Innervation of nociceptor neurons in the spleen promotes germinal center responses and humoral immunity.

Peripheral sensory neurons widely innervate various tissues to continuously monitor and respond to environmental stimuli. Whether peripheral sensory neurons innervate the spleen and modulate splenic immune response remains poorly defined. Here, we demonstrate that nociceptive sensory nerve fibers extensively innervate the spleen along blood vessels and reach B cell zones. The spleen-innervating nociceptors predominantly originate from left T8-T13 dorsal root ganglia (DRGs), promoting the splenic germinal center (GC) response and humoral immunity. Nociceptors can be activated by antigen-induced accumulation of splenic prostaglandin E2 (PGE2) and then release calcitonin gene-related peptide (CGRP), which further promotes the splenic GC response at the early stage. Mechanistically, CGRP directly acts on B cells through its receptor CALCRL-RAMP1 via the cyclic AMP (cAMP) signaling pathway. Activating nociceptors by ingesting capsaicin enhances the splenic GC response and anti-influenza immunity. Collectively, our study establishes a specific DRG-spleen sensory neural connection that promotes humoral immunity, suggesting a promising approach for improving host defense by targeting the nociceptive nervous system.

Humoral and cellular immune memory to four COVID-19 vaccines.

Multiple COVID-19 vaccines, representing diverse vaccine platforms, successfully protect against symptomatic COVID-19 cases and deaths. Head-to-head comparisons of T cell, B cell, and antibody responses to diverse vaccines in humans are likely to be informative for understanding protective immunity against COVID-19, with particular interest in immune memory. Here, SARS-CoV-2-spike-specific immune responses to Moderna mRNA-1273, Pfizer/BioNTech BNT162b2, Janssen Ad26.COV2.S, and Novavax NVX-CoV2373 were examined longitudinally for 6 months 100% of individuals made memory CD4+ T cells, with cTfh and CD4-CTL highly represented after mRNA or NVX-CoV2373 vaccination. mRNA vaccines and Ad26.COV2.S induced comparable CD8+ T cell frequencies, though only detectable in 60-67% of subjects at 6 months. A differentiating feature of Ad26.COV2.S immunization was a high frequency of CXCR3+ memory B cells. mRNA vaccinees had substantial declines in antibodies, while memory T and B cells were comparatively stable. These results may also be relevant for insights against other pathogens.

Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2.

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants.

Virus-like particle (VLP) and live virus assays were used to investigate neutralizing immunity against Delta and Omicron SARS-CoV-2 variants in 259 samples from 128 vaccinated individuals. Following Delta breakthrough infection, titers against WT rose 57-fold and 3.1-fold compared with uninfected boosted and unboosted individuals, respectively, versus only a 5.8-fold increase and 3.1-fold decrease for Omicron breakthrough infection. Among immunocompetent, unboosted patients, Delta breakthrough infections induced 10.8-fold higher titers against WT compared with Omicron (p = 0.037). Decreased antibody responses in Omicron breakthrough infections relative to Delta were potentially related to a higher proportion of asymptomatic or mild breakthrough infections (55.0% versus 28.6%, respectively), which exhibited 12.3-fold lower titers against WT compared with moderate to severe infections (p = 0.020). Following either Delta or Omicron breakthrough infection, limited variant-specific cross-neutralizing immunity was observed. These results suggest that Omicron breakthrough infections are less immunogenic than Delta, thus providing reduced protection against reinfection or infection from future variants.

Low-dose Ad26.COV2.S protection against SARS-CoV-2 challenge in rhesus macaques.

We previously reported that a single immunization with an adenovirus serotype 26 (Ad26)-vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. To evaluate reduced doses of Ad26.COV2.S, 30 rhesus macaques were immunized once with 1 × 1011, 5 × 1010, 1.125 × 1010, or 2 × 109 viral particles (vp) Ad26.COV2.S or sham and were challenged with SARS-CoV-2. Vaccine doses as low as 2 × 109 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125 × 1010 vp were required for protection in nasal swabs. Activated memory B cells and binding or neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show enhancement of disease. These data demonstrate that a single immunization with relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques, although a higher vaccine dose may be required for protection in the upper respiratory tract.

Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19.

Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.

Identification of Near-Pan-neutralizing Antibodies against HIV-1 by Deconvolution of Plasma Humoral Responses.

Anti-HIV-1 envelope broadly neutralizing monoclonal antibodies (bNAbs) isolated from memory B cells may not fully represent HIV-1-neutralizing profiles measured in plasma. Accordingly, we characterized near-pan-neutralizing antibodies extracted directly from the plasma of two "elite neutralizers." Circulating anti-gp120 polyclonal antibodies were deconvoluted using proteomics to guide lineage analysis of bone marrow plasma cells. In both subjects, a single lineage of anti-CD4-binding site (CD4bs) antibodies explained the plasma-neutralizing activity. Importantly, members of these lineages potently neutralized 89%-100% of a multi-tier 117 pseudovirus panel, closely matching the specificity and breadth of the circulating antibodies. X-ray crystallographic analysis of one monoclonal, N49P7, suggested a unique ability to bypass the CD4bs Phe43 cavity, while reaching deep into highly conserved residues of Layer 3 of the gp120 inner domain, likely explaining its extreme potency and breadth. Further direct analyses of plasma anti-HIV-1 bNAbs should provide new insights for developing antibody-based antiviral agents and vaccines.

Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies.

Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.

Analysis of antibody binding specificities in twin and SNP-genotyped cohorts reveals that antiviral antibody epitope selection is a heritable trait.

Human immune responses to viral infections are highly variable, but the genetic factors that contribute to this variability are not well characterized. We used VirScan, a high-throughput epitope scanning technology, to analyze pan-viral antibody reactivity profiles of twins and SNP-genotyped individuals. Using these data, we determined the heritability and genomic loci associated with antibody epitope selection, response breadth, and control of Epstein-Barr virus (EBV) viral load. 107 EBV peptide reactivities were heritable and at least two Epstein-Barr nuclear antigen 2 (EBNA-2) reactivities were associated with variants in the MHC class II locus. We identified an EBV serosignature that predicted viral load in peripheral blood mononuclear cells and was associated with variants in the MHC class I locus. Our study illustrates the utility of epitope profiling to investigate the genetics of pathogen immunity, reports heritable features of the antibody response to viruses, and identifies specific HLA loci important for EBV epitope selection.

Antibodies from primary humoral responses modulate the recruitment of naive B cells during secondary responses.

Vaccines generate high-affinity antibodies by recruiting antigen-specific B cells to germinal centers (GCs), but the mechanisms governing the recruitment to GCs on secondary challenges remain unclear. Here, using preclinical SARS-CoV and HIV mouse models, we demonstrated that the antibodies elicited during primary humoral responses shaped the naive B cell recruitment to GCs during secondary exposures. The antibodies from primary responses could either enhance or, conversely, restrict the GC participation of naive B cells: broad-binding, low-affinity, and low-titer antibodies enhanced recruitment, whereas, by contrast, the high titers of high-affinity, mono-epitope-specific antibodies attenuated cognate naive B cell recruitment. Thus, the directionality and intensity of that effect was determined by antibody concentration, affinity, and epitope specificity. Circulating antibodies can, therefore, be important determinants of antigen immunogenicity. Future vaccines may need to overcome-or could, alternatively, leverage-the effects of circulating primary antibodies on subsequent naive B cell recruitment.

Phage display of environmental protein toxins and virulence factors reveals the prevalence, persistence, and genetics of antibody responses.

Microbial exposures are crucial environmental factors that impact healthspan by sculpting the immune system and microbiota. Antibody profiling via Phage ImmunoPrecipitation Sequencing (PhIP-Seq) provides a high-throughput, cost-effective approach for detecting exposure and response to microbial protein products. We designed and constructed a library of 95,601 56-amino acid peptide tiles spanning 14,430 proteins with "toxin" or "virulence factor" keyword annotations. We used PhIP-Seq to profile the antibodies of ∼1,000 individuals against this "ToxScan" library. In addition to enumerating immunodominant antibody epitopes, we studied the age-dependent stability of the ToxScan profile and used a genome-wide association study to find that the MHC-II locus modulates bacterial epitope selection. We detected previously described anti-flagellin antibody responses in a Crohn's disease cohort and identified an association between anti-flagellin antibodies and juvenile dermatomyositis. PhIP-Seq with the ToxScan library is thus an effective tool for studying the environmental determinants of health and disease at cohort scale.

Interleukin-21, acting beyond the immunological synapse, independently controls T follicular helper and germinal center B cells.

Germinal centers (GCs), transient structures within B cell follicles and central to affinity maturation, require the coordinated behavior of T and B cells. IL-21, a pleiotropic T cell-derived cytokine, is key to GC biology through incompletely understood mechanisms. By genetically restricting production and receipt of IL-21 in vivo, we reveal how its independent actions on T and B cells combine to regulate the GC. IL-21 established the magnitude of the GC B cell response by promoting CD4+ T cell expansion and differentiation in a dose-dependent manner and with paracrine activity. Within GC, IL-21 specifically promoted B cell centroblast identity and, when bioavailability was high, plasma cell differentiation. Critically, these actions may occur irrespective of cognate T-B interactions, making IL-21 a general promoter of growth as distinct to a mediator of affinity-driven selection via synaptic delivery. This promiscuous activity of IL-21 explains the consequences of IL-21 deficiency on antibody-based immunity.

Development of Tbet- and CD11c-expressing B cells in a viral infection requires T follicular helper cells outside of germinal centers.

Tbet+CD11c+ B cells arise during type 1 pathogen challenge, aging, and autoimmunity in mice and humans. Here, we examined the developmental requirements of this B cell subset. In acute infection, T follicular helper (Tfh) cells, but not Th1 cells, drove Tbet+CD11c+ B cell generation through proximal delivery of help. Tbet+CD11c+ B cells developed prior to germinal center (GC) formation, exhibiting phenotypic and transcriptional profiles distinct from GC B cells. Fate tracking revealed that most Tbet+CD11c+ B cells developed independently of GC entry and cell-intrinsic Bcl6 expression. Tbet+CD11c+ and GC B cells exhibited minimal repertoire overlap, indicating distinct developmental pathways. As the infection resolved, Tbet+CD11c+ B cells localized to the marginal zone where splenic retention depended on integrins LFA-1 and VLA-4, forming a competitive memory subset that contributed to antibody production and secondary GC seeding upon rechallenge. Therefore, Tbet+CD11c+ B cells comprise a GC-independent memory subset capable of rapid and robust recall responses.

Primary germinal center-resident T follicular helper cells are a physiologically distinct subset of CXCR5hiPD-1hi T follicular helper cells.

T follicular helper (Tfh) cells are defined by a Bcl6+CXCR5hiPD-1hi phenotype, but only a minor fraction of these reside in germinal centers (GCs). Here, we examined whether GC-resident and -nonresident Tfh cells share a common physiology and function. Fluorescently labeled, GC-resident Tfh cells in different mouse models were distinguished by low expression of CD90. CD90neg/lo GCTfh cells required antigen-specific, MHCII+ B cells to develop and stopped proliferating soon after differentiation. In contrast, nonresident, CD90hi Tfh (GCTfh-like) cells developed normally in the absence of MHCII+ B cells and proliferated continuously during primary responses. The TCR repertoires of both Tfh subsets overlapped initially but later diverged in association with dendritic cell-dependent proliferation of CD90hi GCTfh-like cells, suggestive of TCR-dependency seen also in TCR-transgenic adoptive transfer experiments. Furthermore, the transcriptomes of CD90neg/lo and CD90hi GCTfh-like cells were enriched in different functional pathways. Thus, GC-resident and nonresident Tfh cells have distinct developmental requirements and activities, implying distinct functions.

Mucosal plasma cells are required to protect the upper airway and brain from infection.

While blood antibodies mediate protective immunity in most organs, whether they protect nasal surfaces in the upper airway is unclear. Using multiple viral infection models in mice, we found that blood-borne antibodies could not defend the olfactory epithelium. Despite high serum antibody titers, pathogens infected nasal turbinates, and neurotropic microbes invaded the brain. Using passive antibody transfers and parabiosis, we identified a restrictive blood-endothelial barrier that excluded circulating antibodies from the olfactory mucosa. Plasma cell depletions demonstrated that plasma cells must reside within olfactory tissue to achieve sterilizing immunity. Antibody blockade and genetically deficient models revealed that this local immunity required CD4+ T cells and CXCR3. Many vaccine adjuvants failed to generate olfactory plasma cells, but mucosal immunizations established humoral protection of the olfactory surface. Our identification of a blood-olfactory barrier and the requirement for tissue-derived antibody has implications for vaccinology, respiratory and CNS pathogen transmission, and B cell fate decisions.

Secondary influenza challenge triggers resident memory B cell migration and rapid relocation to boost antibody secretion at infected sites.

Resident memory B (BRM) cells develop and persist in the lungs of influenza-infected mice and humans; however, their contribution to recall responses has not been defined. Here, we used two-photon microscopy to visualize BRM cells within the lungs of influenza -virus immune and reinfected mice. Prior to re-exposure, BRM cells were sparsely scattered throughout the tissue, displaying limited motility. Within 24 h of rechallenge, these cells increased their migratory capacity, localized to infected sites, and subsequently differentiated into plasma cells. Alveolar macrophages mediated this process, in part by inducing expression of chemokines CXCL9 and CXCL10 from infiltrating inflammatory cells. This led to the recruitment of chemokine receptor CXCR3-expressing BRM cells to infected regions and increased local antibody concentrations. Our study uncovers spatiotemporal mechanisms that regulate lung BRM cell reactivation and demonstrates their capacity to rapidly deliver antibodies in a highly localized manner to sites of viral replication.

A Fc engineering approach to define functional humoral correlates of immunity against Ebola virus.

Protective Ebola virus (EBOV) antibodies have neutralizing activity and induction of antibody constant domain (Fc)-mediated innate immune effector functions. Efforts to enhance Fc effector functionality often focus on maximizing antibody-dependent cellular cytotoxicity, yet distinct combinations of functions could be critical for antibody-mediated protection. As neutralizing antibodies have been cloned from EBOV disease survivors, we sought to identify survivor Fc effector profiles to help guide Fc optimization strategies. Survivors developed a range of functional antibody responses, and we therefore applied a rapid, high-throughput Fc engineering platform to define the most protective profiles. We generated a library of Fc variants with identical antigen-binding fragments (Fabs) from an EBOV neutralizing antibody. Fc variants with antibody-mediated complement deposition and moderate natural killer (NK) cell activity demonstrated complete protective activity in a stringent in vivo mouse model. Our findings highlight the importance of specific effector functions in antibody-mediated protection, and the experimental platform presents a generalizable resource for identifying correlates of immunity to guide therapeutic antibody design.

Indoleamine 2,3-dioxygenase 1 is essential for sustaining durable antibody responses.

Humoral immunity is essential for protection against pathogens, emphasized by the prevention of 2-3 million deaths worldwide annually by childhood immunizations. Long-term protective immunity is dependent on the continual production of neutralizing antibodies by the subset of long-lived plasma cells (LLPCs). LLPCs are not intrinsically long-lived, but require interaction with LLPC niche stromal cells for survival. However, it remains unclear which and how these interactions sustain LLPC survival and long-term humoral immunity. We now have found that the immunosuppressive enzyme indoleamine 2,3- dioxygenase 1 (IDO1) is required to sustain antibody responses and LLPC survival. Activation of IDO1 occurs upon the engagement of CD80/CD86 on the niche dendritic cells by CD28 on LLPC. Kynurenine, the product of IDO1 catabolism, activates the aryl hydrocarbon receptor in LLPC, reinforcing CD28 expression and survival signaling. These findings expand the immune function of IDO1 and uncover a novel pathway for sustaining LLPC survival and humoral immunity.

COVID-19 immune signatures reveal stable antiviral T cell function despite declining humoral responses.

Cellular and humoral immunity to SARS-CoV-2 is critical to control primary infection and correlates with severity of disease. The role of SARS-CoV-2-specific T cell immunity, its relationship to antibodies, and pre-existing immunity against endemic coronaviruses (huCoV), which has been hypothesized to be protective, were investigated in 82 healthy donors (HDs), 204 recovered (RCs), and 92 active COVID-19 patients (ACs). ACs had high amounts of anti-SARS-CoV-2 nucleocapsid and spike IgG but lymphopenia and overall reduced antiviral T cell responses due to the inflammatory milieu, expression of inhibitory molecules (PD-1, Tim-3) as well as effector caspase-3, -7, and -8 activity in T cells. SARS-CoV-2-specific T cell immunity conferred by polyfunctional, mainly interferon-γ-secreting CD4+ T cells remained stable throughout convalescence, whereas humoral responses declined. Immune responses toward huCoV in RCs with mild disease and strong cellular SARS-CoV-2 T cell reactivity imply a protective role of pre-existing immunity against huCoV.

Limited access to antigen drives generation of early B cell memory while restraining the plasmablast response.

Cell fate decisions during early B cell activation determine the outcome of responses to pathogens and vaccines. We examined the early B cell response to T-dependent antigen in mice by single-cell RNA sequencing. Early after immunization, a homogeneous population of activated precursors (APs) gave rise to a transient wave of plasmablasts (PBs), followed a day later by the emergence of germinal center B cells (GCBCs). Most APs rapidly exited the cell cycle, giving rise to non-GC-derived early memory B cells (eMBCs) that retained an AP-like transcriptional profile. Rapid decline of antigen availability controlled these events; provision of excess antigen precluded cell cycle exit and induced a new wave of PBs. Fate mapping revealed a prominent contribution of eMBCs to the MBC pool. Quiescent cells with an MBC phenotype dominated the early response to immunization in primates. A reservoir of APs/eMBCs may enable rapid readjustment of the immune response when failure to contain a threat is manifested by increased antigen availability.