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1.
J Intern Med ; 2020 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-33034095

RESUMO

BACKGROUND: Convalescent plasma therapy for COVID-19 relies on transfer of anti-viral antibody from donors to recipients via plasma transfusion. The relationship between clinical characteristics and antibody response to COVID-19 is not well defined. We investigated predictors of convalescent antibody production and quantified recipient antibody response in a convalescent plasma therapy clinical trial. METHODS: Multivariable analysis of clinical and serological parameters in 103 confirmed COVID-19 convalescent plasma donors 28 days or more following symptom resolution was performed. Mixed effects regression models with piecewise linear trends were used to characterize serial antibody responses in 10 convalescent plasma recipients with severe COVID-19. RESULTS: Donor antibody titers ranged from 0 to 1:3,892 (anti-receptor binding domain (RBD)) and 0 to 1:3,289 (anti-spike). Higher anti-RBD and anti-spike titers were associated with increased age, hospitalization for COVID-19, fever, and absence of myalgia (all p<0.05). Fatigue was significantly associated with anti-RBD (p=0.03). In pairwise comparison among ABO blood types, AB donors had higher anti-RBD and anti-spike than O donors (p<0.05). No toxicity was associated with plasma transfusion. Non-ECMO recipient anti-RBD antibody titer increased on average 31% per day during the first three days post-transfusion (p=0.01) and anti-spike antibody titer by 40.3% (p=0.02). CONCLUSION: Advanced age, fever, absence of myalgia, fatigue, blood type and hospitalization were associated with higher convalescent antibody titer to COVID-19. Despite variability in donor titer, 80% of convalescent plasma recipients showed significant increase in antibody levels post-transfusion. A more complete understanding of the dose-response effect of plasma transfusion among COVID-19 patients is needed.

2.
Immunity ; 2020 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-33096040

RESUMO

Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we found mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes were polyreactive. Polyreactive mAbs were preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augmented mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal that polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.

3.
J Virol ; 2020 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-32907980

RESUMO

Humoral immune responses to influenza virus vaccines in elderly individuals are poorly adapted towards new antigenically drifted influenza virus strains. Instead, older individuals respond in an original antigenic sin-fashion and produce much more cross-reactive but less potent antibodies. Here, we investigated four influenza B hemagglutinin head specific, hemagglutination inhibition inactive monoclonal antibodies (mAbs) from elderly individuals. We found that they were broadly reactive within the B/Victoria/2/1987-like lineage and two were highly cross-reactive with B/Yamagata/16/1988-like lineage viruses. The mAbs were found to be neutralizing, utilize Fc-effector functions and protective against lethal viral challenge in a mouse model. In order to identify residues on the influenza B virus hemagglutinin interacting with the mAbs, we generated escape mutant viruses. Interestingly, escape from these mAbs led to numerous HA mutations within the head domain, including the defined antigenic sites. We observed that each individual escape mutant virus was able to avoid neutralization from its respective mAb along with other mAbs in the panel, although in many cases binding activity was maintained. Point mutant viruses indicated that K90 is critical for neutralization of two mAbs, while escape from the other two mAbs require a combination of mutations in the hemagglutinin. Three out of four escape mutant viruses had increased lethality in the DBA2/J mouse model. Our work indicates that these cross-reactive antibodies have the potential to cause antigenic drift in the viral population by driving mutations that increase virus fitness. However, binding activity and cross-neutralization was maintained by a majority of antibodies in the panel suggesting that this drift may not lead to escape from antibody mediated protection.IMPORTANCE Understanding the immune response that older individuals mount to influenza virus vaccination and infection is critical in order to design better vaccines for this age group. Here we show that older individuals make broadly-neutralizing antibodies that have no hemagglutination inhibiting activity and are less potent than strain specific antibodies. These antibodies could drive viral escape from neutralization but did not result in escape from binding. Given their different mechanisms of action, they might retain protective activity even against escape variants.

4.
Vaccine ; 38(45): 7129-7137, 2020 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-32943267

RESUMO

The influenza virus neuraminidase (NA) plays an integral role in the influenza virus life cycle through the release of virions from infected cells. NA-specific antibodies can impede virus replication by binding to the NA and blocking its enzymatic activity, providing significant protection from influenza-associated morbidity and mortality. NA included in current seasonal influenza virus vaccines exhibits low immunogenicity, potentially caused by compromised antigenic integrity during vaccine production. To determine how certain types of "stress" could influence the antigenicity of NA we performed a series of in vitro experiments where we treated NA with formalin, EDTA or heat and measured the impact of these treatments on NA enzymatic activity and structural integrity. We found that increasing concentrations of formalin or EDTA and increasing temperature abolished the enzymatic activity of both H1N1, H3N2, and influenza B purified viruses and recombinant NA proteins. However, formalin and EDTA treatment did not drastically affect conformational epitopes found on the NA, whereas heat treatment abolished conformational epitopes. We next performed a vaccination experiment, where mice were vaccinated with recombinant N2 NA treated with 0.3% formalin or 0.125 M EDTA (which both inactivated NA activity) were protected from virus challenge while animals vaccinated with heat treated NA were not. We next tested the protective effect of monomeric (no enzymatic activity) versus tetrameric (highly active) N1 NA. Again, only the tetrameric form protected mice from challenge while the monomeric form did not. Together, our data demonstrate that enzymatically active NA is not required to induce protective antibody responses as a vaccine, however a correctly folded NA is essential.

5.
Immunol Rev ; 296(1): 191-204, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32666572

RESUMO

Natural influenza virus infections and seasonal vaccinations often do not confer broadly neutralizing immunity across diverse influenza strains. In addition, the virus is capable of rapid antigenic drift in order to evade pre-existing immunity. The surface glycoproteins, hemagglutinin, and neuraminidase can easily mutate their immunodominant epitopes without impacting fitness. Skewing human antibody repertoires to target more conserved epitopes is thus an expanding area of research: Many groups are attempting to produce universal influenza vaccines that can protect across a wide variety of strains. Achieving this goal will require a detailed understanding of how infection history impacts humoral responses. It will also require the ability to manipulate or enhance B cell selection in order to expand clones that can recognize subdominant but protective epitopes. In this review, we will discuss what immune imprinting means to immunologists and describe efforts to overcome or silence imprinting in order to improve vaccination efficiency.

6.
PLoS Pathog ; 16(4): e1008409, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32287326

RESUMO

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.


Assuntos
Doenças Transmissíveis Emergentes/veterinária , Doenças do Cão/virologia , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Zoonoses/virologia , Animais , Doenças Transmissíveis Emergentes/transmissão , Doenças Transmissíveis Emergentes/virologia , Doenças do Cão/transmissão , Cães , Furões , Cobaias , Humanos , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N8/classificação , Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Humana/transmissão , Influenza Humana/virologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Estados Unidos , Zoonoses/transmissão
7.
Cell ; 180(1): 18-20, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31951517

RESUMO

Victora and colleagues challenge current perceptions that memory B cells readily participate in secondary germinal center reactions, allowing further modification of specificity upon reactivation. Rather, naïve B cells are the predominant B cell type that populate secondary germinal centers. This work has important basic immunological and translational implications.


Assuntos
Centro Germinativo , Memória Imunológica , Linfócitos B
8.
Proc Natl Acad Sci U S A ; 117(6): 2767-2769, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31988118

RESUMO

While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying combinations of escape mutations, we developed an exponential protein barcoding system which can label 128 clones using seven antibodies. This study provides a proof of principle for a strategy to create protein-level cell barcodes that can be used in vivo in mice to track clonal populations.


Assuntos
Anticorpos Monoclonais/análise , Rastreamento de Células/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Animais , Rastreamento de Células/instrumentação , Feminino , Citometria de Fluxo/métodos , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Melanoma/química , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Orthomyxoviridae/química , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo
9.
Cell Immunol ; 348: 103998, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31733824

RESUMO

Immunosenescence is defined as the progressive deterioration of the immune system with aging. Immunosenescence stifles the generation of protective B and T cell-mediated adaptive immunity in response to various pathogens, resulting in increased disease susceptibility and severity in the elderly population. In particular, immunosenescence has major impacts on the phenotype, function, and receptor repertoire of B and T cells in the elderly, hindering protective responses induced by seasonal influenza virus vaccination. In order to overcome the detrimental impacts of immunosenescence on protective immunity to influenza viruses, we review our current understanding of the effects of aging on adaptive immune responses to influenza and discuss current and future avenues of vaccine research for eliciting more potent anti-influenza immunity in the elderly.

10.
Lancet Infect Dis ; 20(1): 80-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31630990

RESUMO

BACKGROUND: Influenza viruses cause substantial annual morbidity and mortality globally. Current vaccines protect against influenza only when well matched to the circulating strains. However, antigenic drift can cause considerable mismatches between vaccine and circulating strains, substantially reducing vaccine effectiveness. Moreover, current seasonal vaccines are ineffective against pandemic influenza, and production of a vaccine matched to a newly emerging virus strain takes months. Therefore, there is an unmet medical need for a broadly protective influenza virus vaccine. We aimed to test the ability of chimeric H1 haemagglutinin-based universal influenza virus vaccine candidates to induce broadly cross-reactive antibodies targeting the stalk domain of group 1 haemagglutinin-expressing influenza viruses. METHODS: We did a randomised, observer-blinded, phase 1 study in healthy adults in two centres in the USA. Participants were randomly assigned to one of three prime-boost, chimeric haemagglutinin-based vaccine regimens or one of two placebo groups. The vaccine regimens included a chimeric H8/1, intranasal, live-attenuated vaccine on day 1 followed by a non-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine on day 85; the same regimen but with the inactivated vaccine being adjuvanted with AS03; and an AS03-adjuvanted, chimeric H8/1, intramuscular, inactivated vaccine followed by an AS03-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine. In this planned interim analysis, the primary endpoints of reactogenicity and safety were assessed by blinded study group. We also assessed anti-H1 haemagglutinin stalk, anti-H2, anti-H9, and anti-H18 IgG antibody titres and plasmablast and memory B-cell responses in peripheral blood. This trial is registered with ClinicalTrials.gov, number NCT03300050. FINDINGS: Between Oct 10, 2017, and Nov 27, 2017, 65 participants were enrolled and randomly assigned. The adjuvanted inactivated vaccine, but not the live-attenuated vaccine, induced a substantial serum IgG antibody response after the prime immunisation, with a seven times increase in anti-H1 stalk antibody titres on day 29. After boost immunisation, all vaccine regimens induced detectable anti-H1 stalk antibody (2·2-5·6 times induction over baseline), cross-reactive serum IgG antibody, and peripheral blood plasmablast responses. An unsolicited adverse event was reported for 29 (48%) of 61 participants. Solicited local adverse events were reported in 12 (48%) of 25 participants following prime vaccination with intramuscular study product or placebo, in 12 (33%) of 36 after prime immunisation with intranasal study product or placebo, and in 18 (32%) of 56 following booster doses of study product or placebo. Solicited systemic adverse events were reported in 14 (56%) of 25 after prime immunisation with intramuscular study product or placebo, in 22 (61%) of 36 after immunisation with intranasal study product or placebo, and in 21 (38%) of 56 after booster doses of study product or placebo. Disaggregated safety data were not available at the time of this interim analysis. INTERPRETATION: The tested chimeric haemagglutinin-based, universal influenza virus vaccine regimens elicited cross-reactive serum IgG antibodies that targeted the conserved haemagglutinin stalk domain. This is the first proof-of-principle study to show that high anti-stalk titres can be induced by a rationally designed vaccine in humans and opens up avenues for further development of universal influenza virus vaccines. On the basis of the blinded study group, the vaccine regimens were tolerable and no safety concerns were observed. FUNDING: Bill & Melinda Gates Foundation.

11.
Cell Rep ; 29(13): 4460-4470.e8, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875553

RESUMO

Antibodies targeting the receptor binding site (RBS) of the influenza virus hemagglutinin (HA) protein are usually not broadly reactive because their footprints are typically large and extend to nearby variable HA residues. Here, we identify several human H3N2 HA RBS-targeting monoclonal antibodies (mAbs) that are sensitive to substitutions in conventional antigenic sites and are therefore not broadly reactive. However, we also identify an H3N2 HA RBS-targeting mAb that is exceptionally broadly reactive despite being sensitive to substitutions in residues outside of the RBS. We show that similar antibodies are present at measurable levels in the sera of some individuals but that they are inefficiently elicited by conventional vaccines. Our data indicate that HA RBS-targeting antibodies can be effective against variable viral strains even when they are somewhat sensitive to substitutions in HA residues adjacent to the RBS.


Assuntos
Anticorpos Antivirais/química , Epitopos/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Vacinação , Substituição de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , Especificidade de Anticorpos , Sítios de Ligação , Epitopos/imunologia , Epitopos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Soros Imunes/química , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas
12.
Cell ; 178(6): 1313-1328.e13, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31491384

RESUMO

Emerging evidence indicates a central role for the microbiome in immunity. However, causal evidence in humans is sparse. Here, we administered broad-spectrum antibiotics to healthy adults prior and subsequent to seasonal influenza vaccination. Despite a 10,000-fold reduction in gut bacterial load and long-lasting diminution in bacterial diversity, antibody responses were not significantly affected. However, in a second trial of subjects with low pre-existing antibody titers, there was significant impairment in H1N1-specific neutralization and binding IgG1 and IgA responses. In addition, in both studies antibiotics treatment resulted in (1) enhanced inflammatory signatures (including AP-1/NR4A expression), observed previously in the elderly, and increased dendritic cell activation; (2) divergent metabolic trajectories, with a 1,000-fold reduction in serum secondary bile acids, which was highly correlated with AP-1/NR4A signaling and inflammasome activation. Multi-omics integration revealed significant associations between bacterial species and metabolic phenotypes, highlighting a key role for the microbiome in modulating human immunity.

13.
J Virol ; 93(21)2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31434733

RESUMO

Vaccination is the best measure of protection against influenza virus infection. Vaccine-induced antibody responses target mainly the hemagglutinin (HA) surface glycoprotein, composed of the head and the stalk domains. Recently two novel vaccine platforms have been developed for seasonal influenza vaccination: a recombinant HA vaccine produced in insect cells (Flublok) and Flucelvax, prepared from virions produced in mammalian cells. In order to compare the fine specificity of the antibodies induced by these two novel vaccine platforms, we characterized 42 Flublok-induced monoclonal antibodies (MAbs) and 38 Flucelvax-induced MAbs for avidity, cross-reactivity, and any selectivity toward the head versus the stalk domain. These studies revealed that Flublok induced a greater proportion of MAbs targeting epitopes near the receptor-binding domain on HA head (hemagglutinin inhibition-positive MAbs) than Flucelvax, while the two vaccines induced similar low frequencies of stalk-reactive MAbs. Finally, mice immunized with Flublok and Flucelvax also induced similar frequencies of stalk-reactive antibody-secreting cells, showing that HA head immunodominance is independent of immune memory bias. Collectively, our results suggest that these vaccine formulations are similarly immunogenic but differ in the preferences of the elicited antibodies toward the receptor-binding domain on the HA head.IMPORTANCE There are ongoing efforts to increase the efficacy of influenza vaccines and to promote production strategies that can rapidly respond to newly emerging viruses. It is important to understand if current alternative seasonal vaccines, such as Flublok and Flucelvax, that use alternate production strategies can induce protective influenza-specific antibodies and to evaluate what type of epitopes are targeted by distinct vaccine formulations.

14.
Elife ; 82019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31452511

RESUMO

A longstanding question is how influenza virus evolves to escape human immunity, which is polyclonal and can target many distinct epitopes. Here, we map how all amino-acid mutations to influenza's major surface protein affect viral neutralization by polyclonal human sera. The serum of some individuals is so focused that it selects single mutations that reduce viral neutralization by over an order of magnitude. However, different viral mutations escape the sera of different individuals. This individual-to-individual variation in viral escape mutations is not present among ferrets that have been infected just once with a defined viral strain. Our results show how different single mutations help influenza virus escape the immunity of different members of the human population, a phenomenon that could shape viral evolution and disease susceptibility.


Assuntos
Anticorpos Antivirais/sangue , Variação Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Evasão da Resposta Imune , Influenza Humana/virologia , Mutação , Orthomyxoviridae/imunologia , Animais , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Influenza Humana/imunologia , Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Soro/imunologia
15.
Nat Microbiol ; 4(12): 2216-2225, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31406333

RESUMO

A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines.

16.
J Immunol ; 202(10): 2907-2923, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30962292

RESUMO

Nur77 (Nr4a1) belongs to a small family of orphan nuclear receptors that are rapidly induced by BCR stimulation, yet little is known about its function in B cells. We have previously characterized a reporter of Nr4a1 transcription, Nur77-eGFP, in which GFP expression faithfully detects Ag encounter by B cells in vitro and in vivo. In this study, we report that Nur77 expression correlates with the degree of self-reactivity, counterselection, and anergy among individual B cell clones from two distinct BCR transgenic mouse models but is dispensable for all of these tolerance mechanisms. However, we identify a role for Nur77 in restraining survival of self-reactive B cells in the periphery under conditions of competition for a limited supply of the survival factor BAFF. We find that Nur77 deficiency results in the progressive accumulation of self-reactive B cells in the mature repertoire with age and is sufficient to break B cell tolerance in VH3H9 H chain transgenic mice. We thus propose that Nur77 is upregulated in self-reactive B cells in response to chronic Ag stimulation and selectively restricts the survival of these cells, gradually pruning self-reactivity from the mature repertoire to impose a novel layer of peripheral B cell tolerance.


Assuntos
Antígenos/farmacologia , Linfócitos B/imunologia , Tolerância Imunológica/efeitos dos fármacos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Antígenos/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Camundongos , Camundongos Knockout , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/imunologia , Receptores de Antígenos de Linfócitos B/genética
17.
Cell Host Microbe ; 25(3): 357-366.e6, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30795982

RESUMO

Influenza is a leading cause of death in the elderly, and the vaccine protects only a fraction of this population. A key aspect of antibody-mediated anti-influenza virus immunity is adaptation to antigenically distinct epitopes on emerging strains. We examined factors contributing to reduced influenza vaccine efficacy in the elderly and uncovered a dramatic reduction in the accumulation of de novo immunoglobulin gene somatic mutations upon vaccination. This reduction is associated with a significant decrease in the capacity of antibodies to target the viral glycoprotein, hemagglutinin (HA), and critical protective epitopes surrounding the HA receptor-binding domain. Immune escape by antigenic drift, in which viruses generate mutations in key antigenic epitopes, becomes highly exaggerated. Because of this reduced adaptability, most B cells activated in the elderly cohort target highly conserved but less potent epitopes. Given these findings, vaccines driving immunoglobulin gene somatic hypermutation should be a priority to protect elderly individuals.


Assuntos
Linfócitos B/imunologia , Epitopos/imunologia , Imunidade Humoral , Vacinas contra Influenza/imunologia , Orthomyxoviridae/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Epitopos/genética , Voluntários Saudáveis , Humanos , Vacinas contra Influenza/administração & dosagem , Pessoa de Meia-Idade , Mutação , Orthomyxoviridae/genética , Adulto Jovem
18.
J Clin Invest ; 129(1): 93-105, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30457979

RESUMO

Vaccines are among the most effective public health tools for combating certain infectious diseases such as influenza. The role of the humoral immune system in vaccine-induced protection is widely appreciated; however, our understanding of how antibody specificities relate to B cell function remains limited due to the complexity of polyclonal antibody responses. To address this, we developed the Spec-seq framework, which allows for simultaneous monoclonal antibody (mAb) characterization and transcriptional profiling from the same single cell. Here, we present the first application of the Spec-seq framework, which we applied to human plasmablasts after influenza vaccination in order to characterize transcriptional differences governed by B cell receptor (BCR) isotype and vaccine reactivity. Our analysis did not find evidence of long-term transcriptional specialization between plasmablasts of different isotypes. However, we did find enhanced transcriptional similarity between clonally related B cells, as well as distinct transcriptional signatures ascribed by BCR vaccine recognition. These data suggest IgG and IgA vaccine-positive plasmablasts are largely similar, whereas IgA vaccine-negative cells appear to be transcriptionally distinct from conventional, terminally differentiated, antigen-induced peripheral blood plasmablasts.


Assuntos
Vacinas contra Influenza/imunologia , Plasmócitos/imunologia , Transcrição Genética/imunologia , Vacinação , Anticorpos Antivirais/imunologia , Feminino , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Vacinas contra Influenza/administração & dosagem , Masculino , Plasmócitos/citologia , Receptores de Antígenos de Linfócitos B/imunologia , Transcrição Genética/efeitos dos fármacos
19.
Methods Mol Biol ; 1904: 109-145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30539468

RESUMO

In the age of personalized medicine, an efficient method to generate monoclonal antibodies (mAbs) is essential for biomedical and immunotherapeutic research. Numerous aspects of basic B-cell biology can be studied at the monoclonal level, including B-cell development, antibody responses to infection or vaccination, and autoimmune responses. Single-cell B-cell receptor cloning allows for the rapid generation of antigen-specific mAbs in a matter of several weeks. In this chapter, we provide an efficient method to generate mAbs from peripheral blood plasmablasts and memory B cells induced by infection and vaccination. Additionally, we provide a protocol on how to optimize single-cell B-cell sorting for both single-cell B-cell receptor cloning and single-cell RNA-sequencing, for the application of studying B-cell specificity and function (spec-seq). This protocol can be easily adapted for other B-cell populations, B cells in tissues, and B cells from other organisms.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Engenharia de Proteínas , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Formação de Anticorpos/genética , Biomarcadores , Humanos , Imunidade Humoral , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Memória Imunológica , Imunofenotipagem , Plasmócitos/imunologia , Plasmócitos/metabolismo , Reação em Cadeia da Polimerase
20.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30518651

RESUMO

Hemagglutinin (HA) stalk-reactive antibodies are the basis of several current "one-shot" universal influenza vaccine efforts because they protect against a wide spectrum of influenza virus strains. The appreciated mechanism of protection by HA stalk-reactive antibodies is to inhibit HA stalk reconfiguration, blocking viral fusion and entry. This study shows that HA stalk-reactive antibodies also inhibit neuraminidase (NA) enzymatic activity, prohibiting viral egress. NA inhibition (NI) was evident for an attached substrate but not for unattached small-molecule cleavage of sialic acid. This finding suggests that the antibodies inhibit NA enzymatic activity through steric hindrance, thus limiting NA access to sialic acids when adjacent to HA on whole virions. Consistently, F(ab')2 fragments that occupied reduced area without loss of avidity or disrupted HA/NA interactions showed significantly reduced NI activity. Notably, HA stalk-binding antibodies lacking NI activity were unable to neutralize viral infection via microneutralization assays. This work suggests that NI activity is an important component of protection mediated by HA stalk-reactive antibodies.IMPORTANCE This study reports a new mechanism of protection mediated by influenza hemagglutinin stalk-reactive antibodies, i.e., inhibition of neuraminidase activity by steric hindrance, blocking access of neuraminidase to sialic acids when it abuts hemagglutinin on whole virions.


Assuntos
Hemaglutininas/imunologia , Neuraminidase/metabolismo , Orthomyxoviridae/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Proteção Cruzada , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Hemaglutininas/metabolismo , Humanos , Imunização Passiva , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/virologia , Neuraminidase/química , Neuraminidase/imunologia , Testes de Neutralização , Infecções por Orthomyxoviridae/virologia , Proteínas Virais/química
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