Segmentation of the influenza A virus (IAV) genome enables rapid gene reassortment at the cost of complicating the task of assembling the full viral genome. By simultaneously probing for the expression of multiple viral proteins in MDCK cells infected at a low multiplicity with IAV, we observe that the majority of infected cells lack detectable expression of one or more essential viral proteins. Consistent with this observation, up to 90% of IAV-infected cells fail to release infectious progeny, indicating that many IAV virions scored as noninfectious by traditional infectivity assays are capable of single-round infection. This fraction was not significantly affected by target or producer cell type but varied widely between different IAV strains. These data indicate that IAV exists primarily as a swarm of complementation-dependent semi-infectious virions, and thus traditional, propagation-dependent assays of infectivity may drastically misrepresent the true infectious potential of a virus population.
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, such as the fact that each B cell contains a distinct antibody sequence encoded in its genome, that the antibody repertoire is not constant but changes over time, and the high similarity between antibody sequences. We have addressed this challenge 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 pre-vaccination 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.
The nature of the immunoglobulin light chain affects peripheral B cell tolerance and autoreactivity.
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.
During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV disfavours homotypic bivalent antibody binding1–3. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development4, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients5 with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.
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 (IC50: 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.
anthrax; Anthrax Vaccine Adsorbed; human monoclonal antibodies; passive immunotherapeutics; protective antigen
Since its first administration in the 1940s, the influenza vaccine has provided tremendous relief against influenza infections. However, time has revealed the vaccine’s ultimate limit and the call for its reinvention has now come, just as we are beginning to appreciate the antibody immune responses vital in preventing infections. New strategies to design the influenza vaccine rely on selectively inducing broadly neutralizing antibodies that are specific for highly conserved viral epitopes. Such approaches take us away from the limited range of protection provided by current seasonal influenza vaccines and towards a future with a pan-influenza vaccine capable of providing universal strain coverage.
Humoral immune responses are thought to play a major role in dengue virus-induced immunopathology; however, little is known about the plasmablasts producing these antibodies during an ongoing infection. Herein we present an analysis of plasmablast responses in patients with acute dengue virus infection. We found very potent plasmablast responses that often increased more than 1,000-fold over the baseline levels in healthy volunteers. In many patients, these responses made up as much 30% of the peripheral lymphocyte population. These responses were largely dengue virus specific and almost entirely made up of IgG-secreting cells, and plasmablasts reached very high numbers at a time after fever onset that generally coincided with the window where the most serious dengue virus-induced pathology is observed. The presence of these large, rapid, and virus-specific plasmablast responses raises the question as to whether these cells might have a role in dengue immunopathology during the ongoing infection. These findings clearly illustrate the need for a detailed understanding of the repertoire and specificity of the antibodies that these plasmablasts produce.
The discovery of lymphocytes bearing two light chains in mice carrying self-reactive antibody transgenes has challenged the “one lymphocyte–one antibody” rule. However, the extent and nature of allelically included cells in normal mice is unknown. We show that 10% of mature B cells coexpress both Igκ alleles. These cells are not the result of failure in allelic exclusion per se, but arise through receptor editing. We find that under physiological conditions, editing occurs both by deletion and by inclusion with equal probability. In addition, we demonstrate that B lymphocytes carrying two B-cell receptors are recruited to germinal center reactions, and thus fully participate in humoral immune responses. Our data measure the scope of allelic inclusion and provide a mechanism whereby autoreactive B cells might “escape” central tolerance.
H3N2 influenza viruses have now circulated in the human population for 43 years since the pandemic of 1968, accumulating sequence changes in the hemagglutinin (HA) and neuraminidase (NA) that are believed to be predominantly due to selection for escape from antibodies. Examination of mutations that persist and accumulate led to identification of antigenically significant mutations that are contained in five antigenic sites (A–E) mapped on to the H3 HA. In early H3N2 isolates, antigenic site A appeared to be dominant while in the 1990s site B seemed more important. To obtain experimental evidence for dominance of antigenic sites on modern H3 HAs, we have measured antibodies in plasma of human subjects who received the 2006–07 trivalent subunit influenza vaccine (H3 component A/Wisconsin/67/05) or the 2008–09 formulation (H3 component A/Uruguay/716/07). Plasmas were tested against expressed HA of Wisconsin-like influenza A/Oklahoma/309/06 and site-directed mutants in antigenic site A (NNES121-124ITEG, N126T, N133D, TSSS135-138GSNA, K140I, RSNNS142-146PGSG), and antigenic site B (HL156-157KS, KFK158-160GST, NDQI189-192QEQT, A196V). “Native ELISA” analysis and escape mutant selection with two human monoclonal antibodies demonstrated that antibody E05 binds to antigenic site A and 1_C02 binds to site B. We find that most individuals, after vaccination in seasons 2006–07 and/or 2008–09, showed dominance of antigenic site B recognition over antigenic site A. A minority showed dominance of site A in 2006 but these were reduced in 2008 when the vaccine virus had a site A mutation. A better understanding of immunodominance may allow prediction of future antigenic drift and assist in vaccine strain selection.
It is believed that immunoglobulin-variable region gene (IgV) somatic hypermutation (SHM) is initiated by activation-induced cytidine deaminase (AID) upon deamination of cytidine to deoxyuracil. Patch-excision repair of these lesions involving error prone DNA polymerases such as polη causes mutations at all base positions. If not repaired, the deaminated nucleotides on the coding and noncoding strands result in C-to-T and G-to-A exchanges, respectively. Herein it is reported that IgV gene evolution has been considerably influenced by the need to accommodate extensive C deaminations and the resulting accumulation of C-to-T and G-to-A exchanges. Although seemingly counterintuitive, the precise placement of C and G nucleotides causes most C-to-T and G-to-A mutations to be silent or conservative. We hypothesize that without intricate positioning of C and G nucleotides the efficiency of affinity maturation would be significantly reduced due to a dominance of replacements caused by C and G transition mutations. The complexity of these evolved biases in codon use are compounded by the precise concomitant hotspot/coldspot targeting of AID activity and Polη errors to maximize SHM in the CDRs and minimize mutations in the FWRs.
We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics.
Monoclonal antibody technology has undergone rapid and innovative reinvention over the last 30 years. Application of these technologies to human samples revealed valuable therapeutic and experimental insights. These technologies, each with their own benefits and flaws, have proven indispensable for immunological research and in our fight to provide new treatments and improved vaccines for infectious disease.
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.
The major goal in vaccination is establishment of long-term, prophylactic humoral memory to a pathogen. Two major components to long-lived humoral memory are plasma cells for the production of specific immunoglobulin and memory B cells that survey for their specific antigen in the periphery for later affinity maturation, proliferation, and differentiation. The study of human B cell memory has been aided by the discovery of a general marker for B cell memory, expression of CD27; however, new data suggests the existence of CD27− memory B cells as well. These recently described non-canonical memory populations have increasingly pointed to the heterogeneity of the memory compartment. The novel B memory subsets in humans appear to have unique origins, localization, and functions compared to what was considered to be a “classical” memory B cell. In this article, we review the known B cell memory subsets, the establishment of B cell memory in vaccination and infection, and how understanding these newly described subsets can inform vaccine design and disease treatment.
B cell; memory B cell; antibody; vaccine; B cell subset; influenza; anthrax; immunoglobulin
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.
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 [BND]). These BND 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 BND cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA. Upon B cell receptor cross-linkage, BND cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic. However, intense stimulation causes BND 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.
Pre-existing neutralizing antibody provides the first line of defense against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14 to 21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B cell receptor (BCR) repertoire that in some donors were dominated by only a few B cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over fifty human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high affinity mAbs from humans within a month after vaccination. The panel of influenza virus specific human mAbs allowed us to address the issue of original antigenic sin (OAS) - the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared to the virus strain present in the vaccine1. However, we found that the vast majority of the influenza virus specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal healthy adults receiving influenza vaccination.
Determination of the origin and fate of autoreactive B cells is critical to understanding and treating autoimmune diseases. We report that, despite being derived from healthy people, antibodies from B cells that have class switched to IgD via genetic recombination (and thus become class switched to Cδ [Cδ-CS] cells) are highly reactive to self antigens. Over half of the antibodies from Cδ-CS B cells bind autoantigens on human epithelioma cell line 2 (HEp-2) cells or antinuclear antigens, and a quarter bind double-stranded DNA; both groups of antibodies are frequently polyreactive. Intriguingly, some Cδ-CS B cells have accumulated basic residues in the antibody variable regions that mediate anti-DNA reactivity via somatic hypermutation and selection, while other Cδ-CS B cells are naturally autoreactive. Though the total percentage was appreciably less than for Cδ-CS cells, a surprising 31% of IgG memory cell antibodies were somewhat autoreactive, and as expected, about 24% of naive cell antibodies were autoreactive. We interpret these findings to indicate either that autoreactive B cells can be induced to class switch to IgD or that autoreactive B cells that use IgD as the B cell receptor are not effectively deleted. Determination of the mechanism by which the majority of Cδ-CS B cells are autoreactive may be important in understanding peripheral tolerance mechanisms and may provide insight into the enigmatic function of the IgD antibody.
Current paradigms of peripheral B cell selection suggest that autoreactive B cells are controlled by clonal deletion, anergy, and developmental arrest. We report that changes to the human antibody repertoire likely resulting from these mechanisms both for a well-characterized autoreactivity from antibodies encoded by the VH4-34 gene and for other hallmarks of an autoreactive repertoire are apparent mainly for class-switched B cells and not for IgM germinal center, IgM memory, or IgM plasma cells. Other possible indicators of autoreactivity found selected with immunoglobulin class include JH6 gene segment usage, increased frequency of B cells with long third hypervariable regions, and distal Jκ gene segment bias. Of particular interest is the finding that B cells with these same characteristics are selected into the lineage of B cells that have undergone the unusual class switch from constant region Cμ to Cδ (Cδ-CS). The Cδ-CS population also displays an increased frequency of charged amino acids localized to the complementarity-determining regions, further suggesting autoreactivity, and evidence is presented that these B cells had undergone extensive receptor editing. Thus, the Cδ-CS lineage may be a “sink” for B cells harboring autoreactive specificities in normal humans. A model for a new tolerizing mechanism that could account for the Cδ-CS lineage is presented.
Contrary to the general precepts of the clonal selection theory, several recent studies have provided evidence for the secondary rearrangement of immunoglobulin (Ig) genes in peripheral lymphoid tissues. These analyses typically used transgenic mouse models and have only detected secondary recombination of Ig light chain genes. Although Ig heavy chain variable region (VH) genes encode a substantial element of antibody combining site specificity, there is scant evidence for VH gene rearrangement in the periphery, leaving the physiological importance of peripheral recombination questionable. The extensive somatic mutations and clonality of the IgD+Strictly-IgM−CD38+ human tonsillar B cell subpopulation have now allowed detection of the first clear examples of receptor revision of human VH genes. The revised VDJ genes contain “hybrid” VH gene segments consisting of portions from two separate germline VH genes, a phenomenon previously only detected due to the pressures of a transgenic system.
receptor revision; immunoglobulin heavy chain; variable region; recombination; receptor editing
During a germinal center reaction, random mutations are introduced into immunoglobulin V genes to increase the affinity of antibody molecules and to further diversify the B cell repertoire. Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response. The mutations of Ig genes are typically basepair substitutions, although DNA insertions and deletions have been reported to occur at a low frequency. In this study, we describe five insertion and four deletion events in otherwise somatically mutated VH gene cDNA molecules. Two of these insertions and all four deletions were obtained through the sequencing of 395 cDNA clones (∼110,000 nucleotides) from CD38+IgD− germinal center, and CD38−IgD− memory B cell populations from a single human tonsil. No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor. These six insertions or deletions and three additional insertion events isolated from other sources occurred as triplets or multiples thereof, leaving the transcripts in frame. Additionally, 8 of 9 of these events occurred in the CDR1 or CDR2, following a pattern consistent with selection, and making it unlikely that these events were artifacts of the experimental system. The lack of similar instances in unmutated IgD+CD38− follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014). Close scrutiny of the 9 insertion/deletion events reported here, and of 25 additional insertions or deletions collected from the literature, suggest that secondary structural elements in the DNA sequences capable of producing loop intermediates may be a prerequisite in most instances. Furthermore, these events most frequently involve sequence motifs resembling known intrinsic hotspots of somatic hypermutation. These insertion/deletion events are consistent with models of somatic hypermutation involving an unstable polymerase enzyme complex lacking proofreading capabilities, and suggest a downregulation or alteration of DNA repair at the V locus during the hypermutation process.
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.
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.