Maturation of germinal center B cells after influenza virus vaccination in humans.

Germinal centers (GC) are microanatomical lymphoid structures where affinity-matured memory B cells and long-lived bone marrow plasma cells are primarily generated. It is unclear how the maturation of B cells within the GC impacts the breadth and durability of B cell responses to influenza vaccination in humans. We used fine needle aspiration of draining lymph nodes to longitudinally track antigen-specific GC B cell responses to seasonal influenza vaccination. Antigen-specific GC B cells persisted for at least 13 wk after vaccination in two out of seven individuals. Monoclonal antibodies (mAbs) derived from persisting GC B cell clones exhibit enhanced binding affinity and breadth to influenza hemagglutinin (HA) antigens compared with related GC clonotypes isolated earlier in the response. Structural studies of early and late GC-derived mAbs from one clonal lineage in complex with H1 and H5 HAs revealed an altered binding footprint. Our study shows that inducing sustained GC reactions after influenza vaccination in humans supports the maturation of responding B cells.

Spatiotemporal development of the human T follicular helper cell response to Influenza vaccination.

We profiled blood and draining lymph node (LN) samples from human volunteers after influenza vaccination over two years to define evolution in the T follicular helper cell (TFH) response. We show LN TFH cells expanded in a clonal-manner during the first two weeks after vaccination and persisted within the LN for up to six months. LN and circulating TFH (cTFH) clonotypes overlapped but had distinct kinetics. LN TFH cell phenotypes were heterogeneous and mutable, first differentiating into pre-TFH during the month after vaccination before maturing into GC and IL-10+ TFH cells. TFH expansion, upregulation of glucose metabolism, and redifferentiation into GC TFH cells occurred with faster kinetics after re-vaccination in the second year. We identified several influenza-specific TFH clonal lineages, including multiple responses targeting internal influenza proteins, and show each TFH state is attainable within a lineage. This study demonstrates that human TFH cells form a durable and dynamic multi-tissue network.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses of these vaccines and the development of new variant-derived ones 1-4 . SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells (MBCs) 5-9 . It remains unclear, however, whether the additional doses induce germinal centre (GC) reactions where reengaged B cells can further mature and whether variant-derived vaccines can elicit responses to novel epitopes specific to such variants. Here, we show that boosting with the original SARS- CoV-2 spike vaccine (mRNA-1273) or a B.1.351/B.1.617.2 (Beta/Delta) bivalent vaccine (mRNA-1273.213) induces robust spike-specific GC B cell responses in humans. The GC response persisted for at least eight weeks, leading to significantly more mutated antigen-specific MBC and bone marrow plasma cell compartments. Interrogation of MBC-derived spike-binding monoclonal antibodies (mAbs) isolated from individuals boosted with either mRNA-1273, mRNA-1273.213, or a monovalent Omicron BA.1-based vaccine (mRNA-1273.529) revealed a striking imprinting effect by the primary vaccination series, with all mAbs (n=769) recognizing the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted approach, we isolated mAbs that recognized the spike protein of the SARS-CoV-2 Omicron (BA.1) but not the original SARS-CoV-2 spike from the mRNA-1273.529 boosted individuals. The latter mAbs were less mutated and recognized novel epitopes within the spike protein, suggesting a naïve B cell origin. Thus, SARS-CoV-2 boosting in humans induce robust GC B cell responses, and immunization with an antigenically distant spike can overcome the antigenic imprinting by the primary vaccination series.

Germinal centre-driven maturation of B cell response to mRNA vaccination.

Germinal centres (GC) are lymphoid structures in which B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells1-5 (BMPCs). SARS-CoV-2 mRNA vaccination induces a persistent GC response that lasts for at least six months in humans6-8. The fate of responding GC B cells as well as the functional consequences of such persistence remain unknown. Here, we detected SARS-CoV-2 spike protein-specific MBCs in 42 individuals who had received two doses of the SARS-CoV-2 mRNA vaccine BNT162b2 six month earlier. Spike-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of sequencing the B cell receptors of responding blood plasmablasts and MBCs, lymph node GC B cells and plasma cells and BMPCs from eight individuals and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1,540 spike-specific B cell clones. On average, early blood spike-specific plasmablasts exhibited the lowest SHM frequencies. By contrast, SHM frequencies of spike-specific GC B cells increased by 3.5-fold within six months after vaccination. Spike-specific MBCs and BMPCs accumulated high levels of SHM, which corresponded with enhanced anti-spike antibody avidity in blood and enhanced affinity as well as neutralization capacity of BMPC-derived monoclonal antibodies. We report how the notable persistence of the GC reaction induced by SARS-CoV-2 mRNA vaccination in humans culminates in affinity-matured long-term antibody responses that potently neutralize the virus.

Germinal centre-driven maturation of B cell response to SARS-CoV-2 vaccination.

Germinal centres (GC) are lymphoid structures where vaccine-responding B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells (BMPCs) 1-4 . Induction of the latter is a hallmark of durable immunity after vaccination 5 . SARS-CoV-2 mRNA vaccination induces a robust GC response in humans 6-8 , but the maturation dynamics of GC B cells and propagation of their progeny throughout the B cell diaspora have not been elucidated. Here we show that anti-SARS-CoV-2 spike (S)-binding GC B cells were detectable in draining lymph nodes for at least six months in 10 out of 15 individuals who had received two doses of BNT162b2, a SARS-CoV-2 mRNA vaccine. Six months after vaccination, circulating S-binding MBCs were detected in all participants (n=42) and S-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of single-cell RNA sequencing of responding blood and lymph node B cells from eight participants and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1540 S-specific B cell clones. SHM accumulated along the B cell differentiation trajectory, with early blood plasmablasts showing the lowest frequencies, followed by MBCs and lymph node plasma cells whose SHM largely overlapped with GC B cells. By three months after vaccination, the frequency of SHM within GC B cells had doubled. Strikingly, S + BMPCs detected six months after vaccination accumulated the highest level of SHM, corresponding with significantly enhanced anti-S polyclonal antibody avidity in blood at that time point. This study documents the induction of affinity-matured BMPCs after two doses of SARS-CoV-2 mRNA vaccination in humans, providing a foundation for the sustained high efficacy observed with these vaccines.

A vaccine-induced public antibody protects against SARS-CoV-2 and emerging variants.

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.

SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses.

SARS-CoV-2 mRNA-based vaccines are about 95% effective in preventing COVID-191-5. The dynamics of antibody-secreting plasmablasts and germinal centre B cells induced by these vaccines in humans remain unclear. Here we examined antigen-specific B cell responses in peripheral blood (n = 41) and draining lymph nodes in 14 individuals who had received 2 doses of BNT162b2, an mRNA-based vaccine that encodes the full-length SARS-CoV-2 spike (S) gene1. Circulating IgG- and IgA-secreting plasmablasts that target the S protein peaked one week after the second immunization and then declined, becoming undetectable three weeks later. These plasmablast responses preceded maximal levels of serum anti-S binding and neutralizing antibodies to an early circulating SARS-CoV-2 strain as well as emerging variants, especially in individuals who had previously been infected with SARS-CoV-2 (who produced the most robust serological responses). By examining fine needle aspirates of draining axillary lymph nodes, we identified germinal centre B cells that bound S protein in all participants who were sampled after primary immunization. High frequencies of S-binding germinal centre B cells and plasmablasts were sustained in these draining lymph nodes for at least 12 weeks after the booster immunization. S-binding monoclonal antibodies derived from germinal centre B cells predominantly targeted the receptor-binding domain of the S protein, and fewer clones bound to the N-terminal domain or to epitopes shared with the S proteins of the human betacoronaviruses OC43 and HKU1. These latter cross-reactive B cell clones had higher levels of somatic hypermutation as compared to those that recognized only the SARS-CoV-2 S protein, which suggests a memory B cell origin. Our studies demonstrate that SARS-CoV-2 mRNA-based vaccination of humans induces a persistent germinal centre B cell response, which enables the generation of robust humoral immunity.

SARS-CoV-2 mRNA vaccination induces functionally diverse antibodies to NTD, RBD, and S2.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.

The plasmablast response to SARS-CoV-2 mRNA vaccination is dominated by non-neutralizing antibodies and targets both the NTD and the RBD.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.

Position-Dependent Differential Targeting of Somatic Hypermutation.

Somatic hypermutation (SHM) generates much of the Ab diversity necessary for affinity maturation and effective humoral immunity. The activation-induced cytidine deaminase-induced DNA lesions and error-prone repair that underlie SHM are known to exhibit intrinsic biases when targeting the Ig sequences. Computational models for SHM targeting often model the targeting probability of a nucleotide in a motif-based fashion, assuming that the same DNA motif is equally likely to be targeted regardless of its position along the Ig sequence. The validity of this assumption, however, has not been rigorously studied in vivo. In this study, by analyzing a large collection of 956,157 human Ig sequences while controlling for the confounding influence of selection, we show that the likelihood of a DNA 5-mer motif being targeted by SHM is not the same at different positions in the same Ig sequence. We found position-dependent differential SHM targeting for about three quarters of the 38 and 269 unique motifs from more than half of the 292 and 1912 motif-allele pairs analyzed using productive and nonproductive Ig sequences, respectively. The direction of the differential SHM targeting was largely conserved across individuals with no allele-specific effect within an IgH variable gene family, but was not consistent with general decay of SHM targeting with increasing distance from the transcription start site. However, SHM targeting did correlate positively with the mutability of the wider sequence neighborhood surrounding the motif. These findings provide insights and future directions for computational efforts toward modeling SHM.

Human germinal centres engage memory and naive B cells after influenza vaccination.

Influenza viruses remain a major public health threat. Seasonal influenza vaccination in humans primarily stimulates pre-existing memory B cells, which differentiate into a transient wave of circulating antibody-secreting plasmablasts1-3. This recall response contributes to 'original antigenic sin'-the selective increase of antibody species elicited by previous exposures to influenza virus antigens4. It remains unclear whether such vaccination can also induce germinal centre reactions in the draining lymph nodes, where diversification and maturation of recruited B cells can occur5. Here we used ultrasound-guided fine needle aspiration to serially sample the draining lymph nodes and investigate the dynamics and specificity of germinal centre B cell responses after influenza vaccination in humans. Germinal centre B cells that bind to influenza vaccine could be detected as early as one week after vaccination. In three out of eight participants, we detected vaccine-binding germinal centre B cells up to nine weeks after vaccination. Between 12% and 88% of the responding germinal centre B cell clones overlapped with B cells detected among early circulating plasmablasts. These shared B cell clones had high frequencies of somatic hypermutation and encoded broadly cross-reactive monoclonal antibodies. By contrast, vaccine-induced B cell clones detected only in the germinal centre compartment exhibited significantly lower frequencies of somatic hypermutation and predominantly encoded strain-specific monoclonal antibodies, which suggests a naive B cell origin. Some of these strain-specific monoclonal antibodies recognized epitopes that were not targeted by the early plasmablast response. Thus, influenza virus vaccination in humans can elicit a germinal centre reaction that recruits B cell clones that can target new epitopes, thereby broadening the spectrum of vaccine-induced protective antibodies.

A Potently Neutralizing Antibody Protects Mice against SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for millions of infections and hundreds of thousands of deaths globally. There are no widely available licensed therapeutics against SARS-CoV-2, highlighting an urgent need for effective interventions. The virus enters host cells through binding of a receptor-binding domain within its trimeric spike glycoprotein to human angiotensin-converting enzyme 2. In this article, we describe the generation and characterization of a panel of murine mAbs directed against the receptor-binding domain. One mAb, 2B04, neutralized wild-type SARS-CoV-2 in vitro with remarkable potency (half-maximal inhibitory concentration of <2 ng/ml). In a murine model of SARS-CoV-2 infection, 2B04 protected challenged animals from weight loss, reduced lung viral load, and blocked systemic dissemination. Thus, 2B04 is a promising candidate for an effective antiviral that can be used to prevent SARS-CoV-2 infection.

Overexpression of T-bet in HIV infection is associated with accumulation of B cells outside germinal centers and poor affinity maturation.

Nearly all chronic human infections are associated with alterations in the memory B cell (MBC) compartment, including a large expansion of CD19hiT-bethi MBC in the peripheral blood of HIV-infected individuals with chronic viremia. Despite their prevalence, it is unclear how these B cells arise and whether they contribute to the inefficiency of antibody-mediated immunity in chronic infectious diseases. We addressed these questions by characterizing T-bet-expressing B cells in lymph nodes (LN) and identifying a strong T-bet signature among HIV-specific MBC associated with poor immunologic outcome. Confocal microscopy and quantitative imaging revealed that T-bethi B cells in LN of HIV-infected chronically viremic individuals distinctly accumulated outside germinal centers (GC), which are critical for optimal antibody responses. In single-cell analyses, LN T-bethi B cells of HIV-infected individuals were almost exclusively found among CD19hi MBC and expressed reduced GC-homing receptors. Furthermore, HIV-specific B cells of infected individuals were enriched among LN CD19hiT-bethi MBC and displayed a distinct transcriptome, with features similar to CD19hiT-bethi MBC in blood and LN GC B cells (GCBC). LN CD19hiT-bethi MBC were also related to GCBC by B cell receptor (BCR)-based phylogenetic linkage but had lower BCR mutation frequencies and reduced HIV-neutralizing capacity, consistent with diminished participation in GC-mediated affinity selection. Thus, in the setting of chronic immune activation associated with HIV viremia, failure of HIV-specific B cells to enter or remain in GC may help explain the rarity of high-affinity protective antibodies.

Repertoire-wide phylogenetic models of B cell molecular evolution reveal evolutionary signatures of aging and vaccination.

In order to produce effective antibodies, B cells undergo rapid somatic hypermutation (SHM) and selection for binding affinity to antigen via a process called affinity maturation. The similarities between this process and evolution by natural selection have led many groups to use phylogenetic methods to characterize the development of immunological memory, vaccination, and other processes that depend on affinity maturation. However, these applications are limited by the fact that most phylogenetic models are designed to be applied to individual lineages comprising genetically diverse sequences, while B cell repertoires often consist of hundreds to thousands of separate low-diversity lineages. Further, several features of affinity maturation violate important assumptions in standard phylogenetic models. Here, we introduce a hierarchical phylogenetic framework that integrates information from all lineages in a repertoire to more precisely estimate model parameters while simultaneously incorporating the unique features of SHM. We demonstrate the power of this repertoire-wide approach by characterizing previously undescribed phenomena in affinity maturation. First, we find evidence consistent with age-related changes in SHM hot-spot targeting. Second, we identify a consistent relationship between increased tree length and signs of increased negative selection, apparent in the repertoires of recently vaccinated subjects and those without any known recent infections or vaccinations. This suggests that B cell lineages shift toward negative selection over time as a general feature of affinity maturation. Our study provides a framework for undertaking repertoire-wide phylogenetic testing of SHM hypotheses and provides a means of characterizing dynamics of mutation and selection during affinity maturation.

Cutting Edge: Ig H Chains Are Sufficient to Determine Most B Cell Clonal Relationships.

B cell clonal expansion is vital for adaptive immunity. High-throughput BCR sequencing enables investigating this process but requires computational inference to identify clonal relationships. This inference usually relies on only the BCR H chain, as most current protocols do not preserve H:L chain pairing. The extent to which paired L chains aids inference is unknown. Using human single-cell paired BCR datasets, we assessed the ability of H chain-based clonal clustering to identify clones. Of the expanded clones identified, <20% grouped cells expressing inconsistent L chains. H chains from these misclustered clones contained more distant junction sequences and shared fewer V segment mutations than the accurate clones. This suggests that additional H chain information could be leveraged to refine clonal relationships. Conversely, L chains were insufficient to refine H chain-based clonal clusters. Overall, the BCR H chain alone is sufficient to identify clonal relationships with confidence.

Spatiotemporal segregation of human marginal zone and memory B cell populations in lymphoid tissue.

Human memory B cells and marginal zone (MZ) B cells share common features such as the expression of CD27 and somatic mutations in their IGHV and BCL6 genes, but the relationship between them is controversial. Here, we show phenotypic progression within lymphoid tissues as MZ B cells emerge from the mature naïve B cell pool via a precursor CD27-CD45RBMEM55+ population distant from memory cells. By imaging mass cytometry, we find that MZ B cells and memory B cells occupy different microanatomical niches in organised gut lymphoid tissues. Both populations disseminate widely between distant lymphoid tissues and blood, and both diversify their IGHV repertoire in gut germinal centres (GC), but nevertheless remain largely clonally separate. MZ B cells are therefore not developmentally contiguous with or analogous to classical memory B cells despite their shared ability to transit through GC, where somatic mutations are acquired.

Affinity Maturation Is Impaired by Natural Killer Cell Suppression of Germinal Centers.

Somatic hypermutation of immunoglobulin sequences in germinal center (GC) reactions must be optimized to elicit high-affinity, protective antibodies after vaccination. We expose natural killer (NK) cells as robust negative regulators of somatic hypermutation in antigen-reactive B cells. NK cells restrict follicular helper T cell (TFH) and GC B cell frequencies and titers of antigen-specific immunoglobulin after administration of alum-adjuvanted hapten-protein conjugate vaccines. This inhibition is perforin dependent, suggesting that NK cells kill one or more cells critical for GC development. In the presence of perforin-competent NK cells, antigen-specific GC B cells acquire fewer mutations, including less frequent generation of non-synonymous substitutions and mutations associated with increased antibody affinity. Thus, NK cells limit the magnitude of GC reactions and thereby restrain vaccine elicitation of high-affinity antibodies. Circumventing this activity of NK cells during vaccination has strong potential to enhance humoral immunity and facilitate vaccine-elicited prevention of disease.

Local Clonal Diversification and Dissemination of B Lymphocytes in the Human Bronchial Mucosa.

The efficacy of the adaptive humoral immune response likely requires diverse, yet focused regional B cell antibody production throughout the body. Here we address, in the first study of its kind, the B cell repertoire in the bronchial mucosa, an important barrier to antigens inhaled from the atmosphere. To accomplish this, we have applied high-throughput Adaptive Immune Receptor Repertoire Sequencing (AIRR-Seq) to 10 bronchial biopsies from altogether four different sites in the right lungs from an asthmatic patient and a healthy subject. While the majority of identified B cell clones were restricted to a single site, many were disseminated in multiple sites. Members of a clone were shared more between adjacent biopsies than between distal biopsies, suggesting local mucosal migration and/or a homing mechanism for B cells through the blood or lymph. A smaller fraction of clones spanned the bronchial mucosa and peripheral blood, suggesting ongoing trafficking between these compartments. The bronchial mucosal B cell repertoire in the asthmatic patient was geographically more variable but less diverse compared to that of the healthy subject, suggesting an ongoing, antigen-driven humoral immune response in atopic asthma. Whether this is a feature of atopy or disease status remains to be clarified in future studies. We observed a subset of highly mutated and antigen-selected IgD-only cells in the bronchial mucosa. These cells were found in relative high abundance in the asthmatic individual but also, albeit at lower abundance, in the healthy subject. This novel finding merits further exploration using a larger cohort of subjects.

The natural killer cell response to West Nile virus in young and old individuals with or without a prior history of infection.

West Nile virus (WNV) typically leads to asymptomatic infection but can cause severe neuroinvasive disease or death, particularly in the elderly. Innate NK cells play a critical role in antiviral defenses, yet their role in human WNV infection is poorly defined. Here we demonstrate that NK cells mount a robust, polyfunctional response to WNV characterized by cytolytic activity, cytokine and chemokine secretion. This is associated with downregulation of activating NK cell receptors and upregulation of NK cell activating ligands for NKG2D. The NK cell response did not differ between young and old WNV-naïve subjects, but a history of symptomatic infection is associated with more IFN-γ producing NK cell subsets and a significant decline in a specific NK cell subset. This NK repertoire skewing could either contribute to or follow heightened immune pathogenesis from WNV infection, and suggests that NK cells could play an important role in WNV infection in humans.