RESUMO
SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Imunidade/imunologia , SARS-CoV-2/imunologia , Células T Auxiliares Foliculares/imunologia , Vacinação , Vacinas Sintéticas/imunologia , Vacinas de mRNA/imunologia , Adulto , Linfócitos B/imunologia , Vacina BNT162/imunologia , COVID-19/sangue , Células Clonais , Estudos de Coortes , Citocinas/metabolismo , Feminino , Centro Germinativo/imunologia , Cadeias beta de HLA-DP/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Células Jurkat , Linfonodos/metabolismo , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/metabolismo , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
The differentiation and specificity of human CD4+ T follicular helper cells (TFH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4+ TFH cell response. The first vaccination induced an increase in the frequency of circulating TFH (cTFH) and LN TFH cells at week 1 postvaccination. This increase was transient for cTFH cells, whereas the LN TFH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of TFH cells in the LN, including pre-TFH cells, memory TFH cells, germinal center (GC) TFH cells and interleukin-10+ TFH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC TFH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific TFH cell clonal lineages, including multiple responses targeting internal influenza virus proteins, and found that each TFH cell state was attainable within a clonal lineage. Thus, human TFH cells form a durable and dynamic multitissue network.
RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and mRNA vaccination induce robust CD4+ T cell responses. Using single-cell transcriptomics, here, we evaluated CD4+ T cells specific for the SARS-CoV-2 spike protein in the blood and draining lymph nodes (dLNs) of individuals 3 months and 6 months after vaccination with the BNT162b2 mRNA vaccine. We analyzed 1,277 spike-specific CD4+ T cells, including 238 defined using Trex, a deep learning-based reverse epitope mapping method to predict antigen specificity. Human dLN spike-specific CD4+ follicular helper T (TFH) cells exhibited heterogeneous phenotypes, including germinal center CD4+ TFH cells and CD4+IL-10+ TFH cells. Analysis of an independent cohort of SARS-CoV-2-infected individuals 3 months and 6 months after infection found spike-specific CD4+ T cell profiles in blood that were distinct from those detected in blood 3 months and 6 months after BNT162b2 vaccination. Our findings provide an atlas of human spike-specific CD4+ T cell transcriptional phenotypes in the dLNs and blood following SARS-CoV-2 vaccination or infection.
RESUMO
In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.
Assuntos
Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , RNA Mensageiro/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Ligação Competitiva , Humanos , Imunoglobulina G/metabolismo , Mutação/genética , Domínios Proteicos , Hipermutação Somática de Imunoglobulina/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of human infections. One limitation to the evaluation of potential therapies and vaccines to inhibit SARS-CoV-2 infection and ameliorate disease is the lack of susceptible small animals in large numbers. Commercially available laboratory strains of mice are not readily infected by SARS-CoV-2 because of species-specific differences in their angiotensin-converting enzyme 2 (ACE2) receptors. Here, we transduced replication-defective adenoviruses encoding human ACE2 via intranasal administration into BALB/c mice and established receptor expression in lung tissues. hACE2-transduced mice were productively infected with SARS-CoV-2, and this resulted in high viral titers in the lung, lung pathology, and weight loss. Passive transfer of a neutralizing monoclonal antibody reduced viral burden in the lung and mitigated inflammation and weight loss. The development of an accessible mouse model of SARS-CoV-2 infection and pathogenesis will expedite the testing and deployment of therapeutics and vaccines.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Betacoronavirus/imunologia , Infecções por Coronavirus/terapia , Modelos Animais de Doenças , Pneumonia Viral/terapia , Enzima de Conversão de Angiotensina 2 , Animais , COVID-19 , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Feminino , Células HEK293 , Humanos , Imunização Passiva/métodos , Pulmão/metabolismo , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , SARS-CoV-2 , Transdução Genética , Células Vero , Carga Viral/imunologiaRESUMO
There is growing appreciation for neuraminidase (NA) as an influenza vaccine target; however, its antigenicity remains poorly characterized. In this study, we isolated three broadly reactive N2 antibodies from the plasmablasts of a single vaccinee, including one that cross-reacts with NAs from seasonal H3N2 strains spanning five decades. Although these three antibodies have diverse germline usages, they recognize similar epitopes that are distant from the NA active site and instead involve the highly conserved underside of NA head domain. We also showed that all three antibodies confer prophylactic and therapeutic protection in vivo, due to both Fc effector functions and NA inhibition through steric hindrance. Additionally, the contribution of Fc effector functions to protection in vivo inversely correlates with viral growth inhibition activity in vitro. Overall, our findings advance the understanding of NA antibody response and provide important insights into the development of a broadly protective influenza vaccine.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Influenza Humana , Infecções por Orthomyxoviridae , Humanos , Influenza Humana/prevenção & controle , Neuraminidase , Infecções por Orthomyxoviridae/prevenção & controle , Vírus da Influenza A Subtipo H3N2 , Epitopos , Anticorpos Antivirais , Anticorpos Monoclonais , Vacinação , Glicoproteínas de Hemaglutininação de Vírus da InfluenzaRESUMO
Neuraminidase (NA) is one of the two influenza virus surface glycoproteins, and antibodies that target it are an independent correlate of protection. However, our current understanding of NA antigenicity is incomplete. Here, we describe human monoclonal antibodies (mAbs) from a patient with a pandemic H1N1 virus infection in 2009. Two mAbs exhibited broad reactivity and inhibited NA enzyme activity of seasonal H1N1 viruses circulating before and after 2009, as well as viruses with avian or swine N1s. The mAbs provided robust protection from lethal challenge with human H1N1 and avian H5N1 viruses in mice, and both target an epitope on the lateral face of NA. In summary, we identified two broadly protective NA antibodies that share a novel epitope, inhibited NA activity, and provide protection against virus challenge in mice. Our work reaffirms that NA should be included as a target in future broadly protective or universal influenza virus vaccines.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Neuraminidase , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , Neuraminidase/química , Neuraminidase/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Microscopia Crioeletrônica , Epitopos , Camundongos Endogâmicos BALB C , Animais , Camundongos , Influenza Humana/tratamento farmacológico , Modelos Animais de DoençasRESUMO
The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Linfócitos B/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Centro Germinativo/imunologia , Pulmão/virologia , SARS-CoV-2/fisiologia , Animais , Células Cultivadas , Células Clonais , Cricetinae , Modelos Animais de Doenças , Humanos , Testes de Neutralização , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Carga ViralRESUMO
Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus in vitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.
Assuntos
Anticorpos Antivirais/imunologia , Domínio Catalítico/imunologia , Vírus da Influenza B/imunologia , Neuraminidase/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Cães , Feminino , Células HEK293 , Humanos , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Leucócitos Mononucleares/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Infecções por Orthomyxoviridae/imunologiaRESUMO
The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.
Assuntos
Linfócitos B , Vacinas contra COVID-19 , COVID-19 , Centro Germinativo , Imunização Secundária , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Plasmócitos/citologia , Plasmócitos/imunologia , Células B de Memória/citologia , Células B de Memória/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologiaRESUMO
Germinal centres (GC) are lymphoid structures in which B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells1-5 (BMPCs). SARS-CoV-2 mRNA vaccination induces a persistent GC response that lasts for at least six months in humans6-8. The fate of responding GC B cells as well as the functional consequences of such persistence remain unknown. Here, we detected SARS-CoV-2 spike protein-specific MBCs in 42 individuals who had received two doses of the SARS-CoV-2 mRNA vaccine BNT162b2 six month earlier. Spike-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of sequencing the B cell receptors of responding blood plasmablasts and MBCs, lymph node GC B cells and plasma cells and BMPCs from eight individuals and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1,540 spike-specific B cell clones. On average, early blood spike-specific plasmablasts exhibited the lowest SHM frequencies. By contrast, SHM frequencies of spike-specific GC B cells increased by 3.5-fold within six months after vaccination. Spike-specific MBCs and BMPCs accumulated high levels of SHM, which corresponded with enhanced anti-spike antibody avidity in blood and enhanced affinity as well as neutralization capacity of BMPC-derived monoclonal antibodies. We report how the notable persistence of the GC reaction induced by SARS-CoV-2 mRNA vaccination in humans culminates in affinity-matured long-term antibody responses that potently neutralize the virus.
Assuntos
Linfócitos B , Vacina BNT162 , Centro Germinativo , Vacinação , Anticorpos Monoclonais , Anticorpos Antivirais , Linfócitos B/citologia , Linfócitos B/imunologia , Vacina BNT162/administração & dosagem , Vacina BNT162/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Humanos , RNA Mensageiro/genética , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Long-lived bone marrow plasma cells (BMPCs) are a persistent and essential source of protective antibodies1-7. Individuals who have recovered from COVID-19 have a substantially lower risk of reinfection with SARS-CoV-28-10. Nonetheless, it has been reported that levels of anti-SARS-CoV-2 serum antibodies decrease rapidly in the first few months after infection, raising concerns that long-lived BMPCs may not be generated and humoral immunity against SARS-CoV-2 may be short-lived11-13. Here we show that in convalescent individuals who had experienced mild SARS-CoV-2 infections (n = 77), levels of serum anti-SARS-CoV-2 spike protein (S) antibodies declined rapidly in the first 4 months after infection and then more gradually over the following 7 months, remaining detectable at least 11 months after infection. Anti-S antibody titres correlated with the frequency of S-specific plasma cells in bone marrow aspirates from 18 individuals who had recovered from COVID-19 at 7 to 8 months after infection. S-specific BMPCs were not detected in aspirates from 11 healthy individuals with no history of SARS-CoV-2 infection. We show that S-binding BMPCs are quiescent, which suggests that they are part of a stable compartment. Consistently, circulating resting memory B cells directed against SARS-CoV-2 S were detected in the convalescent individuals. Overall, our results indicate that mild infection with SARS-CoV-2 induces robust antigen-specific, long-lived humoral immune memory in humans.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , COVID-19/imunologia , Plasmócitos/citologia , Plasmócitos/imunologia , Adulto , Idoso , Sobrevivência Celular , Feminino , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto JovemRESUMO
SARS-CoV-2 mRNA-based vaccines are about 95% effective in preventing COVID-191-5. The dynamics of antibody-secreting plasmablasts and germinal centre B cells induced by these vaccines in humans remain unclear. Here we examined antigen-specific B cell responses in peripheral blood (n = 41) and draining lymph nodes in 14 individuals who had received 2 doses of BNT162b2, an mRNA-based vaccine that encodes the full-length SARS-CoV-2 spike (S) gene1. Circulating IgG- and IgA-secreting plasmablasts that target the S protein peaked one week after the second immunization and then declined, becoming undetectable three weeks later. These plasmablast responses preceded maximal levels of serum anti-S binding and neutralizing antibodies to an early circulating SARS-CoV-2 strain as well as emerging variants, especially in individuals who had previously been infected with SARS-CoV-2 (who produced the most robust serological responses). By examining fine needle aspirates of draining axillary lymph nodes, we identified germinal centre B cells that bound S protein in all participants who were sampled after primary immunization. High frequencies of S-binding germinal centre B cells and plasmablasts were sustained in these draining lymph nodes for at least 12 weeks after the booster immunization. S-binding monoclonal antibodies derived from germinal centre B cells predominantly targeted the receptor-binding domain of the S protein, and fewer clones bound to the N-terminal domain or to epitopes shared with the S proteins of the human betacoronaviruses OC43 and HKU1. These latter cross-reactive B cell clones had higher levels of somatic hypermutation as compared to those that recognized only the SARS-CoV-2 S protein, which suggests a memory B cell origin. Our studies demonstrate that SARS-CoV-2 mRNA-based vaccination of humans induces a persistent germinal centre B cell response, which enables the generation of robust humoral immunity.
Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Centro Germinativo/imunologia , Plasmócitos/imunologia , Vacinas Sintéticas/imunologia , Adulto , Idoso , Animais , Anticorpos Antivirais/imunologia , Vacina BNT162 , COVID-19/prevenção & controle , Chlorocebus aethiops , Células Clonais/citologia , Células Clonais/imunologia , Centro Germinativo/citologia , Voluntários Saudáveis , Humanos , Pessoa de Meia-Idade , Plasmócitos/citologia , SARS-CoV-2/imunologia , Fatores de Tempo , Células Vero , Vacinas de mRNARESUMO
Influenza viruses remain a major public health threat. Seasonal influenza vaccination in humans primarily stimulates pre-existing memory B cells, which differentiate into a transient wave of circulating antibody-secreting plasmablasts1-3. This recall response contributes to 'original antigenic sin'-the selective increase of antibody species elicited by previous exposures to influenza virus antigens4. It remains unclear whether such vaccination can also induce germinal centre reactions in the draining lymph nodes, where diversification and maturation of recruited B cells can occur5. Here we used ultrasound-guided fine needle aspiration to serially sample the draining lymph nodes and investigate the dynamics and specificity of germinal centre B cell responses after influenza vaccination in humans. Germinal centre B cells that bind to influenza vaccine could be detected as early as one week after vaccination. In three out of eight participants, we detected vaccine-binding germinal centre B cells up to nine weeks after vaccination. Between 12% and 88% of the responding germinal centre B cell clones overlapped with B cells detected among early circulating plasmablasts. These shared B cell clones had high frequencies of somatic hypermutation and encoded broadly cross-reactive monoclonal antibodies. By contrast, vaccine-induced B cell clones detected only in the germinal centre compartment exhibited significantly lower frequencies of somatic hypermutation and predominantly encoded strain-specific monoclonal antibodies, which suggests a naive B cell origin. Some of these strain-specific monoclonal antibodies recognized epitopes that were not targeted by the early plasmablast response. Thus, influenza virus vaccination in humans can elicit a germinal centre reaction that recruits B cell clones that can target new epitopes, thereby broadening the spectrum of vaccine-induced protective antibodies.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Memória Imunológica/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Adulto , Animais , Células Clonais/imunologia , Mapeamento de Epitopos , Feminino , Centro Germinativo/citologia , Humanos , Masculino , CamundongosRESUMO
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/metabolismoRESUMO
The latent reservoir of HIV-1 is a major barrier for viral eradication. Potent HIV-1 broadly neutralizing antibodies (bNabs) have been used to prevent and treat HIV-1 infections in animal models and clinical trials. Combination of bNabs and latency-reversing agents (LRAs) is considered a promising approach for HIV-1 eradication. PCR-based assays that can rapidly and specifically measure singly spliced HIV-1 vpu/env mRNA are needed to evaluate the induction of the viral envelope production at the transcription level and bNab-mediated reservoir clearance. Here we reported a PCR-based method to accurately quantify the production of intracellular HIV-1 vpu/env mRNA. With the vpu/env assay, we determined the LRA combinations that could effectively induce vpu/env mRNA production in CD4+ T cells from ART-treated individuals. None of the tested LRAs were effective alone. A comparison between the quantitative viral outgrowth assay (Q-VOA) and the vpu/env assay showed that vpu/env mRNA production was closely associated with the reactivation of replication-competent HIV-1, suggesting that vpu/env mRNA was mainly produced by intact viruses. Finally, antibody-mediated in vitro killing in HIV-1-infected humanized mice demonstrated that the vpu/env assay could be used to measure the reduction of infected cells in tissues and was more accurate than the commonly used gag-based PCR assay which measured unspliced viral genomic RNA. In conclusion, the vpu/env assay allows convenient and accurate assessment of HIV-1 latency reversal and bNab-mediated therapeutic strategies.ImportanceHIV-1 persists in individuals on antiretroviral therapy (ART) due to the long-lived cellular reservoirs that contain dormant viruses. Recent discoveries of HIV-1-specific broadly neutralizing antibodies (bNabs) targeting HIV-1 Env protein rekindled the interest in antibody-mediated elimination of latent HIV-1. Latency-reversing agents (LRAs) together with HIV-1 bNabs is a possible strategy to clear residual viral reservoirs, which makes the evaluation of HIV-1 Env expression upon LRA treatment critical. We developed a PCR-based assay to quantify the production of intracellular HIV-1 vpu/env mRNA. Using patient CD4+ T cells, we found that induction of HIV-1 vpu/env mRNA required a combination of different LRAs. Using in vitro, ex vivo and humanized mouse models, we showed that the vpu/env assay could be used to measure antibody efficacy in clearing HIV-1 infection. These results suggest that the vpu/env assay can accurately evaluate HIV-1 reactivation and bNab-based therapeutic interventions.
RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for millions of infections and hundreds of thousands of deaths globally. There are no widely available licensed therapeutics against SARS-CoV-2, highlighting an urgent need for effective interventions. The virus enters host cells through binding of a receptor-binding domain within its trimeric spike glycoprotein to human angiotensin-converting enzyme 2. In this article, we describe the generation and characterization of a panel of murine mAbs directed against the receptor-binding domain. One mAb, 2B04, neutralized wild-type SARS-CoV-2 in vitro with remarkable potency (half-maximal inhibitory concentration of <2 ng/ml). In a murine model of SARS-CoV-2 infection, 2B04 protected challenged animals from weight loss, reduced lung viral load, and blocked systemic dissemination. Thus, 2B04 is a promising candidate for an effective antiviral that can be used to prevent SARS-CoV-2 infection.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , COVID-19 , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Mapeamento de Epitopos , Feminino , Células HEK293 , Humanos , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção , Células VeroRESUMO
BACKGROUND: Cellular immune responses are not well characterized during the initial days of acute symptomatic influenza infection. METHODS: We developed a prospective cohort of human subjects with confirmed influenza illness of varying severity who presented within a week after symptom onset. We characterized lymphocyte and monocyte populations as well as antigen-specific CD8+ T-cell and B-cell responses from peripheral blood mononuclear cells using flow cytometry and enzyme-linked immunospot assays. RESULTS: We recruited 68 influenza-infected individuals on average 3.5 days after the onset of symptoms. Three patients required mechanical ventilation. Influenza-specific CD8+ T-cell responses expanded before the appearance of plasmablast B cells. However, the influenza-specific CD8+ T-cell response was lower in infected subjects than responses seen in uninfected control subjects. Circulating populations of inflammatory monocytes were increased in most subjects compared with healthy controls. Inflammatory monocytes were significantly reduced in the 3 subjects requiring mechanical ventilation. Inflammatory monocytes were also reduced in a separate validation cohort of mechanically ventilated patients. CONCLUSIONS: Antigen-specific CD8+ T cells respond early during acute influenza infection at magnitudes that are lower than responses seen in uninfected individuals. Circulating inflammatory monocytes increase during acute illness and low absolute numbers are associated with very severe disease.
Assuntos
Linfócitos T CD8-Positivos/patologia , Imunidade Celular , Influenza Humana/sangue , Influenza Humana/patologia , Adulto , Idoso , Feminino , Humanos , Influenza Humana/imunologia , Contagem de Leucócitos , Contagem de Linfócitos , Linfócitos/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Estudos Prospectivos , Respiração Artificial , Índice de Gravidade de DoençaRESUMO
Salinity is a major factor limiting crop productivity. Rice (Oryza sativa), a staple crop for the majority of the world, is highly sensitive to salinity stress. To discover novel sources of genetic variation for salt tolerance-related traits in rice, we screened 390 diverse accessions under 14 days of moderate (9 dS·m-1) salinity. In this study, shoot growth responses to moderate levels of salinity were independent of tissue Na+ content. A significant difference in root Na+ content was observed between the major subpopulations of rice, with indica accessions displaying higher root Na+ and japonica accessions exhibiting lower root Na+ content. The genetic basis of the observed variation in phenotypes was elucidated through genome-wide association (GWA). The strongest associations were identified for root Na+:K+ ratio and root Na+ content in a region spanning ~575 Kb on chromosome 4, named Root Na+ Content 4 (RNC4). Two Na+ transporters, HKT1;1 and HKT1;4 were identified as candidates for RNC4. Reduced expression of both HKT1;1 and HKT1;4 through RNA interference indicated that HKT1;1 regulates shoot and root Na+ content, and is likely the causal gene underlying RNC4. Three non-synonymous mutations within HKT1;1 were present at higher frequency in the indica subpopulation. When expressed in Xenopus oocytes the indica-predominant isoform exhibited higher inward (negative) currents and a less negative voltage threshold of inward rectifying current activation compared to the japonica-predominant isoform. The introduction of a 4.5kb fragment containing the HKT1;1 promoter and CDS from an indica variety into a japonica background, resulted in a phenotype similar to the indica subpopulation, with higher root Na+ and Na+:K+. This study provides evidence that HKT1;1 regulates root Na+ content, and underlies the divergence in root Na+ content between the two major subspecies in rice.
Assuntos
Potenciais de Ação , Proteínas de Transporte de Cátions/genética , Oryza/genética , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Polimorfismo Genético , Sódio/metabolismo , Simportadores/genética , Alelos , Animais , Proteínas de Transporte de Cátions/metabolismo , Especiação Genética , Transporte de Íons , Oryza/classificação , Fenótipo , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Potássio/metabolismo , Simportadores/metabolismo , XenopusRESUMO
Chloroplast division is initiated by assembly of a mid-chloroplast FtsZ (Z) ring comprising two cytoskeletal proteins, FtsZ1 and FtsZ2. The division-site regulators ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3), MinD1, and MinE1 restrict division to the mid-plastid, but their roles are poorly understood. Using genetic analyses in Arabidopsis thaliana, we show that ARC3 mediates division-site placement by inhibiting Z-ring assembly, and MinD1 and MinE1 function through ARC3. ftsZ1 null mutants exhibited some mid-plastid FtsZ2 rings and constrictions, whereas neither constrictions nor FtsZ1 rings were observed in mutants lacking FtsZ2, suggesting FtsZ2 is the primary determinant of Z-ring assembly in vivo. arc3 ftsZ1 double mutants exhibited multiple parallel but no mid-plastid FtsZ2 rings, resembling the Z-ring phenotype in arc3 single mutants and showing that ARC3 affects positioning of FtsZ2 rings as well as Z rings. ARC3 overexpression in the wild type and ftsZ1 inhibited Z-ring and FtsZ2-ring assembly, respectively. Consistent with its effects in vivo, ARC3 interacted with FtsZ2 in two-hybrid assays and inhibited FtsZ2 assembly in a heterologous system. Our studies are consistent with a model wherein ARC3 directly inhibits Z-ring assembly in vivo primarily through interaction with FtsZ2 in heteropolymers and suggest that ARC3 activity is spatially regulated by MinD1 and MinE1 to permit Z-ring assembly at the mid-plastid.