Immunity In Mouse Model Research Articles

Sensory neurons regulate stimulus-dependent humoral immunity in mouse models of bacterial infection and asthma

Sensory neurons sense pathogenic infiltration to drive innate immune responses, but their role in humoral immunity is unclear. Here, using mouse models of Streptococcus pneumoniae infection and Alternaria alternata asthma, we show that sensory neurons are required for B cell recruitment and antibody production. In response to S. pneumoniae, sensory neuron depletion increases bacterial burden and reduces B cell numbers, IgG release, and neutrophil stimulation. Meanwhile, during A. alternata-induced airway inflammation, sensory neuron depletion decreases B cell population sizes, IgE levels, and asthmatic characteristics. Mechanistically, during bacterial infection, sensory neurons preferentially release vasoactive intestinal polypeptide (VIP). In response to asthma, sensory neurons release substance P. Administration of VIP into sensory neuron-depleted mice suppresses bacterial burden, while VIPR1 deficiency increases infection. Similarly, exogenous substance P delivery aggravates asthma in sensory neuron-depleted mice, while substance P deficiency ameliorates asthma. Our data, thus demonstrate that sensory neurons release select neuropeptides which target B cells dependent on the immunogen.

Enhanced Immune Responses in Mice by Combining the Mpox Virus B6R-Protein and Aluminum Hydroxide-CpG Vaccine Adjuvants.

Novel adjuvants and innovative combinations of adjuvants (Adjuvant Systems) have facilitated the development of enhanced and new vaccines against re-emerging and challenging pathogenic microorganisms. Nonetheless, the efficacy of adjuvants is influenced by various factors, and the same adjuvant may generate entirely different immune responses when paired with different antigens. Herein, we combined the MPXV-B6R antigen with BC02, a novel adjuvant with proprietary technology, to assess its capability to induce both cellular and humoral immunity in mouse models. Mice received two intramuscular injections of B6R-BC02, which resulted in the production of MPXV-specific IgG, IgG1, and IgG2a antibodies. Additionally, it elicited strong MPXV-specific Th1-oriented cellular immunity and persistent effector memory B-cell responses. The advantages of BC02 were further validated, including rapid initiation of the immune response, robust recall memory, and sustained immune response induction. Although the potential of immunized mice to produce serum-neutralizing antibodies against the vaccinia virus requires further improvement, the exceptional performance of BC02 as an adjuvant for the MPXV-B6R antigen has been consistently demonstrated.

Anti-CRMP2 antibody induces anxiety-like behavior and increases pyramidal neuron excitability in mice

BackgroundWe have previously identified auto-antibody (Ab) to collapsin response mediator protein 2 (CRMP2) in patients with encephalitis. The present study aims to evaluate the pathogenic effects of anti-CRMP2 Ab. MethodsRecombinant CRMP2 protein was injected subcutaneously into mice to establish an active immune mouse model with anti-CRMP2 Ab. Behavioral assessments, histopathological staining, and electrophysiological testing were performed to identify any pathogenic changes. ResultsThe mice exhibited signs of impaired motor coordination four weeks post-immunization of CRMP2 protein. Moreover, CRMP2 immunized mice for eight weeks showed anxiety-like behaviors indicating by tests of open field and the elevated plus maze. After incubating the CA1 region of hippocampal brain section with the sera from CRMP2 immunized mice, the whole-cell path-clamp recordings showed increased excitability of pyramidal neurons. However, no obvious inflammation and infiltration of immune cells were observed by histopathological analysis. Western blot showed that the phosphorylation levels of CRMP2-Thr514 and -Ser522 were not affected. ConclusionIn an active immunization model with CRMP2 protein, impaired coordination and anxiety-like behaviors were observed. Also, anti-CRMP2 Abs containing sera heightened the excitability of hippocampal pyramidal neurons in vitro, which imply the pathogenic effects of anti-CRMP2 Ab.

Distal Immunization and Systemic Cytokines Establish a Transient Immune Alert State in the Intestine.

Conventionally, immune responses are studied in the context of inflamed tissues and their corresponding draining lymph nodes (LNs). However, little is known about the effects of systemic inflammatory signals generated during local inflammation on distal tissues and nondraining LNs. Using a mouse model of cutaneous immunization, we found that systemic inflammatory stimuli triggered a rapid and selective distal response in the small intestine and the mesenteric LN (mesLN). This consisted of increased permeability of intestinal blood vessels and lymphatic drainage of bloodborne solutes into the mesLN, enhanced activation and migration of intestinal dendritic cells, as well as amplified T cell responses in the mesLNs to systemic but not orally derived Ags. Mechanistically, we found that the small intestine endothelial cells preferentially expressed molecules involved in TNF-α signaling and that TNF-α blockade markedly diminished distal intestinal responses to cutaneous immunization. Together, these findings reveal that the intestinal immune system is rapidly and selectively activated in response to inflammatory cues regardless of their origin, thus identifying an additional layer of defense and enhanced surveillance of a key barrier organ at constant risk of pathogen encounter.

Abstract 5301: Tumor-targeted activation of CD137 using Bicycles: New insights into mechanism of action and discovery of BT7455, a clinical candidate for the treatment of EphA2-expressing cancers

Abstract Introduction: Clinical studies in cancer patients have validated CD137 agonism as an activator of the immune system to enable tumor rejection. We have demonstrated that small, chemically synthetic bicyclic peptides can drive tumor-localized agonism of CD137 and anti-tumor immunity in mouse models. Here, we report the next stage of our work - delving into the mechanism of action of these novel agents and extending our program to serve patients whose tumors express EphA2. Experimental Procedures: MultiOmyx™ hyperplexed immunofluorescence assay was used to evaluate the expression of CD137 and EphA2 in head and neck squamous cell carcinoma samples. Human PBMC/tumor cell co-culture assays were used to assess CD137 Bicycle TICA™ in vitro bioactivity and syngeneic mouse tumor models were used to evaluate CD137 Bicycle TICA anti-tumor activity as mono- or combination therapies. Pharmacodynamic activity was evaluated by transcriptional profiling using NanoString assays or single cell RNA sequencing. Summary of the Data: Studies utilizing a syngeneic mouse model and deep RNA sequencing of tumors from CD137 Bicycle TICA-treated mice implicated intratumoral dendritic cells in addition to T cells as early responders to localized CD137 agonism and potential contributors to the anti-tumor response. Furthermore, a mouse efficacy model demonstrated synergy with checkpoint inhibitor therapy. Multiplex imaging revealed that EphA2 and CD137 are co-expressed in human tumors of high unmet medical need and therefore, using our highly modular Bicycle platform, we discovered BT7455, a Bicycle TICA that engages EphA2 and CD137 with high affinity, resulting in potent EphA2-dependent activity in vitro and robust anti-tumor activity in vivo with intermittent dosing in mouse models. Gene expression profiling of tumors revealed that BT7455 led to increased production of cytokines and chemokines known to drive T cell infiltration, the extent of which differentiated it from both a checkpoint inhibitor and an anti-CD137 monoclonal antibody agonist. BT7455 was well tolerated in preclinical species and liver function tests indicated no evidence of hepatic toxicity. Statement of Conclusions: In summary, we have identified EphA2 as a promising target to pair with a CD137 agonist. In addition to CD8+ T cells, tumor-resident dendritic cells are a likely contributor to anti-tumor immunity following treatment with Bicycle® TICAs, supporting utility for Bicycle® tumor-targeted CD137 agonists in solid tumors beyond those that are highly T cell-infiltrated. We have advanced a clinical development candidate, BT7455, to realize this potential for patients with EphA2-expressing cancers. Citation Format: Johanna Lahdenranta, Kristen Hurov, Heather Cohen, Lia Luus, Cara Bray, Peter Brown, Anna Devlen, Carly Campbell, Matthew Gray, Mike Kelly, Gemma Mudd, Punit Upadhyaya, Sailaja Battula, Kelvin Zhang, Anne-Sophie Dugast, Kevin McDonnell, Phil Brandish, Nicholas Keen. Tumor-targeted activation of CD137 using Bicycles: New insights into mechanism of action and discovery of BT7455, a clinical candidate for the treatment of EphA2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5301.

Abstract 1532: Improved reproducibility in multiplex immunofluorescence analysis of the tumor microenvironment using a novel quenching solution

Abstract Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used in histopathology because the fixed tissues are stable and easy to store. In the context of immunofluorescence detection, however, endogenous autofluorescence of FFPE tissues can significantly reduce or mask immunofluorescent signals. The autofluorescence of FFPE tissues is caused by a variety of factors, including the presence of endogenous fluorophores, tissue fixation, and processing. While there are several methods that can be used to quench autofluorescence in FFPE tissues, chemical quenchers, which absorb or scatter light, are most frequently used for multiplex IF. Examples of chemical quenchers commonly used in manual multiplex staining include Sudan Black B and TrueBlack; however, these are not chemically compatible for use on automated platforms such as the Leica Biosystems BOND RX. Due to the need for increased throughput and decreased hands-on time, automation has risen in popularity versus manual staining, leading to a need for a versatile quenching solution compatible with automation. Here we demonstrate that NovaPlex Quenching Solution (NPQS), a novel proprietary quenching agent, reduces the background caused by autofluorescence and enhances the signal of the Cell IDx UltraPlex multiplex immunofluorescent (mxIF) assay across tissue types. Qualitative studies showed a decrease in autofluorescent signal in FFPE mouse spleen stained with antibodies against B220/Foxp3/CD3e, suggesting the utility of NPQS in mouse models of cancer and immunity. Similarly, nonspecific background was decreased across FFPE human tissue types, including lung adenocarcinoma stained for CD8/Foxp3/CD3e/CD4 and tonsil stained for CD8/Foxp3/PD-1/CD4. Further statistical studies in FFPE human tonsil tissue stained with CD8/Foxp3/PD-1/CD4 showed there was no significant decrease in dye intensity across all four markers and channels, resulting in a significant increase in signal-to-noise ratio for both CD8 at 490nm and PD-1 at 650nm (p<0.05). These experiments using Cell IDx/LBS UltraPlex mxIF technology demonstrate an effective method to improve the signal-to-noise ratios of fluorophores without reducing signal intensity from any of the target proteins in these parallel multiplex immunofluorescent assays. Citation Format: Matt Levin, Jack Heath, April Schrank-Hacker, Shelby Dunn, Minh-Chau Vu, Imane Hamza, David Schwartz. Improved reproducibility in multiplex immunofluorescence analysis of the tumor microenvironment using a novel quenching solution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1532.

Anti-GluA3 autoantibodies define a new sub-population of frontotemporal lobar degeneration patients with distinct neuropathological features

Autoantibodies directed against the GluA3 subunit (anti-GluA3 hIgGs) of AMPA receptors have been identified in 20%–25% of patients with frontotemporal lobar degeneration (FTLD). Data from patients and in vitro/ex vivo pre-clinical studies indicate that anti-GluA3 hIgGs negatively affect glutamatergic neurotransmission. However, whether and how the chronic presence of anti-GluA3 hIgGs triggers synaptic dysfunctions and the appearance of FTLD-related neuropathological and behavioural signature has not been clarified yet. To address this question, we developed and characterized a pre-clinical mouse model of passive immunization with anti-GluA3 hIgGs purified from patients. In parallel, we clinically compared FTLD patients who were positive for anti-GluA3 hIgGs to negative ones.Clinical data showed that the presence of anti-GluA3 hIgGs defined a subgroup of patients with distinct clinical features. In the preclinical model, anti-GluA3 hIgGs administration led to accumulation of phospho-tau in the postsynaptic fraction and dendritic spine loss in the prefrontal cortex. Remarkably, the preclinical model exhibited behavioural disturbances that mostly reflected the deficits proper of patients positive for anti-GluA3 hIgGs. Of note, anti-GluA3 hIgGs-mediated alterations were rescued in the animal model by enhancing glutamatergic neurotransmission with a positive allosteric modulator of AMPA receptors. Overall, our study clarified the contribution of anti-GluA3 autoantibodies to central nervous system symptoms and pathology and identified a specific subgroup of FTLD patients. Our findings will be instrumental in the development of a therapeutic personalised medicine strategy for patients positive for anti-GluA3 hIgGs.

Unique macrophage phenotypes activated by BMP signaling in breast cancer bone metastases.

Metastatic breast cancer (mBC) tissue in bone was systematically profiled to define the composition of the tumor microenvironment. Gene expression identified a high myeloid signature of patients with improved survival outcomes. Bone metastases were profiled by spatial proteomics to examine myeloid populations within the stroma that correlated with macrophage functions. Single-cell spatial analysis uncovered macrophage activation in the stroma of mBC bone lesions. Matched BC patient samples of primary breast tumor and bone metastasis tissues were compared for gene expression in the bone, where bone morphogenetic protein 2 (BMP2) was most significantly upregulated. Immune cell changes from breast to bone demonstrated a loss of lymphoid cells but a consistent population of macrophages. BMP-activated macrophages were increased uniquely in bone. Bone marrow-derived macrophage activation coupled with BMP inhibition increased inflammatory responses. Using experimental mouse models of mBC bone metastasis and trained immunity, we found that BMP inhibition restricts progression of metastases early in the macrophage activation state but not after tumors were established in the bone. This study revealed unique myeloid BMP activation states that are distinctly integrated with bone metastases.

Hybrid LNP Prime Dendritic Cells for Nucleotide Delivery.

The efficient activation of professional antigen-presenting cells-such as dendritic cells (DC)-in tumors and lymph nodes is critical for the design of next-generation cancer vaccines and may be able to provide anti-tumor effects by itself through immune stimulation. The challenge is to stimulate these cells without causing excessive toxicity. It is hypothesized that a multi-pronged combinatorial approach to DC stimulation would allow dose reductions of innate immune receptor-stimulating TLR3 agonists while enhancing drug efficacy. Here, a hybrid lipid nanoparticle (LNP) platform is developed and tested for double-stranded RNA (polyinosinic:polycytidylic acid for TLR3 agonism) and immune modulator (L-CANDI) delivery. This study shows that the ≈120nm hybrid nanoparticles-in-nanoparticles effectively eradicate tumors by themselves and generate long-lasting, durable anti-tumor immunity in mouse models.

Towards a better preclinical cancer model – human immune aging in humanized mice

BackgroundPreclinical models are often used for cancer studies and evaluation of novel therapeutics. The relevance of these models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. Nonetheless, cancer is an age-related disease, and studies often overlook the effects of aging. Here we have established a humanized mouse model of human immune aging to investigate the role of this phenomenon on liver tumor dynamics.MethodsMultiple organs and tissues (blood, thymus, lung, liver, spleen and bone marrow) were harvested from NOD-scid IL2rγ−/− (NIKO) mice reconstituted with human immune cells, over a period of 60 weeks post-birth, for immune profiling. Young and aging immune cells were compared for transcriptomic changes and functional differences. Effect of immune aging was investigated in a liver cancer humanized mouse model.ResultsFocusing on the T cell population, which is central to cancer immunosurveillance and immunotherapy, we showed that the proportion of naïve T cells declined while memory subsets and senescent-like cells increased with age. RNA-sequencing revealed that downregulated genes were related to immune responses and processes, and this was corroborated by reduced cytokine production in aging T cells. Finally, we showed faster liver tumor growth in aging than younger humanized mice, which could be attributed to specific pathways of aging T cell exhaustion.ConclusionOur work improves on existing humanized (immune) mouse model and highlights the importance of considering immune aging in liver cancer modeling.

Sex-dependent behavioral alterations in a poly(I:C)-induced maternal immune activation mouse model without segment filamentous bacteria

Maternal immune activation is one of the environmental risk factors for offspring to develop psychiatric disorders. A synthetic viral mimetic immunogen, polyinosinic-polycytidylic acid (poly(I:C)), is used to induce maternal immune activation in animal models of psychiatric disorders. In the mouse poly(I:C) model, the existence of segment filamentous bacteria (SFB) in the maternal intestine has been reported to be important for the induction of ASD-related behavioral alterations as well as atypical cortical development called cortical patches. This study aimed to elucidate the effect of a single poly(I:C) injection during embryonic day (E) 9 to E16 on offspring’s behavior in the ensured absence of maternal SFB by vancomycin drinking in C57BL/6N mice. The cortical patches were not found at either injection timings with poly(I:C) or PBS vehicle, tested in male or female offspring at postnatal day 0 or 1. Prepulse inhibition was decreased in male adult offspring most strongly at poly(I:C) injection timings later than E11, whereas a modest but significant decrease was observed in female offspring with an injection during E12 to E15. The decrease in social interaction was observed in female offspring most conspicuously at injection timings later than E11, whereas a significant decrease was observed in male offspring with an injection during E12 to E15. In conclusion, this study indicated that behavioral alterations could be induced without maternal SFB. The effect on behavior was substantially different between males and females.

Introduction of a novel chimeric active immunization mouse model of PLA2R1-associated membranous nephropathy

The phospholipase A2 receptor 1 (PLA2R1) is the major target antigen in patients with membranous nephropathy (MN), an antibody-mediated autoimmune glomerular disease. Investigation of MN pathogenesis has been hampered by the lack of reliable animal models. Here, we overcome this issue by generating a transgenic mouse line expressing a chimeric PLA2R1 (chPLA2R1) consisting of three human PLA2R1 domains (cysteine-rich, fibronectin type-II and CTLD1) and seven murine PLA2R1 domains (CTLD2-8) specifically in podocytes. Mice expressing the chPLA2R1 were healthy at birth and showed no major glomerular alterations when compared to mice with a wild-type PLA2R1 status. Upon active immunization with human PLA2R1 (hPLA2R1), chPLA2R1-positive mice developed anti-hPLA2R1 antibodies, a nephrotic syndrome, and all major histological features of MN, including granular deposition of mouse IgG and complement components in immunofluorescence and subepithelial electron-dense deposits and podocyte foot process effacement in electron microscopy. In order to investigate the role of the complement system in this model, we further crossed chPLA2R1-positive mice with mice lacking the central complement component C3 (C3-/- mice). Upon immunization with hPLA2R1, chPLA2R1-positive C3-/- mice had substantially less severe albuminuria and nephrotic syndrome when compared to chPLA2R1-positive mice with a wild-type C3 status. In conclusion, we introduce a novel active immunization model of PLA2R1-associated MN and demonstrate a pathogenic role of the complement system in this model.

Hematopoietic stem and progenitor cells confer cross-protective trained immunity in mouse models

Recent studies suggest that infection reprograms hematopoietic stem and progenitor cells (HSPCs) to enhance innate immune responses upon secondary infectious challenge, a process called "trained immunity." However, the specificity and cell types responsible for this response remain poorly defined. We established a model of trained immunity in mice in response to Mycobacterium avium infection. scRNA-seq analysis revealed that HSPCs activate interferon gamma-response genes heterogeneously upon primary challenge, while rare cell populations expand. Macrophages derived from trained HSPCs demonstrated enhanced bacterial killing and metabolism, and a single dose of recombinant interferon gamma exposure was sufficient to induce similar training. Mice transplanted with influenza-trained HSPCs displayed enhanced immunity against M.avium challenge and vice versa, demonstrating cross protection against antigenically distinct pathogens. Together, these results indicate that heterogeneous responses to infection by HSPCs can lead to long-term production of bone marrow derived macrophages with enhanced function and confer cross-protection against alternative pathogens.

Autoimmune Atrial Fibrillation.

Atrial fibrillation (AF) is by far the most common cardiac arrhythmia. In about 3% of individuals, AF develops as a primary disorder without any identifiable trigger (idiopathic or historically termed lone AF). In line with the emerging field of autoantibody-related cardiac arrhythmias, the objective of this study was to explore whether autoantibodies targeting cardiac ion channels can underlie unexplained AF. Peptide microarray was used to screen patient samples for autoantibodies. We compared patients with unexplained AF (n=37 pre-existent AF; n=14 incident AF on follow-up) to age- and sex-matched controls (n=37). Electrophysiological properties of the identified autoantibody were then tested in vitro with the patch clamp technique and in vivo with an experimental mouse model of immunization. A common autoantibody response against Kir3.4 protein was detected in patients with AF and even before the development of clinically apparent AF. Kir3.4 protein forms a heterotetramer that underlies the cardiac acetylcholine-activated inwardly rectifying K+ current, IKACh. Functional studies on human induced pluripotent stem cell-derived atrial cardiomyocytes showed that anti-Kir3.4 IgG purified from patients with AF shortened action potentials and enhanced the constitutive form of IKACh, both key mediators of AF. To establish a causal relationship, we developed a mouse model of Kir3.4 autoimmunity. Electrophysiological study in Kir3.4-immunized mice showed that Kir3.4 autoantibodies significantly reduced atrial effective refractory period and predisposed animals to a 2.8-fold increased susceptibility to AF. To our knowledge, this is the first report of an autoimmune pathogenesis of AF with direct evidence of Kir3.4 autoantibody-mediated AF.

Cyto-IL-15 synergizes with the STING agonist ADU-S100 to eliminate prostate tumors and confer durable immunity in mouse models.

Prostate cancer is one of the most commonly diagnosed malignancies in men with high mortality rates. Despite the recent therapeutic advances, such as immunotherapies, survival of patients with advance disease remains significantly low. Blockade of immune checkpoints has led to low response rates in these patients probably due to the immunosuppressive microenvironment and low mutation burden of prostate tumors. Combination of multiple immunotherapeutic regimes has also been unsatisfactory due to augmented adverse effects. To activate multiple immune-stimulatory pathways in the hostile prostate cancer microenvironment, we used a combination of cytotopically modified interleukin-15 (cyto-IL-15) with the stimulator of interferon genes (STING) agonist, ADU-S100. To determine whether this combination regime could lead to both local and systemic anti-tumor effects, intratumoral administration of these agents was used in murine models of prostate cancer. Tumor growth and mouse survival were monitored, and ex vivo analyses, and RNA sequencing were performed on the tumors. Intratumorally injected ADU-S100 and cyto-IL-15 synergized to eliminate tumors in 58-67% of mice with unilateral tumors and promoted abscopal immunity in 50% of mice with bilateral tumors treated only at one side. Moreover, this combination regime offered immunoprotection against tumor rechallenge in 83% of cured mice. The efficacy of the combination treatment was associated with a strong innate and adaptive immune activation and induction of apoptotic and necrotic cell death. Cytokines, including type I and II interferons, and cytokine signalling pathways were activated, NK and T cell mediated cytotoxicity was increased, and B cells were activated both locally and systemically. While ADU-S100 led to an ulcerative pathology at the injection site, no other adverse effects were observed. Localised administration of a STING agonist together with cyto-IL-15 can confer significant systemic benefits and long-lasting immunity against prostate tumors while reducing immune related toxicities.

Antibiotic-induced gut bacteria depletion has no effect on HBV replication in HBV immune tolerance mouse model

Commensal microbiota is closely related to Hepatitis B virus (HBV) infection. Gut bacteria maturation accelerates HBV immune clearance in hydrodynamic injection (HDI) HBV mouse model. However, the effect of gut bacteria on HBV replication in recombinant adeno-associated virus (AAV)-HBV mouse model with immune tolerance remains obscure. We aim to investigate its role on HBV replication in AAV-HBV mouse model. C57BL/6 mice were administrated with broad-spectrum antibiotic mixtures (ABX) to deplete gut bacteria and intravenously injected with AAV-HBV to establish persistent HBV replication. Gut microbiota community was analyzed by fecal qPCR assay and 16S ribosomal RNA (rRNA) gene sequencing. HBV replication markers in blood and liver were determined by ELISA, qPCR assay and Western blot at indicated time points. Immune response in AAV-HBV mouse model was activated through HDI of HBV plasmid or poly(I:C) and then detected by quantifying the percentage of IFN-γ+/CD8+ T cells in the spleen via flow cytometry as well as the splenic IFN-γ mRNA level via qPCR assay. We found that antibiotic exposure remarkably decreased gut bacteria abundance and diversity. Antibiotic treatment failed to alter the levels of serological HBV antigens, intrahepatic HBV RNA transcripts and HBc protein in AAV-HBV mouse model, but contributed to HBsAg increase after breaking of immune tolerance. Overall, our data uncovered that antibiotic-induced gut bacteria depletion has no effect on HBV replication in immune tolerant AAV-HBV mouse model, providing new thoughts for elucidating the correlation between gut bacteria dysbiosis by antibiotic abuse and clinical chronic HBV infection.

Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

Sialophorin is an essential host element for vaccine immunity against pulmonary fungal infections

Abstract The global incidence of fungal infections is alarming due to the increasing immunocompromised population. Vaccine measures are necessary to prevent fungal diseases. Identifying a protective host element as a functional phenotypic marker is immensely valuable as a target to enhance vaccine responses, measure vaccine efficacy, and assess disease outcomes. We identified a host element, O-glycosylated sialophorin, preferentially associated with antifungal memory Tc17 (IL-17A +) cells. Using a mouse model of fungal vaccine immunity during CD4 +T-cell deficiency, we found that sialophorin was essential to bolster vaccine-induced CD8 +T-cell responses and immunity to pulmonary infection. Sialophorin-sufficient CD8 +T cells were superior in responses than the deficient cells under a similar inflammatory micromilieu. Supplementation of sialophorin mAb that binds to O-glycosylated form potentiated the CD8 +T cell responses to fungal antigens, and an inhibitory mAb that binds to non-O-glycosylated form selectively reduced Tc1 (IFNγ +) but not Tc17 cell responses. Sialophorin-mediated immunity was independent of T cell trafficking into the lung. Finally, using fungal antigens vaccination, we show that O-glycosylated sialophorin is a marker of vaccine-potency and immunity. Our study revealed that sialophorin is an essential host element to bolster vaccine responses and serves as a potential biomarker of fungal immunity. NIAID-NIH 5R01AI153522

Adenosine deaminase augments SARS-CoV-2 specific cellular and humoral responses in aged mouse models of immunization and challenge

Abstract Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity and mortality. Furthermore, the aged patient population has suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mouse models. Aged mice had altered cellular responses, including decreased IFNγ secretion and increased TNFα secretion as compared to young mice. Aged mice had significantly decreased binding and neutralizing antibodies in their serum compared to their young counterparts. Co-immunization with the plasmid-encoded molecular adjuvant adenosine deaminase-1 (pADA) enhanced cellular responses and expanded the breadth and affinity of humoral responses in aged mice. pADA co-delivery also altered the gene expression profiles of lymph node lymphocytes in aged animals. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a strong T H1 gene profile and decreased FoxP3 gene expression. Upon challenge pADA co-immunization decreased viral loads and age-associated morbidity and mortality in mouse-adapted and ACE2 transgenic SARS-CoV-2 challenge models. These data support the use of aged mice as a model for age-associated decreases in vaccine immunogenicity and increased in infection-mediated morbidity and mortality in the context of SARS-CoV-2. These studies provide further support for the use of adenosine deaminase as a molecular adjuvant in the elderly. This work was supported by NIH NCI award T32 CA09171 (ENG) and NIH NIAID award T32-AI-055400 (EMP) and The Wistar Institute coronavirus discovery fund with additional support from CEPI/Inovio Pharmaceuticals.

Regulation of CD8 T-cell signaling, metabolism, and cytotoxic activity by extracellular lysophosphatidic acid.

Lysophosphatidic acid (LPA) is an endogenous bioactive lipid that is produced extracellularly and signals to cells via cognate LPA receptors, which are G-protein coupled receptors (GPCRs). Mature lymphocytes in mice and humans express three LPA receptors, LPA2 , LPA5, and LPA6 , and work from our group has determined that LPA5 signaling by T lymphocytes inhibits specific antigen-receptor signaling pathways that ultimately impair lymphocyte activation, proliferation, and function. In this review, we discuss previous and ongoing work characterizing the ability of an LPA-LPA5 axis to serve as a peripheral immunological tolerance mechanism that restrains adaptive immunity but is subverted during settings of chronic inflammation. Specifically, LPA-LPA5 signaling is found to regulate effector cytotoxic CD8 T cells by (at least) two mechanisms: (i) regulating the actin-microtubule cytoskeleton in a manner that impairs immunological synapse formation between an effector CD8 T cell and antigen-specific target cell, thus directly impairing cytotoxic activity, and (ii) shifting T-cell metabolism to depend on fatty-acid oxidation for mitochondrial respiration and reducing metabolic efficiency. The in vivo outcome of LPA5 inhibitory activity impairs CD8 T-cell killing and tumor immunity in mouse models providing impetus to consider LPA5 antagonism for the treatment of malignancies and chronic infections.