Publications by authors named "Nai-Ying Zheng"

37 Publications

SARS-CoV-2 Infection Severity Is Linked to Superior Humoral Immunity against the Spike.

mBio 2021 01 19;12(1). Epub 2021 Jan 19.

Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, Illinois, USA

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity of the antibody response mounted against this novel virus is not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and nonstructural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike. With the ongoing pandemic, it is critical to understand how natural immunity against SARS-CoV-2 and COVID-19 develops. We have identified that subjects with more severe COVID-19 disease mount a more robust and neutralizing antibody response against SARS-CoV-2 spike protein. Subjects who mounted a larger response against the spike also mounted antibody responses against other viral antigens, including the nucleocapsid protein and ORF8. Additionally, this study reveals that subjects with more severe disease mount a larger memory B cell response against the spike. These data suggest that subjects with more severe COVID-19 disease are likely better protected from reinfection with SARS-CoV-2.
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http://dx.doi.org/10.1128/mBio.02940-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845638PMC
January 2021

Preexisting immunity shapes distinct antibody landscapes after influenza virus infection and vaccination in humans.

Sci Transl Med 2020 12;12(573)

Committee on Immunology, University of Chicago, Chicago, IL 60637, USA.

Humans are repeatedly exposed to variants of influenza virus throughout their lifetime. As a result, preexisting influenza-specific memory B cells can dominate the response after infection or vaccination. Memory B cells recalled by adulthood exposure are largely reactive to conserved viral epitopes present in childhood strains, posing unclear consequences on the ability of B cells to adapt to and neutralize newly emerged strains. We sought to investigate the impact of preexisting immunity on generation of protective antibody responses to conserved viral epitopes upon influenza virus infection and vaccination in humans. We accomplished this by characterizing monoclonal antibodies (mAbs) from plasmablasts, which are predominantly derived from preexisting memory B cells. We found that, whereas some influenza infection-induced mAbs bound conserved and neutralizing epitopes on the hemagglutinin (HA) stalk domain or neuraminidase, most of the mAbs elicited by infection targeted non-neutralizing epitopes on nucleoprotein and other unknown antigens. Furthermore, most infection-induced mAbs had equal or stronger affinity to childhood strains, indicating recall of memory B cells from childhood exposures. Vaccination-induced mAbs were similarly induced from past exposures and exhibited substantial breadth of viral binding, although, in contrast to infection-induced mAbs, they targeted neutralizing HA head epitopes. Last, cocktails of infection-induced mAbs displayed reduced protective ability in mice compared to vaccination-induced mAbs. These findings reveal that both preexisting immunity and exposure type shape protective antibody responses to conserved influenza virus epitopes in humans. Natural infection largely recalls cross-reactive memory B cells against non-neutralizing epitopes, whereas vaccination harnesses preexisting immunity to target protective HA epitopes.
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http://dx.doi.org/10.1126/scitranslmed.abd3601DOI Listing
December 2020

Polyreactive Broadly Neutralizing B cells Are Selected to Provide Defense against Pandemic Threat Influenza Viruses.

Immunity 2020 Dec 22;53(6):1230-1244.e5. Epub 2020 Oct 22.

Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, IL 60637, USA; Committee on Immunology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.immuni.2020.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772752PMC
December 2020

Distinct B cell subsets give rise to antigen-specific antibody responses against SARS-CoV-2.

Res Sq 2020 Sep 25. Epub 2020 Sep 25.

Discovery of durable memory B cell (MBC) subsets against neutralizing viral epitopes is critical for determining immune correlates of protection from SARS-CoV-2 infection. Here, we identified functionally distinct SARS-CoV-2-reactive B cell subsets by profiling the repertoire of convalescent COVID-19 patients using a high-throughput B cell sorting and sequencing platform. Utilizing barcoded SARS-CoV-2 antigen baits, we isolated thousands of B cells that segregated into discrete functional subsets specific for the spike, nucleocapsid protein (NP), and open reading frame (ORF) proteins 7a and 8. Spike-specific B cells were enriched in canonical MBC clusters, and monoclonal antibodies (mAbs) from these cells were potently neutralizing. By contrast, B cells specific to ORF8 and NP were enriched in naïve and innate-like clusters, and mAbs against these targets were exclusively non-neutralizing. Finally, we identified that B cell specificity, subset distribution, and affinity maturation were impacted by clinical features such as age, sex, and symptom duration. Together, our data provide a comprehensive tool for evaluating B cell immunity to SARS-CoV-2 infection or vaccination and highlight the complexity of the human B cell response to SARS-CoV-2.
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http://dx.doi.org/10.21203/rs.3.rs-80476/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523131PMC
September 2020

SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike.

bioRxiv 2020 Sep 13. Epub 2020 Sep 13.

Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, IL 60637, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity and kinetics of the antibody response mounted against this novel virus are not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and non-structural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.
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http://dx.doi.org/10.1101/2020.09.12.294066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491512PMC
September 2020

Antibiotics-Driven Gut Microbiome Perturbation Alters Immunity to Vaccines in Humans.

Cell 2019 09;178(6):1313-1328.e13

Institute for Immunity, Transplantation and Infection, School of Medicine, Stanford University, Stanford, CA 94305, USA; Department of Pathology, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USA; Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2019.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750738PMC
September 2019

Monoclonal Antibody Responses after Recombinant Hemagglutinin Vaccine versus Subunit Inactivated Influenza Virus Vaccine: a Comparative Study.

J Virol 2019 11 15;93(21). Epub 2019 Oct 15.

Department of Medicine, Section of Rheumatology, The University of Chicago, Chicago, Illinois, USA

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. 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.
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http://dx.doi.org/10.1128/JVI.01150-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803255PMC
November 2019

The neuraminidase of A(H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A/Hong Kong/4801/2014 vaccine strain.

Nat Microbiol 2019 12 12;4(12):2216-2225. Epub 2019 Aug 12.

Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA.

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.
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http://dx.doi.org/10.1038/s41564-019-0522-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879794PMC
December 2019

Influenza Virus Vaccination Elicits Poorly Adapted B Cell Responses in Elderly Individuals.

Cell Host Microbe 2019 03 19;25(3):357-366.e6. Epub 2019 Feb 19.

Department of Medicine, Section of Rheumatology, The University of Chicago, Chicago, IL 60637, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chom.2019.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452894PMC
March 2019

Spec-seq unveils transcriptional subpopulations of antibody-secreting cells following influenza vaccination.

J Clin Invest 2019 01 19;129(1):93-105. Epub 2018 Nov 19.

The Committee on Immunology.

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.
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http://dx.doi.org/10.1172/JCI121341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307935PMC
January 2019

Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies.

Cell 2018 04;173(2):417-429.e10

Department of Medicine, Section of Rheumatology, the Knapp Center for Lupus and Immunology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.
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http://dx.doi.org/10.1016/j.cell.2018.03.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890936PMC
April 2018

Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection.

Cell Host Microbe 2016 Jun;19(6):800-13

The Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology Research, The University of Chicago, Chicago, IL 60637, USA. Electronic address:

Pathogenic H7N9 avian influenza viruses continue to represent a public health concern, and several candidate vaccines are currently being developed. It is vital to assess if protective antibodies are induced following vaccination and to characterize the diversity of epitopes targeted. Here we characterized the binding and functional properties of twelve H7-reactive human antibodies induced by a candidate A/Anhui/1/2013 (H7N9) vaccine. Both neutralizing and non-neutralizing antibodies protected mice in vivo during passive transfer challenge experiments. Mapping the H7 hemagglutinin antigenic sites by generating escape mutant variants against the neutralizing antibodies identified unique epitopes on the head and stalk domains. Further, the broadly cross-reactive non-neutralizing antibodies generated in this study were protective through Fc-mediated effector cell recruitment. These findings reveal important properties of vaccine-induced antibodies and provide a better understanding of the human monoclonal antibody response to influenza in the context of vaccines.
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http://dx.doi.org/10.1016/j.chom.2016.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901526PMC
June 2016

B Cell Responses during Secondary Dengue Virus Infection Are Dominated by Highly Cross-Reactive, Memory-Derived Plasmablasts.

J Virol 2016 06 27;90(12):5574-85. Epub 2016 May 27.

Department of Pediatrics, Division of Infectious Disease, Emory University School of Medicine, Atlanta, Georgia, USA Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia, USA

Unlabelled: Dengue virus (DENV) infection results in the production of both type-specific and cross-neutralizing antibodies. While immunity to the infecting serotype is long-lived, heterotypic immunity wanes a few months after infection. Epidemiological studies link secondary heterotypic infections with more severe symptoms, and cross-reactive, poorly neutralizing antibodies have been implicated in this increased disease severity. To understand the cellular and functional properties of the acute dengue virus B cell response and its role in protection and immunopathology, we characterized the plasmablast response in four secondary DENV type 2 (DENV2) patients. Dengue plasmablasts had high degrees of somatic hypermutation, with a clear preference for replacement mutations. Clonal expansions were also present in each donor, strongly supporting a memory origin for these acutely induced cells. We generated 53 monoclonal antibodies (MAbs) from sorted patient plasmablasts and found that DENV-reactive MAbs were largely envelope specific and cross neutralizing. Many more MAbs neutralized DENV than reacted to envelope protein, emphasizing the significance of virion-dependent B cell epitopes and the limitations of envelope protein-based antibody screening. A majority of DENV-reactive MAbs, irrespective of neutralization potency, enhanced infection by antibody-dependent enhancement (ADE). Interestingly, even though DENV2 was the infecting serotype in all four patients, several MAbs from two patients neutralized DENV1 more potently than DENV2. Further, half of all type-specific neutralizing MAbs were also DENV1 biased in binding. Taken together, these findings are reminiscent of original antigenic sin (OAS), given that the patients had prior dengue virus exposures. These data describe the ongoing B cell response in secondary patients and may further our understanding of the impact of antibodies in dengue virus pathogenesis.

Importance: In addition to their role in protection, antibody responses have been hypothesized to contribute to the pathology of dengue. Recent studies characterizing memory B cell (MBC)-derived MAbs have provided valuable insight into the targets and functions of B cell responses generated after DENV exposure. However, in the case of secondary infections, such MBC-based approaches fail to distinguish acutely induced cells from the preexisting MBC pool. Our characterization of plasmablasts and plasmablast-derived MAbs provides a focused analysis of B cell responses activated during ongoing infection. Additionally, our studies provide evidence of OAS in the acute-phase dengue virus immune response, providing a basis for future work examining the impact of OAS phenotype antibodies on protective immunity and disease severity in secondary infections.
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http://dx.doi.org/10.1128/JVI.03203-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886779PMC
June 2016

High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination.

PLoS One 2015 7;10(5):e0125618. Epub 2015 May 7.

Committee on Immunology, The University of Chicago, Chicago, Illinois, United States of America; Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology, The University of Chicago, Chicago, Illinois, United States of America.

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125618PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423960PMC
April 2016

Preexisting human antibodies neutralize recently emerged H7N9 influenza strains.

J Clin Invest 2015 Mar 17;125(3):1255-68. Epub 2015 Feb 17.

The emergence and seasonal persistence of pathogenic H7N9 influenza viruses in China have raised concerns about the pandemic potential of this strain, which, if realized, would have a substantial effect on global health and economies. H7N9 viruses are able to bind to human sialic acid receptors and are also able to develop resistance to neuraminidase inhibitors without a loss in fitness. It is not clear whether prior exposure to circulating human influenza viruses or influenza vaccination confers immunity to H7N9 strains. Here, we demonstrate that 3 of 83 H3 HA-reactive monoclonal antibodies generated by individuals that had previously undergone influenza A virus vaccination were able to neutralize H7N9 viruses and protect mice against homologous challenge. The H7N9-neutralizing antibodies bound to the HA stalk domain but exhibited a difference in their breadth of reactivity to different H7 influenza subtypes. Mapping viral escape mutations suggested that these antibodies bind at least two different epitopes on the stalk region. Together, these results indicate that these broadly neutralizing antibodies may contribute to the development of therapies against H7N9 strains and may also be effective against pathogenic H7 strains that emerge in the future.
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http://dx.doi.org/10.1172/JCI74374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362269PMC
March 2015

Staphylococcus aureus infection induces protein A-mediated immune evasion in humans.

J Exp Med 2014 Nov 27;211(12):2331-9. Epub 2014 Oct 27.

Committee on Immunology; Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology Research; Department of Microbiology; and Department of Pathology, The University of Chicago, Chicago, IL 60637 Committee on Immunology; Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology Research; Department of Microbiology; and Department of Pathology, The University of Chicago, Chicago, IL 60637

Staphylococcus aureus bacterial infection commonly results in chronic or recurrent disease, suggesting that humoral memory responses are hampered. Understanding how S. aureus subverts the immune response is critical for the rescue of host natural humoral immunity and vaccine development. S. aureus expresses the virulence factor Protein A (SpA) on all clinical isolates, and SpA has been shown in mice to expand and ablate variable heavy 3 (VH3) idiotype B cells. The effects of SpA during natural infection, however, have not been addressed. Acutely activated B cells, or plasmablasts (PBs), were analyzed to dissect the ongoing immune response to infection through the production of monoclonal antibodies (mAbs). The B cells that were activated by infection had a highly limited response. When screened against multiple S. aureus antigens, only high-affinity binding to SpA was observed. Consistently, PBs underwent affinity maturation, but their B cell receptors demonstrated significant bias toward the VH3 idiotype. These data suggest that the superantigenic activity of SpA leads to immunodominance, limiting host responses to other S. aureus virulence factors that would be necessary for protection and memory formation.
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http://dx.doi.org/10.1084/jem.20141404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4235641PMC
November 2014

Potential antigenic explanation for atypical H1N1 infections among middle-aged adults during the 2013-2014 influenza season.

Proc Natl Acad Sci U S A 2014 Nov 20;111(44):15798-803. Epub 2014 Oct 20.

Wistar Institute, Philadelphia, PA 19104; Institute for Immunology, Department of Microbiology, and

Influenza viruses typically cause the most severe disease in children and elderly individuals. However, H1N1 viruses disproportionately affected middle-aged adults during the 2013-2014 influenza season. Although H1N1 viruses recently acquired several mutations in the hemagglutinin (HA) glycoprotein, classic serological tests used by surveillance laboratories indicate that these mutations do not change antigenic properties of the virus. Here, we show that one of these mutations is located in a region of HA targeted by antibodies elicited in many middle-aged adults. We find that over 42% of individuals born between 1965 and 1979 possess antibodies that recognize this region of HA. Our findings offer a possible antigenic explanation of why middle-aged adults were highly susceptible to H1N1 viruses during the 2013-2014 influenza season. Our data further suggest that a drifted H1N1 strain should be included in future influenza vaccines to potentially reduce morbidity and mortality in this age group.
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http://dx.doi.org/10.1073/pnas.1409171111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226110PMC
November 2014

Lineage structure of the human antibody repertoire in response to influenza vaccination.

Sci Transl Med 2013 Feb;5(171):171ra19

Department of Bioengineering, Stanford University School of Medicine, Stanford, CA 94305, USA.

The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cell's genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals' repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system's clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.
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http://dx.doi.org/10.1126/scitranslmed.3004794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699344PMC
February 2013

Global analysis of B cell selection using an immunoglobulin light chain-mediated model of autoreactivity.

J Exp Med 2013 Jan 24;210(1):125-42. Epub 2012 Dec 24.

Section of Rheumatology, Department of Medicine, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637, USA.

The important subtleties of B cell tolerance are best understood in a diverse immunoglobulin (Ig) repertoire context encoding a full spectrum of autoreactivity. To achieve this, we used mice expressing Igκ transgenes that confer varying degrees of autoreactivity within a diverse heavy chain (HC) repertoire. These transgenes, coupled with a biomarker to identify receptor-edited cells and combined with expression cloning of B cell receptors, allowed us to analyze tolerance throughout B cell development. We found that both the nature of the autoantigen and the Ig HC versus light chain (LC) contribution to autoreactivity dictate the developmental stage and mechanism of tolerance. Furthermore, although selection begins in the bone marrow, over one third of primary tolerance occurs in the periphery at the late transitional developmental stage. Notably, we demonstrate that the LC has profound effects on tolerance and can lead to exacerbated autoantibody production.
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http://dx.doi.org/10.1084/jem.20120525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549719PMC
January 2013

Fully human monoclonal antibodies from antibody secreting cells after vaccination with Pneumovax®23 are serotype specific and facilitate opsonophagocytosis.

Immunobiology 2013 May 3;218(5):745-54. Epub 2012 Sep 3.

Oklahoma Medical Research Foundation, Arthritis and Clinical Immunology Research Program, Oklahoma City, OK 73104, USA.

B lymphocyte memory generates antibody-secreting cells (ASCs) that represent a source of protective antibodies that may be exploited for therapeutics. Here we vaccinated four donors with Pneumovax®23 and produced human monoclonal antibodies (hmAbs) from ASCs. We have cloned 137 hmAbs and the specificities of these antibodies encompass 19 of the 23 serotypes in the vaccine, as well as cell wall polysaccharide (CWPS). Although the majority of the antibodies are serotype specific, 12% cross-react with two serotypes. The Pneumovax®23 ASC antibody sequences are highly mutated and clonal, indicating an anamnestic response, even though this was a primary vaccination. Hmabs from 64% of the clonal families facilitate opsonophagocytosis. Although 9% of the total antibodies bind to CWPS impurity in the vaccine, none of these clonal families showed opsonophagocytic activity. Overall, these studies have allowed us to address unanswered questions in the field of human immune responses to polysaccharide vaccines, including the cross-reactivity of individual antibodies between serotypes and the percentage of antibodies that are protective after vaccination with Pneumovax®23.
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http://dx.doi.org/10.1016/j.imbio.2012.08.278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556204PMC
May 2013

Antibody pressure by a human monoclonal antibody targeting the 2009 pandemic H1N1 virus hemagglutinin drives the emergence of a virus with increased virulence in mice.

mBio 2012 29;3(3). Epub 2012 May 29.

Laboratory of Infectious Diseases, NIAID, Bethesda, Maryland, USA.

Unlabelled: In 2009, a novel H1N1 influenza A virus (2009 pH1N1) emerged and caused a pandemic. A human monoclonal antibody (hMAb; EM4C04), highly specific for the 2009 pH1N1 virus hemagglutinin (HA), was isolated from a severely ill 2009 pH1N1 virus-infected patient. We postulated that under immune pressure with EM4C04, the 2009 pH1N1 virus would undergo antigenic drift and mutate at sites that would identify the antibody binding site. To do so, we infected MDCK cells in the presence of EM4C04 and generated 11 escape mutants, displaying 7 distinct amino acid substitutions in the HA. Six substitutions greatly reduced MAb binding (K123N, D131E, K133T, G134S, K157N, and G158E). Residues 131, 133, and 134 are contiguous with residues 157 and 158 in the globular domain structure and contribute to a novel pH1N1 antibody epitope. One mutation near the receptor binding site, S186P, increased the binding affinity of the HA to the receptor. 186P and 131E are present in the highly virulent 1918 virus HA and were recently identified as virulence determinants in a mouse-passaged pH1N1 virus. We found that pH1N1 escape variants expressing these substitutions enhanced replication and lethality in mice compared to wild-type 2009 pH1N1 virus. The increased virulence of these viruses was associated with an increased affinity for α2,3 sialic acid receptors. Our study demonstrates that antibody pressure by an hMAb targeting a novel epitope in the Sa region of 2009 pH1N1 HA is able to inadvertently drive the development of a more virulent virus with altered receptor binding properties. This broadens our understanding of antigenic drift.

Importance: Influenza viruses accumulate amino acid substitutions to evade the antibody response in a process known as antigenic drift, making it necessary to vaccinate against influenza annually. Mapping human monoclonal antibody (hMAb) epitopes is a necessary step towards understanding antigenic drift in humans. We defined the specificity of an hMAb that specifically targeted the 2009 pH1N1 virus and describe a novel epitope. In addition, we identified a previously unappreciated potential for antibody escape to enhance the pathogenicity of a virus. The escape mutation that we identified with in vitro immune pressure was independently reported by other investigators using in vivo selection in nonimmune mice. Although in vitro generation of escape mutants is unlikely to recapitulate antigenic drift in its entirety, the data demonstrate that pressure by a human monoclonal antibody targeting a novel epitope in the hemagglutinin of the 2009 pandemic H1N1 virus can inadvertently drive the development of escape mutants, of which a subset have increased virulence and altered receptor binding properties.
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http://dx.doi.org/10.1128/mBio.00120-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372962PMC
August 2012

Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells.

Proc Natl Acad Sci U S A 2012 Jun 21;109(23):9047-52. Epub 2012 May 21.

Emory Vaccine Center, Department of Microbiology and Immunology, School of Medicine, Emory University, Atlanta, GA 30322, USA.

We have previously shown that broadly neutralizing antibodies reactive to the conserved stem region of the influenza virus hemagglutinin (HA) were generated in people infected with the 2009 pandemic H1N1 strain. Such antibodies are rarely seen in humans following infection or vaccination with seasonal influenza virus strains. However, the important question remained whether the inactivated 2009 pandemic H1N1 vaccine, like the infection, could also induce these broadly neutralizing antibodies. To address this question, we analyzed B-cell responses in 24 healthy adults immunized with the pandemic vaccine in 2009. In all cases, we found a rapid, predominantly IgG-producing vaccine-specific plasmablast response. Strikingly, the majority (25 of 28) of HA-specific monoclonal antibodies generated from the vaccine-specific plasmablasts neutralized more than one influenza strain and exhibited high levels of somatic hypermutation, suggesting they were derived from recall of B-cell memory. Indeed, memory B cells that recognized the 2009 pandemic H1N1 HA were detectable before vaccination not only in this cohort but also in samples obtained before the emergence of the pandemic strain. Three antibodies demonstrated extremely broad cross-reactivity and were found to bind the HA stem. Furthermore, one stem-reactive antibody recognized not only H1 and H5, but also H3 influenza viruses. This exceptional cross-reactivity indicates that antibodies capable of neutralizing most influenza subtypes might indeed be elicited by vaccination. The challenge now is to improve upon this result and design influenza vaccines that can elicit these broadly cross-reactive antibodies at sufficiently high levels to provide heterosubtypic protection.
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http://dx.doi.org/10.1073/pnas.1118979109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384143PMC
June 2012

Human monoclonal antibodies generated following vaccination with AVA provide neutralization by blocking furin cleavage but not by preventing oligomerization.

Vaccine 2012 Jun 14;30(28):4276-83. Epub 2012 Mar 14.

Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.

In order to identify the combination of antibody-mediated mechanisms of neutralization that result from vaccination with anthrax vaccine adsorbed (AVA), we isolated antibody secreting cells from a single donor seven days after booster vaccination with AVA and generated nine fully human monoclonal antibodies (hmAb) with high specificity for protective antigen (PA). Two of the antibodies were able to neutralize lethal toxin in vitro at low concentrations (IC(50): p6C01, 0.12 μg/ml and p6F01, 0.45 μg/ml). Passive transfer of either of these hmAbs to A/J mice prior to challenge with lethal toxin conferred 80-90% protection. We demonstrate that hmAb p6C01 is neutralizing by preventing furin cleavage of PA in a dose-dependent manner, but the mechanism of p6F01 is unclear. Three additional antibodies were found to bind to domain 3 of PA and prevent oligomerization, although they did not confer significant protection in vivo and showed a significant prozone-like effect in vitro. These fully human antibodies provide insight into the neutralizing response to AVA for future subunit vaccine and passive immunotherapeutic cocktail design.
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http://dx.doi.org/10.1016/j.vaccine.2012.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367042PMC
June 2012

High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions.

Nat Med 2012 Feb 26;18(3):441-5. Epub 2012 Feb 26.

Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway.

Celiac disease is an immune-mediated disorder in which mucosal autoantibodies to the enzyme transglutaminase 2 (TG2) are generated in response to the exogenous antigen gluten in individuals who express human leukocyte antigen HLA-DQ2 or HLA-DQ8 (ref. 3). We assessed in a comprehensive and nonbiased manner the IgA anti-TG2 response by expression cloning of the antibody repertoire of ex vivo-isolated intestinal antibody-secreting cells (ASCs). We found that TG2-specific plasma cells are markedly expanded within the duodenal mucosa in individuals with active celiac disease. TG2-specific antibodies were of high affinity yet showed little adaptation by somatic mutations. Unlike infection-induced peripheral blood plasmablasts, the TG2-specific ASCs had not recently proliferated and were not short-lived ex vivo. Altogether, these observations demonstrate that there is a germline repertoire with high affinity for TG2 that may favor massive generation of autoreactive B cells. TG2-specific antibodies did not block enzymatic activity and served as substrates for TG2-mediated crosslinking when expressed as IgD or IgM but not as IgA1 or IgG1. This could result in preferential recruitment of plasma cells from naive IgD- and IgM-expressing B cells, thus possibly explaining why the antibody response to TG2 bears signs of a primary immune response despite the disease chronicity.
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http://dx.doi.org/10.1038/nm.2656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4533878PMC
February 2012

Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies.

J Clin Invest 2011 Aug 25;121(8):3109-19. Epub 2011 Jul 25.

Department of Immunology and Microbiology, Stanford University School of Medicine, Stanford, California, USA.

During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies.
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http://dx.doi.org/10.1172/JCI57834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3148747PMC
August 2011

Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection.

J Exp Med 2011 Jan 10;208(1):181-93. Epub 2011 Jan 10.

Emory Vaccine Center, School of Medicine, Emory University, Atlanta, GA 30322, USA.

The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates.
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http://dx.doi.org/10.1084/jem.20101352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023136PMC
January 2011

Rapid generation of rotavirus-specific human monoclonal antibodies from small-intestinal mucosa.

J Immunol 2010 Nov 8;185(9):5377-83. Epub 2010 Oct 8.

Center for Immune Regulation, Oslo University Hospital, Oslo, Norway.

The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03-0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with K(d) < 1 × 10(-10)). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity.
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http://dx.doi.org/10.4049/jimmunol.1001587DOI Listing
November 2010

Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen.

Nat Protoc 2009 ;4(3):372-84

Department of Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. The expressed antibodies can then be purified and assayed for binding and neutralization. This method uses established techniques but is novel in their combination and application. This protocol can be completed with as little as 20 ml of human blood and in as little as 28 d when optimal. Although previous methodologies to produce hmAbs, including B-cell immortalization or phage display, can be used to isolate the rare specific antibody even years after immunization, in comparison, these approaches are inefficient, resulting in few relevant antibodies. Although dependent on having an ongoing immune response, the approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time.
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http://dx.doi.org/10.1038/nprot.2009.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2750034PMC
April 2009

Functional anergy in a subpopulation of naive B cells from healthy humans that express autoreactive immunoglobulin receptors.

J Exp Med 2009 Jan 22;206(1):139-51. Epub 2008 Dec 22.

Immunobiology and Cancer, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

Self-reactive B cells not controlled by receptor editing or clonal deletion may become anergic. We report that fully mature human B cells negative for surface IgM and retaining only IgD are autoreactive and functionally attenuated (referred to as naive IgD(+)IgM(-) B cells [B(ND)]). These B(ND) cells typically make up 2.5% of B cells in the peripheral blood, have antibody variable region genes in germline (unmutated) configuration, and, by all current measures, are fully mature. Analysis of 95 recombinant antibodies expressed from the variable genes of single B(ND) cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA. Upon B cell receptor cross-linkage, B(ND) cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic. However, intense stimulation causes B(ND) cells to fully respond, suggesting that these cells could be the precursors of autoantibody secreting plasma cells in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. This is the first identification of a distinct mature human B cell subset that is naturally autoreactive and controlled by the tolerizing mechanism of functional anergy.
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http://dx.doi.org/10.1084/jem.20080611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2626668PMC
January 2009