) in some endemic regions of Sub-Saharan Africa is worse than ever, with multiple virus strains and genotypes currently circulating in a given area. Despite the recent advances on ASF vaccine development, there are no commercial vaccines yet, and most of the promising vaccine prototypes available today have been specifically designed to fight the genotype II strains currently circulating in Europe, Asia, and Oceania. Previous results from our laboratory have demonstrated the ability of BA71∆CD2, a recombinant LAV lacking CD2v, to confer protection against homologous (BA71) and heterologous genotype I (E75) and genotype II (Georgia2007/01) ASFV strains, both belonging to same clade (clade C). Here, we extend these results using BA71∆CD2 as a tool trying to understand ASFV cross-protection, using phylogenetically distant ASFV strains. We first observed that five out of six (83.3%) of the pigs immunized once with 106 PFU of BA71∆CD2 survived the tick-bite challenge using Ornithodoros sp. soft ticks naturally infected with RSA/11/2017 strain (genotype XIX, clade D). Second, only two out of six (33.3%) survived the challenge with Ken06.Bus (genotype IX, clade A), which is phylogenetically more distant to BA71∆CD2 than the RSA/11/2017 strain. On the other hand, homologous prime-boosting with BA71∆CD2 only improved the survival rate to 50% after Ken06.Bus challenge, all suffering mild ASF-compatible clinical signs, while 100% of the pigs immunized with BA71∆CD2 and boosted with the parental BA71 virulent strain survived the lethal challenge with Ken06.Bus, without almost no clinical signs of the disease. Our results confirm that cross-protection is a multifactorial phenomenon that not only depends on sequence similarity. We believe that understanding this complex phenomenon will be useful for designing future vaccines for ASF-endemic areas.


Assuntos
Vírus da Febre Suína Africana/imunologia , Febre Suína Africana/imunologia , Febre Suína Africana/virologia , Proteção Cruzada/imunologia , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Febre Suína Africana/prevenção & controle , Vírus da Febre Suína Africana/genética , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Células COS , Linhagem Celular , Chlorocebus aethiops , Genótipo , Imunização , Imunoglobulina G/imunologia , Suínos , Proteínas Virais/imunologia
8.
PLoS Pathog ; 16(12): e1009103, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33315937

RESUMO

The antibody molecule comprises a variable domain conferring antigen specificity and affinity distinct from the heavy chain constant (CH) domains dictating effector functions. We here interrogate this paradigm by evaluating the unique influence of the CH1α domain on epitope specificity and functions using two mucosal gp41-specific Fab-IgAs (FabA) derived from HIV-1 highly-exposed but persistently seronegative individuals (HESN). These HESN develop selectively affinity-matured HIV-1-specific mucosal IgA that target the gp41 viral envelope and might provide protection although by unclear mechanisms. Isotype-switching FabAs into Fab-IgGs (FabGs) results in a >10-fold loss in affinity for HIV-1 clade A, B, and C gp41, together with reduced neutralization of HIV-1 cross-clade. The FabA conformational epitopes map selectively on gp41 in 6-Helix bundle and pre-fusion conformations cross-clade, unlike FabGs. Finally, we designed in silico, a 12 amino-acid peptide recapitulating one FabA conformational epitope that inhibits the FabA binding to gp41 cross-clade and its neutralizing activity. Altogether, our results reveal that the CH1α domain shapes the antibody paratope through an allosteric effect, thereby strengthening the antibody specificity and functional activities. Further, they clarify the mechanisms by which these HESN IgAs might confer protection against HIV-1-sexual acquisition. The IgA-specific epitope we characterized by reverse vaccinology could help designing a mucosal HIV-1 vaccine.


Assuntos
Especificidade de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunoglobulina A/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Soronegatividade para HIV/imunologia , Humanos , Imunoglobulina A/química , Imunoglobulina G/imunologia , Domínios Proteicos/imunologia
9.
Nat Commun ; 11(1): 4956, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009383

RESUMO

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.


Assuntos
5-Metilcitosina/análogos & derivados , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , 5-Metilcitosina/metabolismo , Animais , Especificidade de Anticorpos/imunologia , Sequência de Bases , Corpos Embrioides/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
10.
Cell Rep ; 33(4): 108322, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33091382

RESUMO

Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Sondas Moleculares/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Especificidade de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Biotinilação , Microscopia Crioeletrônica , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(37): 22815-22822, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868420

RESUMO

The sensitive and accurate quantification of protein biomarkers plays important roles in clinical diagnostics and biomedical research. Sandwich ELISA and its variants accomplish the capture and detection of a target protein via two antibodies that tightly bind at least two distinct epitopes of the same antigen and have been the gold standard for sensitive protein quantitation for decades. However, existing antibody-based assays cannot distinguish between signal arising from specific binding to the protein of interest and nonspecific binding to assay surfaces or matrix components, resulting in significant background signal even in the absence of the analyte. As a result, they generally do not achieve single-molecule sensitivity, and they require two high-affinity antibodies as well as stringent washing to maximize sensitivity and reproducibility. Here, we show that surface capture with a high-affinity antibody combined with kinetic fingerprinting using a dynamically binding, low-affinity fluorescent antibody fragment differentiates between specific and nonspecific binding at the single-molecule level, permitting the direct, digital counting of single protein molecules with femtomolar-to-attomolar limits of detection (LODs). We apply this approach to four exemplary antigens spiked into serum, demonstrating LODs 55- to 383-fold lower than commercially available ELISA. As a real-world application, we establish that endogenous interleukin-6 (IL-6) can be quantified in 2-µL serum samples from chimeric antigen receptor T cell (CAR-T cell) therapy patients without washing away excess serum or detection probes, as is required in ELISA-based approaches. This kinetic fingerprinting thus exhibits great potential for the ultrasensitive, rapid, and streamlined detection of many clinically relevant proteins.


Assuntos
Ligação Proteica/fisiologia , Imagem Individual de Molécula/métodos , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Especificidade de Anticorpos/fisiologia , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Cinética , Limite de Detecção , Nanotecnologia , Proteínas , Reprodutibilidade dos Testes
13.
Nat Commun ; 11(1): 3971, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769993

RESUMO

Efficacy evaluation through human trials is crucial for advancing a vaccine candidate to clinics. Next-generation sequencing (NGS) can be used to quantify B cell repertoire response and trace antibody lineages during vaccination. Here, we demonstrate this application with a case study of Hecolin®, the licensed vaccine for hepatitis E virus (HEV). Four subjects are administered the vaccine following a standard three-dose schedule. Vaccine-induced antibodies exhibit a high degree of clonal diversity, recognize five conformational antigenic sites of the genotype 1 HEV p239 antigen, and cross-react with other genotypes. Unbiased repertoire sequencing is performed for seven time points over six months of vaccination, with maturation pathways characterize for a set of vaccine-induced antibodies. In addition to dynamic repertoire profiles, NGS analysis reveals differential patterns of HEV-specific antibody lineages and highlights the necessity of the long vaccine boost. Together, our study presents a quantitative strategy for vaccine evaluation in small-scale human studies.


Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Vírus da Hepatite E/imunologia , Vacinação , Vacinas contra Hepatite Viral/imunologia , Adulto , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Linfócitos B/imunologia , Epitopos/imunologia , Genótipo , Vírus da Hepatite E/genética , Humanos , Fatores de Tempo , Doadores de Tecidos , Adulto Jovem
14.
PLoS One ; 15(7): e0236172, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726321

RESUMO

There are several broadly neutralizing monoclonal antibodies that neutralize influenza viruses with different mechanisms from traditional polyclonal antibodies induced by vaccination. CT149, which is one of the broadly neutralizing antibodies, was also previously reported to neutralize group 2 and some of group 1 influenza viruses (13 out of 13 tested group 2 viruses and 5 out of 11 group 1 viruses). In this study, we developed another antibody with the aim of compensating partial coverage of CT149 against group 1 influenza viruses. CT120 was screened among different antibody candidates and mixed with CT149. Importantly, although the binding sites of CT120 and CT149 are close to each other, the two antibodies do not interfere. The mixture of CT120 and CT149, which we named as CT-P27, showed broad efficacy by neutralizing 37 viruses from 11 different subtypes, of both group 1 and 2 influenza A viruses. Moreover, CT-P27 showed in vivo therapeutic efficacy, long prophylactic potency, and synergistic effect with oseltamivir in influenza virus-challenged mouse models. Our findings provide a novel therapeutic opportunity for more efficient treatment of influenza.


Assuntos
Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/farmacologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Antígenos Virais/imunologia , Hemaglutinação/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Camundongos , Testes de Neutralização , Vacinação
15.
Proc Natl Acad Sci U S A ; 117(30): 17957-17964, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661157

RESUMO

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.


Assuntos
Adjuvantes Imunológicos , Linfócitos B/imunologia , Reações Cruzadas/imunologia , Memória Imunológica , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Linfócitos B/metabolismo , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Imunização Secundária , Masculino , Plasmócitos/imunologia , Plasmócitos/metabolismo
16.
Immunity ; 52(5): 842-855.e6, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32353250

RESUMO

B cell subsets expressing the transcription factor T-bet are associated with humoral immune responses and autoimmunity. Here, we examined the anatomic distribution, clonal relationships, and functional properties of T-bet+ and T-bet- memory B cells (MBCs) in the context of the influenza-specific immune response. In mice, both T-bet- and T-bet+ hemagglutinin (HA)-specific B cells arose in germinal centers, acquired memory B cell markers, and persisted indefinitely. Lineage tracing and IgH repertoire analyses revealed minimal interconversion between T-bet- and T-bet+ MBCs, and parabionts showed differential tissue residency and recirculation properties. T-bet+ MBCs could be subdivided into recirculating T-betlo MBCs and spleen-resident T-bethi MBCs. Human MBCs displayed similar features. Conditional gene deletion studies revealed that T-bet expression in B cells was required for nearly all HA stalk-specific IgG2c antibodies and for durable neutralizing titers to influenza. Thus, T-bet expression distinguishes MBC subsets that have profoundly different homing, residency, and functional properties, and mediate distinct aspects of humoral immune memory.


Assuntos
Especificidade de Anticorpos/imunologia , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Memória Imunológica/imunologia , Especificidade de Órgãos/imunologia , Proteínas com Domínio T/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Anticorpos Anti-HIV/imunologia , Humanos , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(23): 12693-12699, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457160

RESUMO

Natural environments can present diverse challenges, but some genotypes remain fit across many environments. Such "generalists" can be hard to evolve, outcompeted by specialists fitter in any particular environment. Here, inspired by the search for broadly neutralizing antibodies during B cell affinity maturation, we demonstrate that environmental changes on an intermediate timescale can reliably evolve generalists, even when faster or slower environmental changes are unable to do so. We find that changing environments on timescales comparable with evolutionary transients in a population enhance the rate of evolving generalists from specialists, without enhancing the reverse process. The yield of generalists is further increased in more complex dynamic environments, such as a "chirp" of increasing frequency. Our work offers design principles for how nonequilibrium fitness "seascapes" can dynamically funnel populations to genotypes unobtainable in static environments.


Assuntos
Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/genética , Meio Ambiente , Evolução Molecular , Modelos Genéticos , Animais , Anticorpos Neutralizantes/genética , Especificidade de Anticorpos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular , Genótipo , Humanos
19.
PLoS One ; 15(4): e0230782, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294093

RESUMO

Understanding immune responses to native antigens in response to natural infections can lead to improved approaches to vaccination. This study sought to characterize the humoral immune response to anthrax toxin components, capsule and spore antigens in individuals (n = 46) from the Kayseri and Malatya regions of Turkey who had recovered from mild or severe forms of cutaneous anthrax infection, compared to regional healthy controls (n = 20). IgG antibodies to each toxin component, the poly-γ-D-glutamic acid capsule, the Bacillus collagen-like protein of anthracis (BclA) spore antigen, and the spore carbohydrate anthrose, were detected in the cases, with anthrax toxin neutralization and responses to Protective Antigen (PA) and Lethal Factor (LF) being higher following severe forms of the disease. Significant correlative relationships among responses to PA, LF, Edema Factor (EF) and capsule were observed among the cases. Though some regional control sera exhibited binding to a subset of the tested antigens, these samples did not neutralize anthrax toxins and lacked correlative relationships among antigen binding specificities observed in the cases. Comparison of serum binding to overlapping decapeptides covering the entire length of PA, LF and EF proteins in 26 cases compared to 8 regional controls revealed that anthrax toxin-neutralizing antibody responses elicited following natural cutaneous anthrax infection are directed to conformational epitopes. These studies support the concept of vaccination approaches that preserve conformational epitopes.


Assuntos
Antraz/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Epitopos/imunologia , Dermatopatias Bacterianas/imunologia , Adulto , Vacinas contra Antraz/imunologia , Especificidade de Anticorpos/imunologia , Bacillus anthracis/imunologia , Feminino , Humanos , Imunidade Humoral/imunologia , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Testes de Neutralização/métodos , Turquia , Adulto Jovem
20.
PLoS One ; 15(4): e0229196, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294099

RESUMO

Citrus mosaic virus (CiMV) is one of the causal viruses of citrus mosaic disease in satsuma mandarins (Citrus unshiu). Prompt detection of trees infected with citrus mosaic disease is important for preventing the spread of this disease. Although rabbit monoclonal antibodies (mAbs) exhibit high specificity and affinity, their applicability is limited by technical difficulties associated with the hybridoma-based technology used for raising these mAbs. Here, we demonstrate a feasible CiMV detection system using a specific rabbit mAb against CiMV coat protein. A conserved peptide fragment of the small subunit of CiMV coat protein was designed and used to immunize rabbits. Antigen-specific antibody-producing cells were identified by the immunospot array assay on a chip method. After cloning of variable regions in heavy or light chain by RT-PCR from these cells, a gene set of 33 mAbs was constructed and these mAbs were produced using Expi293F cells. Screening with the AlphaScreen system revealed eight mAbs exhibiting strong interaction with the antigen peptide. From subsequent sequence analysis, they were grouped into three mAbs denoted as No. 4, 9, and 20. Surface plasmon resonance analysis demonstrated that the affinity of these mAbs for the antigen peptide ranged from 8.7 × 10-10 to 5.5 × 10-11 M. In addition to CiMV, mAb No. 9 and 20 could detect CiMV-related viruses in leaf extracts by ELISA. Further, mAb No. 20 showed a high sensitivity to CiMV and CiMV-related viruses, simply by dot blot analysis. The anti-CiMV rabbit mAbs obtained in this study are envisioned to be extremely useful for practical applications of CiMV detection, such as in a virus detection kit.


Assuntos
Anticorpos Monoclonais/biossíntese , Citrus/virologia , Vírus do Mosaico/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos/imunologia , Proteínas do Capsídeo/imunologia , Cinética , Folhas de Planta/virologia , Coelhos
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