ABSTRACT
Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to prime and elicit antibody responses against the conserved fusion peptide (FP). GC responses and antibody specificities were tracked longitudinally using lymph node fine-needle aspirates and electron microscopy polyclonal epitope mapping (EMPEM), respectively, to show antibody responses to the FP/N611 glycan hole region were primed, although exhibited limited neutralization breadth. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.
ABSTRACT
The upper airway is an important site of infection, but immune memory in the human upper airway is poorly understood, with implications for COVID-19 and many other human diseases1-4. Here we demonstrate that nasal and nasopharyngeal swabs can be used to obtain insights into these challenging problems, and define distinct immune cell populations, including antigen-specific memory B cells and T cells, in two adjacent anatomical sites in the upper airway. Upper airway immune cell populations seemed stable over time in healthy adults undergoing monthly swabs for more than 1 year, and prominent tissue resident memory T (TRM) cell and B (BRM) cell populations were defined. Unexpectedly, germinal centre cells were identified consistently in many nasopharyngeal swabs. In subjects with SARS-CoV-2 breakthrough infections, local virus-specific BRM cells, plasma cells and germinal centre B cells were identified, with evidence of local priming and an enrichment of IgA+ memory B cells in upper airway compartments compared with blood. Local plasma cell populations were identified with transcriptional profiles of longevity. Local virus-specific memory CD4+ TRM cells and CD8+ TRM cells were identified, with diverse additional virus-specific T cells. Age-dependent upper airway immunological shifts were observed. These findings provide new understanding of immune memory at a principal mucosal barrier tissue in humans.
ABSTRACT
Therapeutic monoclonal antibodies (mAbs) have been studied in humans, but the impact on immune memory of mAb treatment during an ongoing infection has remained unclear. We evaluated the effect of infusion of the anti-SARS-CoV-2 spike receptor binding domain (RBD) mAb bamlanivimab on memory B cells (MBCs) in SARS-CoV-2-infected individuals. Bamlanivimab treatment skewed the repertoire of memory B cells targeting Spike towards non-RBD epitopes. Furthermore, the relative affinity of RBD memory B cells was weaker in mAb-treated individuals compared to placebo-treated individuals over time. Subsequently, after mRNA COVID-19 vaccination, memory B cell differences persisted and mapped to a specific reduction in recognition of the class II RBD site, the same RBD epitope recognized by bamlanivimab. These findings indicate a substantial role of antibody feedback in regulating memory B cell responses to infection, and single mAb administration can continue to impact memory B cell responses to additional antigen exposures months later.
ABSTRACT
Little is known about the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or SARS2) vaccine breakthrough infections (BTIs) on the magnitude and breadth of the T cell repertoire after exposure to different variants. We studied samples from individuals who experienced symptomatic BTIs during Delta or Omicron waves. In the pre-BTI samples, 30% of the donors exhibited substantial immune memory against non-S (spike) SARS2 antigens, consistent with previous undiagnosed asymptomatic SARS2 infections. Following symptomatic BTI, we observed (1) enhanced S-specific CD4 and CD8 T cell responses in donors without previous asymptomatic infection, (2) expansion of CD4 and CD8 T cell responses to non-S targets (M, N, and nsps) independent of SARS2 variant, and (3) generation of novel epitopes recognizing variant-specific mutations. These variant-specific T cell responses accounted for 9%-15% of the total epitope repertoire. Overall, BTIs boost vaccine-induced immune responses by increasing the magnitude and by broadening the repertoire of T cell antigens and epitopes recognized.
Subject(s)
CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , COVID-19 Vaccines , COVID-19 , Epitopes, T-Lymphocyte , SARS-CoV-2 , Humans , SARS-CoV-2/immunology , COVID-19/immunology , COVID-19/virology , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/genetics , CD8-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , COVID-19 Vaccines/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , Immunologic Memory/immunology , Female , Adult , Male , Mutation , Middle Aged , T-Lymphocytes/immunology , Breakthrough InfectionsABSTRACT
Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.
Subject(s)
Antibody Affinity , B-Lymphocytes , Germinal Center , HIV Antibodies , HIV-1 , Germinal Center/immunology , Animals , Mice , Humans , B-Lymphocytes/immunology , HIV-1/immunology , HIV Antibodies/immunology , Antibody Affinity/immunology , Antibodies, Neutralizing/immunology , HIV Infections/immunology , AIDS Vaccines/immunology , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, B-Cell/immunology , Gene Knock-In Techniques , Mice, Transgenic , Broadly Neutralizing Antibodies/immunology , Mice, Inbred C57BLABSTRACT
Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.
Subject(s)
AIDS Vaccines , Broadly Neutralizing Antibodies , Complementarity Determining Regions , Germinal Center , HIV Antibodies , Animals , Humans , AIDS Vaccines/immunology , B-Lymphocytes/immunology , Broadly Neutralizing Antibodies/immunology , Complementarity Determining Regions/immunology , Cryoelectron Microscopy , env Gene Products, Human Immunodeficiency Virus/immunology , Germinal Center/immunology , HIV Antibodies/immunology , HIV Infections/immunology , HIV Infections/prevention & control , HIV-1/immunology , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/genetics , Macaca mulatta , Memory B Cells/immunologyABSTRACT
Follicular helper T cells (Tfh) play a crucial role in generating high-affinity antibodies (Abs) and establishing immunological memory. Cytokines, among other functional molecules produced by Tfh, are central to germinal center (GC) reactions. This review focuses on the role of cytokines, including IL-21 and IL-4, in regulating B cell responses within the GC, such as differentiation, affinity maturation, and plasma cell development. Additionally, this review explores the impact of other cytokines like CXCL13, IL-10, IL-9, and IL-2 on GC responses and their potential involvement in autoimmune diseases, allergies, and cancer. This review highlights contributions of Tfh-derived cytokines to both protective immunity and immunopathology across a spectrum of diseases. A deeper understanding of Tfh cytokine biology holds promise for insights into biomedical conditions.
ABSTRACT
Therapeutic anti-SARS-CoV-2 monoclonal antibodies (mAbs) have been extensively studied in humans, but the impact on immune memory of mAb treatment during an ongoing immune response has remained unclear. Here, we evaluated the effect of infusion of the anti-SARS-CoV-2 spike receptor binding domain (RBD) mAb bamlanivimab on memory B cells (MBCs) in SARS-CoV-2-infected individuals. Bamlanivimab treatment skewed the repertoire of memory B cells targeting Spike towards non-RBD epitopes. Furthermore, the relative affinity of RBD memory B cells was weaker in mAb-treated individuals compared to placebo-treated individuals over time. Subsequently, after mRNA COVID-19 vaccination, memory B cell differences persisted and mapped to a specific defect in recognition of the class II RBD site, the same RBD epitope recognized by bamlanivimab. These findings indicate a substantial role of antibody feedback in regulating human memory B cell responses, both to infection and vaccination. These data indicate that mAb administration can promote alterations in the epitopes recognized by the B cell repertoire, and the single administration of mAb can continue to determine the fate of B cells in response to additional antigen exposures months later.
ABSTRACT
Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.
Subject(s)
HIV Infections , Immunity, Humoral , Animals , Germinal Center , Adjuvants, Immunologic/pharmacology , Antigens , Primates , Antibodies, Neutralizing , HIV Antibodies , env Gene Products, Human Immunodeficiency VirusABSTRACT
T cells are involved in protective immunity against numerous viral infections. Limited data have been available regarding roles of human T cell responses controlling SARS-CoV-2 viral clearance in primary COVID-19. Here, we examined longitudinal SARS-CoV-2 upper respiratory tract viral RNA levels and early adaptive immune responses from 95 unvaccinated individuals with acute COVID-19. Acute SARS-CoV-2-specific CD4 and CD8 T cell responses were evaluated in addition to antibody responses. Most individuals with acute COVID-19 developed rapid SARS-CoV-2-specific T cell responses during infection, and both early CD4 T cell and CD8 T cell responses correlated with reduced upper respiratory tract SARS-CoV-2 viral RNA, independent of neutralizing antibody titers. Overall, our findings indicate a distinct protective role for SARS-CoV-2-specific T cells during acute COVID-19.
ABSTRACT
Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to elicit immune responses against the conserved fusion peptide. Antibody specificities and GC responses were tracked longitudinally using electron microscopy polyclonal epitope mapping (EMPEM) and lymph node fine-needle aspirates, respectively. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.
ABSTRACT
BACKGROUND: Monkeypox virus has recently infected more than 88 000 people, raising concerns about our preparedness against this emerging viral pathogen. Licensed and approved for mpox, the JYNNEOS vaccine has fewer side-effects than previous smallpox vaccines and has shown immunogenicity against monkeypox in animal models. This study aims to elucidate human immune responses to JYNNEOS vaccination compared with mpox-induced immunity. METHODS: Peripheral blood mononuclear cells and sera were obtained from ten individuals vaccinated with one or two doses of JYNNEOS and six individuals diagnosed with monkeypox virus infection. Samples were obtained from seven individuals before vaccination to serve as a baseline. We examined the polyclonal serum (ELISA) and single B-cell (heavy chain gene and transcriptome data) antibody repertoires and T-cell responses (activation-induced marker and intracellular cytokine staining assays) induced by the JYNNEOS vaccine versus monkeypox virus infection. FINDINGS: All participants were men between the ages of 21 and 60 years, except for one woman in the group of mpox-convalescent individuals, and none had previous orthopoxvirus exposure. All mpox cases were mild. Vaccinee samples were collected 6-33 days after the first dose and 5-40 days after the second dose. Mpox-convalescent samples were collected 20-102 days after infection. In vaccine recipients, gene-level plasmablast and antibody responses were negligible and sera displayed moderate binding to recombinant orthopoxviral proteins (A29L, A35R, E8L, A30L, A27L, A33R, B18R, and L1R) and native proteins from the 2022 monkeypox outbreak strain. By contrast, recent monkeypox virus infection (within 20-102 days) induced robust serum antibody responses to monkeypox virus proteins and to native monkeypox virus proteins from a viral isolate obtained during the 2022 outbreak. JYNNEOS vaccine recipients presented robust orthopoxviral CD4+ and CD8+ T-cell responses. INTERPRETATION: Infection with monkeypox virus resulted in robust B-cell and T-cell responses, whereas immunisation with JYNNEOS elicited more robust T-cell responses. These data can help to inform vaccine design and policies for preventing mpox in humans. FUNDING: National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), and Icahn School of Medicine.
Subject(s)
Mpox (monkeypox) , Smallpox Vaccine , Vaccines , United States , Animals , Male , Female , Humans , Young Adult , Adult , Middle Aged , Mpox (monkeypox)/prevention & control , Leukocytes, Mononuclear , Vaccination , Monkeypox virusABSTRACT
After resolution of infection, T cells differentiate into long-lived memory cells that recirculate through secondary lymphoid organs or establish residence in tissues. In contrast to CD8+ tissue-resident memory T cells (TRM), the developmental origins and transcriptional regulation of CD4+ TRM remain largely undefined. Here, we investigated the phenotypic, functional, and transcriptional profiles of CD4+ TRM in the small intestine (SI) responding to acute viral infection, revealing a shared gene expression program and chromatin accessibility profile with circulating TH1 and the progressive acquisition of a mature TRM program. Single-cell RNA sequencing identified heterogeneity among established CD4+ TRM, which were predominantly located in the lamina propria, and revealed a population of cells that coexpressed both effector- and memory-associated genes, including the transcriptional regulators Blimp1, Id2, and Bcl6. TH1-associated Blimp1 and Id2 and TFH-associated Bcl6 were required for early TRM formation and development of a mature TRM population in the SI. These results demonstrate a developmental relationship between TH1 effector cells and the establishment of early TRM, as well as highlighted differences in CD4+ versus CD8+ TRM populations, providing insights into the mechanisms underlying the origins, differentiation, and persistence of CD4+ TRM in response to viral infection.
Subject(s)
Immunologic Memory , Virus Diseases , Humans , CD4-Positive T-Lymphocytes , Cell Differentiation , Gene ExpressionABSTRACT
Although the importance of genome organization for transcriptional regulation of cell-fate decisions and function is clear, the changes in chromatin architecture and how these impact effector and memory CD8+ T cell differentiation remain unknown. Using Hi-C, we studied how genome configuration is integrated with CD8+ T cell differentiation during infection and investigated the role of CTCF, a key chromatin remodeler, in modulating CD8+ T cell fates through CTCF knockdown approaches and perturbation of specific CTCF-binding sites. We observed subset-specific changes in chromatin organization and CTCF binding and revealed that weak-affinity CTCF binding promotes terminal differentiation of CD8+ T cells through the regulation of transcriptional programs. Further, patients with de novo CTCF mutations had reduced expression of the terminal-effector genes in peripheral blood lymphocytes. Therefore, in addition to establishing genome architecture, CTCF regulates effector CD8+ T cell heterogeneity through altering interactions that regulate the transcription factor landscape and transcriptome.
Subject(s)
Chromatin , Repressor Proteins , Humans , Binding Sites , CCCTC-Binding Factor/metabolism , CD8-Positive T-Lymphocytes/metabolism , DNA/metabolism , Protein Binding , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
CD8 + T cells with stem cell-like properties (T SCM ) sustain adaptive immunity to intracellular pathogens and tumors. However, the developmental origins and chromatin regulatory factors (CRFs) that establish their differentiation are unclear. Using an RNA interference screen of all CRFs we discovered the histone methylase Mll1 was required during T cell receptor (TCR) stimulation for development of a T SCM precursor state and mature memory (T MEM ) cells, but not short-lived or transitory effector cell-like states, in response to viral infections and tumors. Mll1 was essential for widespread de novo deposition of histone H3 lysine 4 trimethylation (H3K4me3) upon TCR stimulation, which accounted for 70% of all activation-induced sites in mature T MEM cells. Mll1 promoted both H3K4me3 deposition and reduced TCR-induced Pol II pausing at genes whose single-cell transcriptional dynamics explained trajectories into nascent T SCM precursor states during viral infection. Our results suggest Mll1-dependent control of Pol II elongation and H3K4me3 establishes and maintains differentiation of CD8 + T SCM cell states.
ABSTRACT
T follicular helper (TFH) cells are essential for developing protective Ab responses following vaccination. Greater understanding of the genetic program leading to TFH differentiation is needed. Chromatin modifications are central in the control of gene expression. However, detailed knowledge of how chromatin regulators (CRs) regulate differentiation of TFH cells is limited. We screened a large short hairpin RNA library targeting all known CRs in mice and identified the histone methyltransferase mixed lineage leukemia 1 (Mll1) as a positive regulator of TFH differentiation. Loss of Mll1 expression reduced formation of TFH cells following acute viral infection or protein immunization. In addition, expression of the TFH lineage-defining transcription factor Bcl6 was reduced in the absence of Mll1. Transcriptomics analysis identified Lef1 and Tcf7 as genes dependent on Mll1 for their expression, which provides one mechanism for the regulation of TFH differentiation by Mll1. Taken together, CRs such as Mll1 substantially influence TFH differentiation.
Subject(s)
Chromatin , T Follicular Helper Cells , Animals , Mice , Cell Differentiation , Chromatin/metabolism , Gene Expression Regulation , Proto-Oncogene Proteins c-bcl-6/metabolism , T Follicular Helper Cells/metabolism , T-Lymphocytes, Helper-InducerABSTRACT
Communication between CD4 T cells and cognate B cells is key for the former to fully mature into germinal center-T follicular helper (GC-TFH) cells and for the latter to mount a CD4 T cell-dependent humoral immune response. Although this interaction occurs in a B:T synapse-dependent manner, how CD4 T cells transcriptionally regulate B:T synapse formation remains largely unknown. Here, we report that Mef2d, an isoform of the myocyte enhancer factor 2 (Mef2) transcription factor family, is a critical regulator of this process. In CD4 T cells, Mef2d negatively regulates expression of Sh2d1a, which encodes SLAM-associated protein (SAP), a critical regulator of B:T synapses. We found that Mef2d regulates Sh2d1a expression via DNA binding-dependent transcriptional repression, inhibiting SAP-dependent B:T synapse formation and preventing antigen-specific CD4 T cells from differentiating into GC-TFH cells. Mef2d also impeded IL-21 production by CD4 T cells, an important B cell help signaling molecule, via direct repression of the Il21 gene. In contrast, CD4 T cell-specific disruption of Mef2d led to a substantial increase in GC-TFH differentiation in response to protein immunization, concurrent with enhanced SAP expression. MEF2D mRNA expression inversely correlates with human systemic lupus erythematosus (SLE) patient autoimmune parameters, including circulating TFH-like cell frequencies, autoantibodies, and SLEDAI scores. These findings highlight Mef2d as a pivotal rheostat in CD4 T cells for controlling GC formation and antibody production by B cells.
Subject(s)
Immunity, Humoral , T-Lymphocytes, Helper-Inducer , Humans , Transcription Factors/metabolism , Cell Differentiation , MEF2 Transcription Factors/genetics , MEF2 Transcription Factors/metabolismABSTRACT
Background: Mpox (formerly known as monkeypox) outbreaks outside endemic areas peaked in July 2022, infecting > 85,000 people and raising concerns about our preparedness against this emerging viral pathogen. Licensed and approved for mpox, the JYNNEOS vaccine has fewer side effects than previous smallpox vaccines and demonstrated efficacy against mpox infection in humans. Comparing JYNNEOS vaccine- and mpox-induced immunity is imperative to evaluate JYNNEOS' immunogenicity and inform vaccine administration and design. Methods: We examined the polyclonal serum (ELISA) and single B cell (heavy chain gene and transcriptome data) antibody repertoires and T cells (AIM and ICS assays) induced by the JYNNEOS vaccine as well as mpox infection. Findings: Gene-level plasmablast and antibody responses were negligible and JYNNEOS vaccinee sera displayed minimal binding to recombinant mpox proteins and native proteins from the 2022 outbreak strain. In contrast, recent mpox infection (within 20-102 days) induced robust serum antibody responses to A29L, A35R, A33R, B18R, and A30L, and to native mpox proteins, compared to vaccinees. JYNNEOS vaccine recipients presented comparable CD4 and CD8 T cell responses against orthopox peptides to those observed after mpox infection. Interpretation: JYNNEOS immunization does not elicit a robust B cell response, and its immunogenicity may be mediated by T cells. Funding: Research reported in this publication was supported, in part, by the National Cancer Institute of the National Institutes of Health under Award Number U54CA267776, U19AI168631(VS), as well as institutional funds from the Icahn School of Medicine.