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bioRxiv2021

Synthetic Antigen Presenting cells reveal the diversity and functional specialization of extracellular vesicles composing the fourth signal of T cell immunological synapses.

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/445691v3_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@100f3aorg.highwire.dtl.DTLVardef@57adfforg.highwire.dtl.DTLVardef@6052fcorg.highwire.dtl.DTLVardef@1e85c21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles. C_LIO_LIBSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV. C_LIO_LItSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs. C_LI

immunology↗

bioRxiv2021

Multi-layered transcriptomic analyses reveal an immunological overlap between COVID-19 and hemophagocytic lymphohistiocytosis associated with disease severity

Severe COVID-19 patients present a clinical and laboratory overlap with other hyperinflammatory conditions such as hemophagocytic lymphohistiocytosis (HLH). However, the underlying mechanisms of these conditions remain to be explored. Here, we investigated the transcriptome of 1596 individuals, including patients with COVID-19 in comparison to healthy controls, other acute inflammatory states (HLH, multisystem inflammatory syndrome in children [MIS-C], Kawasaky disease [KD]), and different respiratory infections (seasonal coronavirus, influenza, bacterial pneumonia). We observed that COVID-19 and HLH share immunological pathways (cytokine/chemokine signaling and neutrophil-mediated immune responses), including gene signatures that stratify COVID-19 patients admitted to the intensive care unit (ICU) and COVID-19_nonICU patients. Of note, among the common differentially expressed genes (DEG), there is a cluster of neutrophil-associated genes that reflects a generalized hyperinflamatory state since it is also dysregulated in patients with KD and bacterial pneumonia. These genes are dysregulated at protein level across several COVID-19 studies and form an interconnected network with differentially expressed plasma proteins that point to neutrophil hyperactivation in COVID-19 patients admitted to the intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils. Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention.

immunology↗

bioRxiv2021

Analysis of centrosomal area actin reorganization and centrosome polarization upon lymphocyte activation at the immunological synapse

T cell receptor (TCR) and B cell receptor (BCR) stimulation of T and B lymphocytes, by antigen presented on an antigen-presenting cell (APC) induces the formation of the immunological synapse (IS). IS formation is associated with an initial increase in cortical filamentous actin (F-actin) at the IS, followed by a decrease in F-actin density at the central region of the IS, which contains the secretory domain. This is followed by the convergence of secretion vesicles towards the centrosome, and the polarization of the centrosome to the IS. These reversible, cortical actin cytoskeleton reorganization processes occur during lytic granule secretion in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, proteolytic granules secretion in B lymphocytes and during cytokine-containing vesicle secretion in T-helper (Th) lymphocytes. In addition, several findings obtained in T and B lymphocytes forming IS show that actin cytoskeleton reorganization also occurs at the centrosomal area. F-actin reduction at the centrosomal area appears to be associated with centrosome polarization. In this chapter we deal with the analysis of centrosomal area F-actin reorganization, as well as the centrosome polarization analysis towards the IS.

immunology↗

bioRxiv2021

Clinical and immunological signatures of severe COVID-19 in previously healthy patients with clonal hematopoiesis

Identifying additional risk factors for COVID-19 severity in numerous previously healthy patients without canonical clinical risk factors remains challenging. In this study, we investigate whether clonal hematopoiesis of indeterminate potential (CHIP), a common aging-related process that predisposes various inflammatory responses, may exert COVID-19 severity. We examine the clinical impact of CHIP in 143 laboratory-confirmed COVID-19 patients. Both stratified analyses and logistic regression including the interaction between canonical risk factors and CHIP show that CHIP is an independent risk factor for severe COVID-19, especially in previously healthy patients. Analyses of 60,310 single-cell immune transcriptome profiles identify distinct immunological signatures for CHIP (+) severe COVID-19 patients, particularly in classical monocytes, with a marked increase in pro-inflammatory cytokine responses and potent IFN-{gamma} mediated hyperinflammation signature. We further demonstrate that the enhanced expression of CHIP (+) specific IFN-{gamma} response genes is attributed to the CHIP mutation-dependent epigenetic reprogramming of poised or bivalent cis-regulatory elements. Our results highlight a unique immunopathogenic mechanism of CHIP in the progression of severe COVID-19, which could be extended to elucidate how CHIP contributes to a variety of human infectious diseases.

immunology↗

bioRxiv2022

Novel neutrophil subsets associated with sepsis, vascular dysfunction and metabolic alterations identified using systems immunology

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Despite continued efforts to understand the pathophysiology of sepsis, no effective therapies are currently available. While singular components of the aberrant immune response have been investigated, comprehensive studies linking different data layers are lacking. Using an integrated systems immunology approach, we evaluated neutrophil phenotypes and concomitant changes in cytokines and metabolites in sepsis patients. Our findings identify novel differentially expressed immature neutrophil subsets in sepsis patients. These and other subsets correlate with various proteins, metabolites, and lipids, including pentraxin-3, angiopoietin-2 and lysophosphatidylcholines, in sepsis patients. These results enabled the construction of a predictive model based on weighted multi-omics linear regression analysis for patient classification. These findings could help inform earlier patient stratification and treatment options, as well as facilitate future mechanistic studies targeting the trifecta of surface marker expression, cytokines, and metabolites.

immunology↗

bioRxiv2022

Immunological findings in a group of individuals who were non-responders to standard two-dose SARS-CoV-2 vaccines

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic. The virus has infected more than 505 million people and caused more than 6 million deaths. However, data on non-responders to SARS-CoV-2 vaccines in the general population are limited. The objective of the study is to comprehensively compare the immunological characteristics of non-responders to SARS-CoV-2 vaccines in the 18-59 years with that in the 60 years and older using internationally recognized cutoff values. Participants included 627 individuals who received physical examinations and volunteered to participate in COVID-19 vaccination from the general population. The main outcome was an effective seroconversion characterized by anti-SARS-CoV-2 spike IgG level of at least 4-fold increase from baseline. Profiling of naive immune cells was analyzed prior to vaccination to demonstrate baseline immunity. Outcomes of effective seroconversion in the 18-59 years with that in the 60 years and older were compared. The quantitative level of the anti-spike IgG was significantly lower in the 60 years and older and in men among the 18-59 years. There were 7.5% of non-responders among the 18-59 years and 11.7% of non-responders in the 60 years and older using the 4-fold increase parameter. The effective seroconversion rate was significantly related to the level of certain immune cells before vaccination, such as CD4 cells, CD8 cells and B cells and the age. An individual with a titer of anti-SARS-CoV-2 spike IgG that is below 50 BAU/mL might be considered a non-responder between 14-90 days after the last vaccine dose. Booster vaccination or additional protective measures should be recommended for non-responders as soon as possible to reduce disease severity and mortality.

immunology↗

bioRxiv2022

Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida

The liver is a multitasking organ with essential functions for vertebrate health spanning metabolism and immunity. In contrast to mammals, our understanding of liver cellular heterogeneity and its role in regulating immunological status remains poorly defined in fishes. Addressing this knowledge gap, we generated a transcriptomic atlas of 47,432 nuclei isolated from the liver of Atlantic salmon (Salmo salar L.) contrasting control fish with those challenged with a pathogenic strain of Aeromonas salmonicida, a problematic bacterial pathogen in global aquaculture. We identified the major liver cell types and their sub-populations, revealing poor conservation of many hepatic cell marker genes utilized in mammals, while identifying novel heterogeneity within the hepatocyte, lymphoid, and myeloid lineages. This included polyploid hepatocytes, multiple T cell populations including {gamma}{delta} T cells, and candidate populations of monocytes/macrophages/dendritic cells. A dominant hepatocyte population radically remodeled its transcriptome following infection to activate the acute phase response and other defense functions, while repressing routine functions such as metabolism. These defense-specialized hepatocytes showed strong activation of genes controlling protein synthesis and secretion, presumably to support the release of acute phase proteins into circulation. The infection response further involved up-regulation of numerous genes in an immune-cell specific manner, reflecting functions in pathogen recognition and killing, antigen presentation, phagocytosis, regulation of inflammation, B cell differentiation and T cell activation. Overall, this study greatly enhances our understanding of the multifaceted role played by liver cells in immune defense and metabolic remodeling following infection and provides many novel cell-specific marker genes to empower future studies of this organ in fishes.

immunology↗

bioRxiv2022

Global and cell type-specific immunological hallmarks of severe dengue progression

Severe dengue (SD) is a major cause of morbidity and mortality impacting approximately 5 million of the 400 million people infected with dengue virus (DENV) annually. To define DENV target cells and immunological hallmarks of SD progression in childrens blood, we integrated virus-inclusive single cell RNA-Seq 2 (viscRNA-Seq 2) with functional assays. Beyond myeloid cells, in natural infection, B cells harbor replicating DENV capable of infecting permissive cells. Alterations in cell type abundance, gene and protein expression and secretion, and cell-cell communications point towards increased migration and inflammation in SD progressors (SDp). Concurrently, antigen presenting cells from SDp demonstrate intact uptake, yet impaired interferon responses and antigen presentation, in part DENV-modulated. Increased activation, regulation, and exhaustion of effector responses and expansion of HLA-DR-expressing, possibly compensatory, adaptive-like NK cells also characterize SDp. These findings reveal DENV target cells in the human blood and provide insight into SD pathogenesis beyond antibody-mediated enhancement.

immunology↗

bioRxiv2022

Cytokine-expression patterns reveal coordinated immunological programs associated with persistent MRSA bacteremia

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a common, life-threatening infection that imposes up to 30% mortality even when appropriate therapy is used. Despite in vitro efficacy, antibiotics often fail to resolve the infection in vivo, resulting in persistent MRSA bacteremia. Recently, several genetic, epigenetic, and proteomic correlates of persistent outcomes have been identified. However, the extent to which single variables or composite patterns operate as independent predictors of outcome or reflect shared underlying mechanisms of persistence is unknown. To explore this question, we employed a tensor-based integration of host transcriptional and proteomic data across a well-characterized cohort of patients with persistent and resolving MRSA bacteremia outcomes. Tensor-based data integration yielded high correlative accuracy with persistence and revealed immunologic signatures shared across both the transcriptomic and proteomic datasets. We find that elevated proliferation of mature granulocytes associates with resolving bacteremia outcomes. In contrast, patients with persistent bacteremia heterogeneously exhibit correlates of granulocyte dysfunction or immature granulocyte proliferation. Collectively, these results suggest that transcriptional and proteomic correlates of persistent versus resolving bacteremia outcomes are complex and may not be disclosed by conventional modeling. However, a tensor-based integration approach can help to reveal consensus molecular mechanisms in an interpretable manner. Significance StatementWhile antibacterial therapies effectively resolve MRSA in vitro, these treatments often fail to clear MRSA bacteremia in vivo, suggesting that host-pathogen interactions are essential to persistent MRSA bacteremia. Recent studies have identified genetic, transcriptomic, and proteomic determinants of MRSA persistence. These determinants independently, however, provide insufficient mechanistic insight and it is unclear if they indicate unique or overlapping persistence mechanisms. Here, we use tensor-based decomposition to jointly analyze cytokine and transcriptomic measurements from patients with MRSA bacteremia. Results indicate that persistence mechanisms integrated across biological modalities reflect diverging mechanisms of persistent bacteremia. Ultimately, these results may help to identify future therapeutic targets for treating persistent MRSA bacteremia.

immunology↗

bioRxiv2023

Social association predicts immunological similarity in rewilded mice

Environmental influences on immune phenotypes are well-documented, but our understanding of which elements of the environment affect immune systems, and how, remains vague. Behaviors, including socializing with others, are central to an individuals interaction with its environment. We tracked behavior of rewilded laboratory mice of three inbred strains in outdoor enclosures and examined contributions of behavior, including social associations, to immune phenotypes. We found that the more associated two individuals were, the more similar their immune phenotypes were. Social association was particularly predictive of similar memory T and B cell profiles and was more influential than sibling relationships or worm infection status. These results highlight the importance of social networks for immune phenotype and reveal important immunological correlates of social life.

immunology↗

bioRxiv2023

Single-cell RNA sequencing reveals HIF1A as a severity-sensitive immunological scar in circulating monocytes of convalescent comorbidity-free COVID-19 patients

COVID-19, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is characterized by a wide range of clinical symptoms and a poorly predictable disease course. Although in-depth transcriptomic investigations of peripheral blood samples from COVID-19 patients have been performed, the detailed molecular mechanisms underlying an asymptomatic, mild or severe disease course, particularly in patients without relevant comorbidities, remain poorly understood. While previous studies have mainly focused on the cellular and molecular dissection of ongoing COVID-19, we set out to characterize transcriptomic immune cell dysregulation at the single-cell level at different time points in patients without comorbidities after disease resolution to identify signatures of different disease severities in convalescence. With single-cell RNA sequencing we reveal a role for hypoxia-inducible factor 1-alpha (HIF1A) as a severity-sensitive long-term immunological scar in circulating monocytes of convalescent COVID-19 patients. Additionally, circulating complexes formed by monocytes with either T cells or NK cells represent a characteristic cellular marker in convalescent COVID-19 patients irrespective of their preceding symptom severity. Together, these results provide cellular and molecular correlates of recovery from COVID-19 and could help in immune monitoring and in the design of new treatment strategies.

immunology↗

bioRxiv2023

Immune modulation of innate and adaptive responses restores immune surveillance and establishes anti-tumor immunological memory

Current immunotherapies have proven effective in strengthening anti-tumor immune responses but constant opposing signals from tumor cells and surrounding microenvironment eventually lead to immune escape. We hypothesize that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system will provide a robust and long-term anti-tumor effect by creating immunological memory against the tumor. To achieve this, we developed CARG-2020, a virus-like-vesicle (VLV). It is a genetically modified and self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three transgenes: 1) the pleiotropic antitumor cytokine IL-12 in which the subunits (p35 and p40) are tethered together; 2) the extracellular domain (ECD) of the pro-tumor IL-17RA, which can serve as a dominant negative antagonist; and 3) shRNA for PD-L1. Using a mouse model of ovarian cancer, we demonstrate the oncolytic effect and immune modulatory capacities of CARG-2020. By enhancing IL-12 and blocking IL-17 and PD-L1, CARG-2020 successfully reactivates immune surveillance by promoting M1 instead of M2 macrophage differentiation, inhibiting MDSC expansion, and establishing a potent CD8+ T cell mediated anti-tumoral response. Furthermore, we demonstrate that this therapeutic approach provides tumor-specific and long-term protection preventing the establishment of new tumors. Our results provide rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance the anti-tumor response and to prevent recurrence.

immunology↗

bioRxiv2023

Fine needle aspiration of human lymph nodes reveals cell populations and soluble interactors pivotal to immunological priming

Lymph node (LN) fine needle aspiration (LN FNA) represents a powerful technique for minimally invasive sampling of human lymph nodes in vivo and has been used to good effect to directly study aspects of the human germinal center response. However, systematic deep phenotyping of the cellular populations and cell-free proteins recovered by LN FNA has not been performed. Thus, we studied human cervical LN FNAs as a proof-of-concept and used single-cell RNA-sequencing and proteomic analysis to benchmark this compartment, define the purity of LN FNA material, and facilitate future studies into this immunologically pivotal environment. Our data provide evidence that LN FNAs contain bone fide LN-resident innate immune populations, with minimal contamination of cells or proteins from blood. Examination of these populations reveals unique biology not predictable from equivalent blood-derived populations. LN FNA supernatants represent a specific source of lymph- and lymph node-derived proteins, and can, in combination with transcriptomic approaches, identify likely receptor-ligand interactions. This study provides the first description of the types and abundance of immune cell populations and cell-free proteins that can be efficiently studied by LN FNA. These findings are of broad utility for understanding LN physiology both in health and disease, including infectious or autoimmune perturbations, and in the case of cervical nodes, neuroscience.

immunology↗

bioRxiv2023

A Blood Immunological Signature of Subclinical Coronary Artery Atherosclerosis in People Living with HIV-1 Receiving Antiretroviral Therapy

Cardiovascular disease (CVD) remains an important co-morbidity in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Our previous studies performed on the Canadian HIV/Aging Cohort Study (CHACS) (>40 years-old; Framingham Risk Score (FRS) >5%), revealed a 2-3-fold increase in non-calcified coronary artery atherosclerosis (CAA) plaque burden, measured by Computed tomography angiography scan (CTAScan) as total (TPV) and low attenuated plaque volume (LAPV) in ART-treated PLWH (HIV+) versus uninfected controls (HIV-). In an effort to identify novel correlates of subclinical CAA, markers of intestinal damage (sCD14, LBP, FABP2); cell trafficking/inflammation (CCL20, CX3CL1, MIF, CCL25); subsets of Th17-polarized and regulatory (Tregs) CD4+ T-cells, classical/intermediate/non-classical monocytes, and myeloid/plasmacytoid dendritic cells, were studied in relationship with HIV and TPV/LAPV status. The TPV detection/values coincided with higher plasma sCD14, FABP2, CCL20, MIF, CX3CL1 and triglyceride levels, lower Th17/Treg ratios, and classical monocyte expansion. Among HIV+, TPV+ versus TPV- exhibited lower Th17 frequencies, reduced Th17/Treg ratios, higher frequencies of non-classical CCR9lowHLADRhigh monocyte, and increased plasma fibrinogen levels. Finally, Th17/Treg ratios and non-classical CCR9lowHLADRhigh monocyte frequencies remained associated with TPV/LAPV after adjusting for FRS and HIV/ART duration in a logistic regression model. These findings point to Th17 paucity and non-classical monocyte abundance as novel immunological correlates of subclinical CAA that may fuel the CVD risk in ART-treated PLWH.

immunology↗

bioRxiv2024

Heterologous Prime-Boost with Immunologically Orthogonal Protein Nanoparticles for Peptide Immunofocusing

Protein nanoparticles are effective platforms for antigen presentation and targeting effector immune cells in vaccine development. Encapsulins are a class of protein-based microbial nanocompartments that self-assemble into icosahedral structures with external diameters ranging from 24 to 42 nm. Encapsulins from Mxyococcus xanthus were designed to package bacterial RNA when produced in E. coli and were shown to have immunogenic and self-adjuvanting properties enhanced by this RNA. We genetically incorporated a 20-mer peptide derived from a mutant strain of the SARS-CoV-2 receptor binding domain (RBD) into the encapsulin protomeric coat protein for presentation on the exterior surface of the particle. This immunogen elicited conformationally-relevant humoral responses to the SARS-CoV-2 RBD. Immunological recognition was enhanced when the same peptide was presented in a heterologous prime/boost vaccination strategy using the engineered encapsulin and a previously reported variant of the PP7 virus-like particle, leading to the development of a selective antibody response against a SARS-CoV-2 RBD point mutant. While generating epitope-focused antibody responses is an interplay between inherent vaccine properties and B/T cells, here we demonstrate the use of orthogonal nanoparticles to fine-tune the control of epitope focusing. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/581861v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@53407corg.highwire.dtl.DTLVardef@1ae8d0eorg.highwire.dtl.DTLVardef@aec021org.highwire.dtl.DTLVardef@c7e0ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Immunological correlates of protection mediated by a whole organism Cryptococcus neoformans vaccine deficient in chitosan

The global burden of infections due to the pathogenic fungus Cryptococcus is substantial in persons with low CD4+ T cell counts. Previously, we deleted three chitin deacetylase genes from C. neoformans to create a chitosan-deficient, avirulent strain, designated cda1{Delta}2{Delta}3{Delta} which, when used as a vaccine, protected mice from challenge with virulent C. neoformans strain KN99. Here, we explored the immunological basis for protection. Vaccine-mediated protection was maintained in mice lacking B cells or CD8+ T cells. In contrast, protection was lost in mice lacking /{beta} T cells or CD4+ T cells. Moreover, CD4+ T cells from vaccinated mice conferred protection upon adoptive transfer to naive mice. Importantly, while monoclonal antibody-mediated depletion of CD4+ T cells just prior to vaccination resulted in complete loss of protection, significant protection was retained in mice depleted of CD4+ T cells after vaccination, but prior to challenge. Vaccine-mediated protection was lost in mice genetically deficient in IFN{gamma}, TNF, or IL-23p19. A robust influx of leukocytes and IFN{gamma}- and TNF-expressing CD4+ T cells was seen in the lungs of vaccinated and challenged mice. Finally, a higher level of IFN{gamma} production by lung cells stimulated ex vivo correlated with lower fungal burden in the lungs. Thus, while B cells and CD8+ T cells are dispensable, IFN{gamma} and CD4+ T cells have overlapping roles in generating protective immunity prior to cda1{Delta}2{Delta}3{Delta} vaccination. However, once vaccinated, protection becomes less dependent on CD4+ T cells, suggesting a strategy for vaccinating HIV+ persons prior to loss of CD4+ T cells. ImportanceThe fungus Cryptococcus neoformans is responsible for >100,000 deaths annually, mostly in persons with impaired CD4+ T cell function such as AIDS. There are no approved human vaccines. We previously created a genetically engineered avirulent strain of C. neoformans, designated cda1{Delta}2{Delta}3{Delta}. When used as a vaccine, cda1{Delta}2{Delta}3{Delta} protects mice against a subsequent challenge with a virulent C. neoformans strain. Here, we defined components of the immune system responsible for vaccine-mediated protection. We found that while B cells and CD8+ T cells were dispensible, protection was lost in mice genetically deficient in CD4+ T cells, and the cytokines IFN{gamma}, TNF, or IL-23. A robust influx of cytokine-producing CD4+ T cells was seen in the lungs of vaccinated mice following infection. Importantly, protection was retained in mice depleted of CD4+ T cells following vaccination, suggesting a strategy to protect persons who are at risk for future CD4+ T cell dysfunction.

immunology↗

bioRxiv2024

ImPaqT - A Golden Gate-based Immunological Toolkit for Zebrafish Transgenesis

Transgenic animals continue to play an essential role in many aspects of zebrafish research, including the development of disease models. The most widely used system for zebrafish transgenesis is the Tol2 transposon system. Here, we have developed ImPaqT (Immunological toolkit for PaqCI-based Golden Gate Assembly of Tol2 Transgenes), a new Tol2-based transgenesis system that utilizes Golden Gate assembly to facilitate the production of transgenic zebrafish lines. This system allows for rapid assembly of multiple fragments into a single transgene, facile swapping of individual sequences to generate new transgenes and an easy cloning workflow to incorporate new genetic elements into the existing kit. Within this toolkit framework, we have generated a number of reagents to enable gene expression within immune and non-immune cell types, an array of best-in-class fluorescent proteins to visualize cell populations and transgenes as well as tools to simplify genetic manipulation, purification and ablation of targeted cells. Unlike recombination-based systems, the Golden Gate approach is also expandable, allowing the incorporation of complex designs such as multi-fragment promoters within the established modular framework of ImPaqT. Here, we demonstrate the function of this new system by generating a number of novel transgenic immune reporter lines. While our toolkit is focused on the immune system as an emerging area of study within zebrafish research, the ImPaqT approach can be broadly adapted to the construction of almost any zebrafish transgene, offering new tools for the generation of transgenes within the zebrafish community.

immunology↗

bioRxiv2024

Immunologic and Biophysical Features of the BNT162b2 JN.1- and KP.2-Adapted COVID-19 Vaccines

Vaccines remain a vital public health tool to reduce the burden of COVID-19. COVID-19 vaccines that are more closely matched to circulating SARS-CoV-2 lineages elicit more potent and relevant immune responses that translate to improved real-world vaccine effectiveness. The rise in prevalence of the Omicron JN.1 lineage, and subsequent derivative sublineages such as KP.2 and KP.3, coincided with reduced neutralizing activity and effectiveness of Omicron XBB.1.5-adapted vaccines. Here, we characterized the biophysical and immunologic attributes of BNT162b2 JN.1- and KP.2-adapted mRNA vaccine-encoded spike (S) protein immunogens. Biophysical interrogations of S revealed the structural consequences of hallmark amino acid substitutions and a potential molecular mechanism of immune escape employed by JN.1 and KP.2. The vaccine candidates were evaluated for their immunogenicity when administered as fourth or fifth doses in BNT162b2-experienced mice or as a primary series in naive mice. In both vaccine-experienced and naive settings, JN.1- and KP.2-adapted vaccines conferred improved neutralizing responses over the BNT162b2 XBB.1.5 vaccine against a broad panel of emerging JN.1 sublineages, including the predominant KP.3.1.1 and emerging XEC lineages. Antigenic mapping of neutralizing responses indicated greater antigenic overlap of JN.1- and KP.2-adapted vaccine responses with currently circulating sublineages compared to an XBB.1.5-adapted vaccine. CD4+ and CD8+ T cell responses were generally conserved across all three vaccines. Together, the data support the selection of JN.1- or KP.2-adapted vaccines for the 2024-25 COVID-19 vaccine formula. ONE-SENTENCE SUMMARYThe Omicron JN.1- and KP.2-adapted BNT162b2 mRNA vaccines encoding prefusion S proteins elicit similar preclinical neutralizing antibody responses against circulating JN.1 sublineage pseudoviruses that are more potent than those elicited by past iterations of BNT162b2 licensed vaccines, thus demonstrating the importance of annual strain changes to the COVID-19 vaccine.

immunology↗