Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection.

Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.

Type I interferons induce an epigenetically distinct memory B cell subset in chronic viral infection.

Memory B cells (MBCs) are key providers of long-lived immunity against infectious disease, yet in chronic viral infection, they do not produce effective protection. How chronic viral infection disrupts MBC development and whether such changes are reversible remain unknown. Through single-cell (sc)ATAC-seq and scRNA-seq during acute versus chronic lymphocytic choriomeningitis viral infection, we identified a memory subset enriched for interferon (IFN)-stimulated genes (ISGs) during chronic infection that was distinct from the T-bet+ subset normally associated with chronic infection. Blockade of IFNAR-1 early in infection transformed the chromatin landscape of chronic MBCs, decreasing accessibility at ISG-inducing transcription factor binding motifs and inducing phenotypic changes in the dominating MBC subset, with a decrease in the ISG subset and an increase in CD11c+CD80+ cells. However, timing was critical, with MBCs resistant to intervention at 4 weeks post-infection. Together, our research identifies a key mechanism to instruct MBC identity during viral infection.

Aberrant B cell repertoire selection associated with HIV neutralizing antibody breadth.

A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth.

STAT3 gain-of-function mutations connect leukemia with autoimmune disease by pathological NKG2Dhi CD8+ T cell dysregulation and accumulation.

The association between cancer and autoimmune disease is unexplained, exemplified by T cell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8+ T cell clonal expansions and autoimmunity, we analyzed patients and mice with germline STAT3 GOF mutations. STAT3 GOF mutations drove the accumulation of effector CD8+ T cell clones highly expressing NKG2D, the receptor for stress-induced MHC-class-I-related molecules. This subset also expressed genes for granzymes, perforin, interferon-γ, and Ccl5/Rantes and required NKG2D and the IL-15/IL-2 receptor IL2RB for maximal accumulation. Leukocyte-restricted STAT3 GOF was sufficient and CD8+ T cells were essential for lethal pathology in mice. These results demonstrate that STAT3 GOF mutations cause effector CD8+ T cell oligoclonal accumulation and that these rogue cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

Broadening Horizons: New Antibodies Against Influenza.

Seasonal influenza vaccine formulation efforts struggle to keep up with viral antigenic variation. Two studies now report engineered or naturally occurring human antibodies targeting the influenza hemagglutinin (HA) stem, with exceptional neutralizing breadth (Joyce et al., 2016; Kallewaard et al., 2016). Antibodies with similar structural features are elicited in multiple subjects, suggesting that modified vaccine regimens could provide broad protection.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination.

Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody.

Genetic and structural basis of the human anti-α-galactosyl antibody response.

Humans lack the capacity to produce the Galα1-3Galß1-4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.

A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome.

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients' B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders.

RAGging on recombination signal sequence strength for diffusion-mediated recombination.

In this article, we discuss new insights into the distinct mechanisms for V(D)J recombination for different immunoglobulin loci. This follows the recent revelation that recombination signal sequences (RSS) within the IGKV locus have evolved to be more efficient mediators of recombination activating gene (RAG) recombination compared to the same elements in the IGH locus. This difference in RSS strength is proposed to be driven by different molecular mechanisms for RAG-mediated recombination between the two loci.

Isolation and characterisation of PR3-specific B cells and their immunoglobulin sequences.

BACKGROUND: PR3 autoantibodies are essential to the diagnosis and monitoring of granulomatosus with polyangiitis, but to date no PR3 autoantibody sequences have been published. OBJECTIVES: To identify and characterise PR3-specific B cells from the peripheral blood of patients with PR3 autoantibodies. METHODS: Peripheral blood mononuclear cells from seven patients with PR3 autoantibodies were stained with PR3. B cells that bound PR3 underwent single cell sorting, transcriptome sequencing, and their immunoglobulin sequences expressed as antibodies and tested for PR3-specificity by ELISA. RESULTS: We identified 19 PR3-specific B cells from only one PR3-seropositive patient at a low frequency (0.0075 % of B cells) in the peripheral blood. These were polyclonal, IgG+ and enriched for IgG4, lambda pairing, IGHJ6 gene usage, CDRH3 length, IGHE and CD71 expression. They demonstrated relatively low levels of somatic hypermutation and variably reduced PR3 binding when reverted to germline. CONCLUSIONS: Identifying PR3-specific B cells in the peripheral blood is possible but challenging and those we did identify exhibited features suggesting that PR3-self reactivity may occur early in B-cell development.

Establishment and Feasibility of an Immediate Lymphatic Reconstruction Program in a Community Health System.

BACKGROUND: Breast cancer-related lymphedema (BCRL) remains a significant post-surgical complication of breast cancer treatment. Immediate lymphatic reconstruction (ILR) at the time of axillary lymph node dissection (ALND) has shown promise in preventing BCRL. While the primary literature supporting ILR comes from academic institutions, the majority of breast cancer care in the USA occurs in the community setting. This study evaluated a preventative lymphedema program performing ILR at a community health system. PATIENTS AND METHODS: A prospective database including all patients who underwent ALND with concurrently attempted ILR from 2019 to 2021 was retrospectively reviewed. The historical benchmark lymphedema rate was calculated through retrospective review of electronic medical records for all patients who underwent ALND without ILR from 2011 to 2021. RESULTS: Ninety patients underwent ALND with ILR, of which ILR was successful in 69 (76.7%). ILR was more likely to be aborted in smokers (p < 0.05) and those with fewer lymphatic channels (p < 0.05) or a higher body mass index (BMI) (p = 0.08). Patients with successful versus aborted ILR had lower lymphedema rates (10.9% versus 66.7%, p < 0.01) and improved Disability of the Arm, Shoulder, and Hand (DASH) scores (8.7 versus 19.8, p = 0.25), and lower lymphedema rates than the historical benchmark (10.9% versus 50.2%, p < 0.01). Among patients with successful ILR, older patients were more likely to develop lymphedema (p < 0.05). CONCLUSIONS: Successful ILR after ALND significantly reduced the lymphedema rate when compared with patients with aborted ILR and our institution's historical benchmark. Our experience supports the efficacy of ILR and highlights the feasibility of ILR within a community health system.

Comparing Costs: Does Extreme Oncoplastic Breast-Conserving Surgery Confer a Cost Benefit When Compared with Mastectomy and Reconstruction?

INTRODUCTION: Extreme oncoplastic breast-conserving surgery (eOBCS) describes the application of OBCS to patients who would otherwise need a mastectomy, and its safety has been previously described. OBJECTIVE: We aimed to compare the costs of eOBCS and mastectomy. METHODS: We reviewed our institutional database to identify breast cancer patients treated surgically from 2018 to 2023. We included patients with a large disease span (≥5 cm) and multifocal/multicentric disease. Patients were grouped by their surgical approach, i.e. eOBCS or mastectomy. The direct costs of care were determined and compared; however, indirect costs were not included. RESULTS: Eighty-six patients met the inclusion criteria, 10 (11.6%) of whom underwent mastectomy and 76 (88.4%) who underwent eOBCS. Six mastectomy patients (60%) had reconstruction and 6 (60%) underwent external beam radiation therapy (EBRT). Reconstructions were completed in a staged fashion, and the mean cost of the index operation (mastectomy and tissue expander) was $17,816. These patients had one to three subsequent surgeries to complete their reconstruction, at a mean cost of $45,904. The mean cost of EBRT was $5542. Thirty-four eOBCS patients (44.7%) underwent 44 margin re-excisions, including 6 (7.9%) who underwent mastectomy. Sixty (78.9%) of the eOBCS patients had EBRT. The mean cost of their index operation was $6345; the mean cost of a re-excision was $3615; the mean cost of their mastectomies with reconstruction was $49,400; and the mean cost of EBRT was $6807. The cost of care for eOBCS patients remained lower than that for mastectomy patients, i.e. $17,318 versus $57,416. CONCLUSION: eOBCS is associated with a lower cost than mastectomy and had a low conversion rate to mastectomy.

Understanding the challenges of identifying, supporting, and signposting patients with alcohol use disorder in secondary care hospitals, post COVID-19: a qualitative analysis from the North East and North Cumbria, England.

BACKGROUND: Alcohol-related mortality and morbidity increased during the COVID-19 pandemic in England, with people from lower-socioeconomic groups disproportionately affected. The North East and North Cumbria (NENC) region has high levels of deprivation and the highest rates of alcohol-related harm in England. Consequently, there is an urgent need for the implementation of evidence-based preventative approaches such as identifying people at risk of alcohol harm and providing them with appropriate support. Non-alcohol specialist secondary care clinicians could play a key role in delivering these interventions, but current implementation remains limited. In this study we aimed to explore current practices and challenges around identifying, supporting, and signposting patients with Alcohol Use Disorder (AUD) in secondary care hospitals in the NENC through the accounts of staff in the post COVID-19 context. METHODS: Semi-structured qualitative interviews were conducted with 30 non-alcohol specialist staff (10 doctors, 20 nurses) in eight secondary care hospitals across the NENC between June and October 2021. Data were analysed inductively and deductively to identify key codes and themes, with Normalisation Process Theory (NPT) then used to structure the findings. RESULTS: Findings were grouped using the NPT domains 'implementation contexts' and 'implementation mechanisms'. The following implementation contexts were identified as key factors limiting the implementation of alcohol prevention work: poverty which has been exacerbated by COVID-19 and the prioritisation of acute presentations (negotiating capacity); structural stigma (strategic intentions); and relational stigma (reframing organisational logics). Implementation mechanisms identified as barriers were: workforce knowledge and skills (cognitive participation); the perception that other departments and roles were better placed to deliver this preventative work than their own (collective action); and the perceived futility and negative feedback cycle (reflexive monitoring). CONCLUSIONS: COVID-19, has generated additional challenges to identifying, supporting, and signposting patients with AUD in secondary care hospitals in the NENC. Our interpretation suggests that implementation contexts, in particular structural stigma and growing economic disparity, are the greatest barriers to implementation of evidence-based care in this area. Thus, while some implementation mechanisms can be addressed at a local policy and practice level via improved training and support, system-wide action is needed to enable sustained delivery of preventative alcohol work in these settings.

Diversification and expansion of the EBV-reactive cytotoxic T lymphocyte repertoire following autologous haematopoietic stem cell transplant for multiple sclerosis.

Both genetic susceptibility and environmental exposures are thought to be involved in multiple sclerosis (MS) pathogenesis. Of all viruses potentially relevant to MS aetiology, Epstein-Barr virus (EBV) is the best-studied. EBV is a B cell lymphotropic virus which is able to evade the immune system by establishing latent infection in memory B cells, and EBV reactivation is restricted by CD8 cytotoxic T cell (CTL) responses in immune competent individuals. Autologous haematopoietic stem cell transplantation (AHSCT) is considered to be the most effective therapy in the treatment of relapsing MS even though chemotherapy-induced lymphopenia can associate with the re-emergence of latent viruses. Despite the increasing interest in EBV and MS pathogenesis the relationship between AHSCT, EBV and viral immunity in people with MS has not been investigated to date. This study analysed immune responses to EBV in a well characterised cohort of 13 individuals with MS by utilising pre-AHSCT, and 6-, 12- and 24-month post AHSCT bio-banked peripheral blood mononuclear cells and plasma samples. It is demonstrated that the infused stem cell product contains latently EBV-infected memory B cells, and that EBV viremia occurs in the immune-compromised recipient post-transplant. High throughput TCR analysis detected expansion and diversification of the CD8 CTL responses reactive with EBV lytic and latent antigens from 6 to 24 months following AHSCT. Increased levels of latent EBV infection found within the B cell pool following treatment, as measured by EBV genomic detection, did not associate with disease relapse. This is the first study of EBV immunity following application of AHSCT in the treatment of MS and not only raises important questions about the role of EBV infection in MS pathogenesis, but is of clinical importance given the expanding clinical trials of adoptive EBV-specific CTLs in MS.

Tracking the clonal dynamics of SARS-CoV-2-specific T cells in children and adults with mild/asymptomatic COVID-19.

Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV-2-specific naïve T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. A novel population of naïve interferon-activated T cells is expanded in acute COVID-19 and is recruited into the memory compartment during convalescence in adults but not children. This was associated with the development of robust CD4+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection.

Simultaneous Symmetry Procedure in Patients Undergoing Oncoplastic Breast-Conserving Surgery: An Evaluation of Patient Desire and Revision Rates.

INTRODUCTION: In the era of oncoplastic breast conserving-surgery (OBCS), cosmetic outcomes and the desire for symmetry have become essential elements of the surgical management of breast cancer (BC). The timing of contralateral symmetry procedures remains a controversial topic. Simultaneous symmetry procedures (SSP) in OBCS have not been routinely offered due to the perceived risk of delayed asymmetry, potentially increasing the need for delayed cosmetic revision. This study evaluates the rate of revision after SSP in patients undergoing OBCS. METHODS: We reviewed our institutional prospectively maintained database identifying all BC patients treated surgically since our introduction of oncoplastic surgery in 2018. We routinely offer SSP when appropriate. Descriptive statistics evaluated oncoplastic surgical techniques, SSP offerings and procedures, perioperative complications, and revision rates after treatment completion. RESULTS: Between 2018 and 2022, 485 breast cancer patients underwent partial mastectomy, and 396 (82%) underwent OBCS. Of the 313 patients offered SSP, 272 (87%) accepted. The margin reexcision rate of this cohort was 20%. Of the 272 patients with SSP, 152 (56%) underwent intraoperative radiation therapy (IORT), and 105 (39%) had adjuvant external beam radiation therapy. Three patients (1%) experienced complications involving the symmetry side. No patients with complications experienced a delay in adjuvant therapies or requested cosmetic revisions. Three patients (1%) desired surgical revisions due to asymmetry. CONCLUSIONS: Symmetry procedures at the time of OBCS are widely accepted by patients and rarely require delayed cosmetic revision. Simultaneous symmetry procedures should be routinely discussed with patients during the surgical planning of OBCS.

A Look at the Other Side: High-Risk Lesions and Occult Contralateral Malignancy in Symmetry Procedures for Patients Undergoing Oncoplastic Breast-Conserving Surgery.

BACKGROUND: The incidence of occult breast cancer among patients undergoing reduction mammoplasty or risk-reducing mastectomies ranges from 1% to approximately 10%, respectively. Identification of incidental cancer often mandates subsequent mastectomy due to ambiguous margins. This study aimed to determine the incidence of contralateral malignancy among patients undergoing oncoplastic breast-conserving surgery (OBCS) with concurrent symmetry procedures. METHODS: The authors reviewed their prospectively maintained institutional database of patients with unilateral breast cancer who underwent OBCS. Patients who underwent excisional biopsy on the contralateral breast were analyzed separately. Patient demographics, pathologic features, and subsequent disease management were evaluated. RESULTS: Between March 2018 and July 2022, 289 patients underwent OBCS with a symmetry procedure, and 100 patients yielded contralateral breast tissue specimens. For 14 patients, a planned excisional biopsy was performed with their symmetry procedure, and five lesions (36%) were found to be malignant. Of the remaining 86 patients, 92% underwent preoperative breast magnetic resonance imaging (MRI). Four patients (4.7%) had occult malignancies identified on the contralateral breast pathology; three patients with ductal carcinoma in situ and one patient with invasive lobular carcinoma. Three patients had undergone preoperative MRI without suspicious findings. No patients required mastectomy for treatment of the contralateral breast cancer. CONCLUSION: The incidence of occult malignancy among OBCS symmetry procedures approaches 5%. The final pathology of excisional biopsies had a higher upgrade rate than previously reported. All identified malignancies were early-stage disease. The higher incidence of occult breast cancer in this population warrants the routine orientation of all specimens, which allows patients with incidental early-stage cancer the option of breast preservation.

Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans.

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.