Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of ...immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ∼60% of the response. An unexpectedly high fraction of serum antibodies recognized both the H1 and H3 monovalent vaccines. Recombinant versions of these H1 + H3 cross-reactive antibodies showed broad binding to hemagglutinins (HAs) from previously circulating virus strains; several of these antibodies, which were prevalent in the serum of multiple donors, recognized the same conserved epitope in the HA head domain. Although the HA-head-specific H1 + H3 antibodies did not show neutralization activity in vitro, they protected mice against infection with the H1N1 and H3N2 virus strains when administered before or after challenge. Collectively, our data reveal unanticipated insights regarding the serological response to influenza vaccination and raise questions about the added benefits of using a quadrivalent vaccine instead of a trivalent vaccine.
Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an ...antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT ⁺ serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with K d ∼ 10 ⁻⁸–10 ⁻¹⁰ M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3 ⁻CD14 ⁻CD19 ⁺CD27 ⁺⁺CD38 ⁺⁺CD20 ⁻TT ⁺) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the K d). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.
Highlights • New technologies have revolutionized serological antibody analysis. • NextGen sequencing and shotgun proteomics help identify and quantify serum mAb's. • Clonality and dynamics of serum ...mAb repertoire in various states can be addressed. • New tools available for identifying novel antigens that elicit serological response.
Humans are repeatedly exposed to influenza virus via infections and vaccinations. Understanding how multiple exposures and pre-existing immunity impact antibody responses is essential for vaccine ...development. Given the recent prevalence of influenza H1N1 A/California/7/2009 (CA09), we examined the clonal composition and dynamics of CA09 hemagglutinin (HA)-reactive IgG repertoire over 5 years in a donor with multiple influenza exposures. The anti-CA09 HA polyclonal response in this donor comprised 24 persistent antibody clonotypes, accounting for 72.6% ± 10.0% of the anti-CA09 HA repertoire over 5 years. These persistent antibodies displayed higher somatic hypermutation relative to transient serum antibodies detected at one time point. Additionally, persistent antibodies predominantly demonstrated cross-reactivity and potent neutralization toward a phylogenetically distant H5N1 A/Vietnam/1203/2004 (VT04) strain, a feature correlated with HA stem recognition. This analysis reveals how “serological imprinting” impacts responses to influenza and suggests that once elicited, cross-reactive antibodies targeting the HA stem can persist for years.
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•Longitudinal profiling of anti-H1N1 serum antibodies (Abs) reveals persisting Abs•The persistent Abs on average account for >70% of the serum responses over 5 years•Most persistent Abs bind and neutralize a highly divergent H5N1 viral strain•Cross-neutralizing anti-influenza Abs can persist in the circulation
In recent years, there has been a great interest in understanding how repeated exposures to influenza can change the already established host antibody repertoire. Lee et al. quantitatively demonstrate that over multiple years and repeated vaccinations, the serum response to influenza is dominated by a small number of persistent antibodies.
The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following SARS-CoV-2 infection are unknown. Proteomic deconvolution of the IgG ...repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor-binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an N-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multi-donor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.
•Next-generation sequencing of peripheral B cells (BCR-seq) in health and disease states.•Serum antibody proteomics (Ig-seq) complements BCR-seq in analyzing endpoint functionality.•Advances in the ...pairing and NGS of antibody heavy chain and light chain (VH:VL) sequences.•Combined, BCR-seq and Ig-seq provide a clear advance in antibody discovery.
Recent developments of high-throughput technologies are enabling the molecular-level analysis and bioinformatic mining of antibody-mediated (humoral) immunity in humans at an unprecedented level. These approaches explore either the sequence space of B-cell receptor repertoires using next-generation deep sequencing (BCR-seq), or the amino acid identities of antibody in blood using protein mass spectrometry (Ig-seq), or both. Generalizable principles about the molecular composition of the protective humoral immune response are being defined, and as such, the field could supersede traditional methods for the development of diagnostics, vaccines, and antibody therapeutics. Three key challenges remain and have driven recent advances: (1) incorporation of innovative techniques for paired BCR-seq to ascertain the complete antibody variable-domain VH:VL clonotype, (2) integration of proteomic Ig-seq with BCR-seq to reveal how the serum antibody repertoire compares with the antibody repertoire encoded by circulating B cells, and (3) a demand to link antibody sequence data to functional meaning (binding and protection).
We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard ...antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography–high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V H CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V H clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.
Significance Morbilliviruses are a growing concern because of their ability to infect multiple species. The spill-over of canine distemper virus (CDV) from domestic dogs has been associated with ...severe declines in wild carnivores worldwide, and therefore mass dog vaccination has been suggested as a potential control strategy. Focusing on three decades of CDV exposure data in dogs and lions of the Serengeti, we show that cyclic infection dynamics in lions initially driven by dogs became more frequent and asynchronous, suggesting that the wider dog population and other wildlife species drive CDV dynamics. Hence, although widespread dog vaccination reduced the infection in dogs, transmission to lion populations still occurred, warranting further investigation into effective management options of CDV in this species-rich ecosystem.
Morbilliviruses cause many diseases of medical and veterinary importance, and although some (e.g., measles and rinderpest) have been controlled successfully, others, such as canine distemper virus (CDV), are a growing concern. A propensity for host-switching has resulted in CDV emergence in new species, including endangered wildlife, posing challenges for controlling disease in multispecies communities. CDV is typically associated with domestic dogs, but little is known about its maintenance and transmission in species-rich areas or about the potential role of domestic dog vaccination as a means of reducing disease threats to wildlife. We address these questions by analyzing a long-term serological dataset of CDV in lions and domestic dogs from Tanzania’s Serengeti ecosystem. Using a Bayesian state–space model, we show that dynamics of CDV have changed considerably over the past three decades. Initially, peaks of CDV infection in dogs preceded those in lions, suggesting that spill-over from dogs was the main driver of infection in wildlife. However, despite dog-to-lion transmission dominating cross-species transmission models, infection peaks in lions became more frequent and asynchronous from those in dogs, suggesting that other wildlife species may play a role in a potentially complex maintenance community. Widespread mass vaccination of domestic dogs reduced the probability of infection in dogs and the size of outbreaks but did not prevent transmission to or peaks of infection in lions. This study demonstrates the complexity of CDV dynamics in natural ecosystems and the value of long-term, large-scale datasets for investigating transmission patterns and evaluating disease control strategies.
Characterizing the in vivo dynamics of the polyclonal antibody repertoire in serum, such as that which might arise in response to stimulation with an antigen, is difficult due to the presence of many ...highly similar immunoglobulin proteins, each specified by distinct B lymphocytes. These challenges have precluded the use of conventional mass spectrometry for antibody identification based on peptide mass spectral matches to a genomic reference database. Recently, progress has been made using bottom-up analysis of serum antibodies by nanoflow liquid chromatography/high-resolution tandem mass spectrometry combined with a sample-specific antibody sequence database generated by high-throughput sequencing of individual B cell immunoglobulin variable domains (V genes). Here, we describe how intrinsic features of antibody primary structure, most notably the interspersed segments of variable and conserved amino acid sequences, generate recurring patterns in the corresponding peptide mass spectra of V gene peptides, greatly complicating the assignment of correct sequences to mass spectral data. We show that the standard method of decoy-based error modeling fails to account for the error introduced by these highly similar sequences, leading to a significant underestimation of the false discovery rate. Because of these effects, antibody-derived peptide mass spectra require increased stringency in their interpretation. The use of filters based on the mean precursor ion mass accuracy of peptide-spectrum matches is shown to be particularly effective in distinguishing between “true” and “false” identifications. These findings highlight important caveats associated with the use of standard database search and error-modeling methods with nonstandard data sets and custom sequence databases.
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