Enhancement of subunit vaccine delivery with zinc-carnosine coordination polymer through the addition of mannan.

Vaccines represent a pivotal health advancement for preventing infection. However, because carrier systems with repeated administration can invoke carrier-targeted immune responses that diminish subsequent immune responses (e.g., PEG antibodies), there is a continual need to develop novel vaccine platforms. Zinc carnosine microparticles (ZnCar MPs), which are composed of a one-dimensional coordination polymer formed between carnosine and the metal ion zinc, have exhibited efficacy in inducing an immune response against influenza. However, ZnCar MPs' limited suspendability hinders clinical application. In this study, we address this issue by mixing mannan, a polysaccharide derived from yeast, with ZnCar MPs. We show that the addition of mannan increases the suspendability of this promising vaccine formulation. Additionally, since mannan is an adjuvant, we illustrate that the addition of mannan increases the antibody response and T cell response when mixed with ZnCar MPs. Mice vaccinated with mannan + OVA/ZnCar MPs had elevated serum IgG and IgG1 levels in comparison to vaccination without mannan. Moreover, in the mannan + OVA/ZnCar MPs vaccinated group, mucosal washes demonstrated increased IgG, IgG1, and IgG2c titers, and antigen recall assays showed enhanced IFN-γ production in response to MHC-I and MHC-II immunodominant peptide restimulation, compared to the vaccination without mannan. These findings suggest that the use of mannan mixed with ZnCar MPs holds potential for subunit vaccination and its improved suspendability further promotes clinical translation.

Immunogenicity of an adjuvanted broadly active influenza vaccine in immunocompromised and diverse populations.

Influenza virus outbreaks are a major burden worldwide each year. Current vaccination strategies are inadequate due to antigenic drift/shift of the virus and the elicitation of low immune responses. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) immunogens subvert the constantly mutating viruses; however, they are poorly immunogenic on their own. To increase the immunogenicity of subunit vaccines such as this, adjuvants can be delivered with the vaccine. For example, agonists of the stimulator of interferon genes (STING) have proven efficacy as vaccine adjuvants. However, their use in high-risk populations most vulnerable to influenza virus infection has not been closely examined. Here, we utilize a vaccine platform consisting of acetalated dextran microparticles loaded with COBRA HA and the STING agonist cyclic GMP-AMP. We examine the immunogenicity of this platform in mouse models of obesity, aging, and chemotherapy-induced immunosuppression. Further, we examine vaccine efficacy in collaborative cross mice, a genetically diverse population that mimics human genetic heterogeneity. Overall, this vaccine platform had variable efficacy in these populations supporting work to better tailor adjuvants to specific populations.

Development of a broadly active influenza intranasal vaccine adjuvanted with self-assembled particles composed of mastoparan-7 and CpG.

Currently licensed vaccine adjuvants offer limited mucosal immunity, which is needed to better combat respiratory infections such as influenza. Mast cells (MCs) are emerging as a target for a new class of mucosal vaccine adjuvants. Here, we developed and characterized a nanoparticulate adjuvant composed of an MC activator [mastoparan-7 (M7)] and a TLR ligand (CpG). This novel nanoparticle (NP) adjuvant was co-formulated with a computationally optimized broadly reactive antigen (COBRA) for hemagglutinin (HA), which is broadly reactive against influenza strains. M7 was combined at different ratios with CpG and tested for in vitro immune responses and cytotoxicity. We observed significantly higher cytokine production in dendritic cells and MCs with the lowest cytotoxicity at a charge-neutralizing ratio of nitrogen/phosphate = 1 for M7 and CpG. This combination formed spherical NPs approximately 200 nm in diameter with self-assembling capacity. Mice were vaccinated intranasally with COBRA HA and M7-CpG NPs in a prime-boost-boost schedule. Vaccinated mice had significantly higher antigen-specific antibody responses (IgG and IgA) in serum and mucosa compared with controls. Splenocytes from vaccinated mice had significantly increased cytokine production upon antigen recall and the presence of central and effector memory T cells in draining lymph nodes. Finally, co-immunization with NPs and COBRA HA induced influenza H3N2-specific HA inhibition antibody titers across multiple strains and partially protected mice from a challenge against an H3N2 virus. These results illustrate that the M7-CpG NP adjuvant combination can induce a protective immune response with a broadly reactive influenza antigen via mucosal vaccination.

Targeting Bcl6 in the TREX1 D18N murine model ameliorates autoimmunity by modulating T-follicular helper cells and germinal center B cells.

The Three Prime Repair EXonuclease I (TREX1) is critical for degrading post-apoptosis DNA. Mice expressing catalytically inactive TREX1 (TREX1 D18N) develop lupus-like autoimmunity due to chronic sensing of undegraded TREX1 DNA substrates, production of the inflammatory cytokines, and the inappropriate activation of innate and adaptive immunity. This study aimed to investigate Thelper (Th) dysregulation in the TREX1 D18N model system as a potential mechanism for lupus-like autoimmunity. Comparison of immune cells in secondary lymphoid organs, spleen and peripheral lymph nodes (LNs) between TREX1 D18N mice and the TREX1 null mice revealed that the TREX1 D18N mice exhibit a Th1 bias. Additionally, the T-follicular helper cells (Tfh) and the germinal celter (GC) B cells were also elevated in the TREX1 D18N mice. Targeting Bcl6, a lineage-defining transcription factor for Tfh and GC B cells, with a commercially available Bcl6 inhibitor, FX1, attenuated Tfh, GC, and Th1 responses, and rescued TREX1 D18N mice from autoimmunity. The study presents Tfh and GC B-cell responses as potential targets in autoimmunity and that Bcl6 inhibitors may offer therapeutic approach in TREX1-associated or other lupus-like diseases.

TREX1 as a Novel Immunotherapeutic Target.

Mutations in the TREX1 3' → 5' exonuclease are associated with a spectrum of autoimmune disease phenotypes in humans and mice. Failure to degrade DNA activates the cGAS-STING DNA-sensing pathway signaling a type-I interferon (IFN) response that ultimately drives immune system activation. TREX1 and the cGAS-STING DNA-sensing pathway have also been implicated in the tumor microenvironment, where TREX1 is proposed to degrade tumor-derived DNA that would otherwise activate cGAS-STING. If tumor-derived DNA were not degraded, the cGAS-STING pathway would be activated to promote IFN-dependent antitumor immunity. Thus, we hypothesize TREX1 exonuclease inhibition as a novel immunotherapeutic strategy. We present data demonstrating antitumor immunity in the TREX1 D18N mouse model and discuss theory surrounding the best strategy for TREX1 inhibition. Potential complications of TREX1 inhibition as a therapeutic strategy are also discussed.

TREX1 - Apex predator of cytosolic DNA metabolism.

The cytosolic Three prime Repair EXonuclease 1 (TREX1) is a powerful DNA-degrading enzyme required for clearing cytosolic DNA to prevent aberrant inflammation and autoimmunity. In the absence of TREX1 activity, cytosolic DNA pattern recognition receptors of the innate immune system are constitutively activated by undegraded TREX1 substrates. This triggers a chronic inflammatory response in humans expressing mutant TREX1 alleles, eliciting a spectrum of rare autoimmune diseases dependent on the nature of the mutation. The precise origins of cytosolic DNA targeted by TREX1 continue to emerge, but DNA emerging from the nucleus or taken up by the cell could represent potential sources. In this Review, we explore the biochemical and immunological data supporting the role of TREX1 in suppressing cytosolic DNA sensing, and discuss the possibility that TREX1 may contribute to maintenance of genome integrity.

T Cells Produce IFN-α in the TREX1 D18N Model of Lupus-like Autoimmunity.

Autoimmunity can result when cells fail to properly dispose of DNA. Mutations in the three-prime repair exonuclease 1 (TREX1) cause a spectrum of human autoimmune diseases resembling systemic lupus erythematosus. The cytosolic dsDNA sensor, cyclic GMP-AMP synthase (cGAS), and the stimulator of IFN genes (STING) are required for pathogenesis, but specific cells in which DNA sensing and subsequent type I IFN (IFN-I) production occur remain elusive. In this study, we demonstrate that TREX1 D18N catalytic deficiency causes dysregulated IFN-I signaling and autoimmunity in mice. Moreover, we show that bone marrow-derived cells drive this process. We identify both innate immune and, surprisingly, activated T cells as sources of pathological IFN-α production. These findings demonstrate that TREX1 enzymatic activity is crucial to prevent inappropriate DNA sensing and IFN-I production in immune cells, including normally low-level IFN-α-producing cells. These results expand our understanding of DNA sensing and innate immunity in T cells and may have relevance to the pathogenesis of human disease caused by TREX1 mutation.

TREX1 D18N mice fail to process erythroblast DNA resulting in inflammation and dysfunctional erythropoiesis.

Anaemia is commonly observed in chronic inflammatory conditions, including systemic lupus erythematosus (SLE), where ∼50% of patients display clinical signs of anaemia. Mutation at the aspartate residue 18 of the three prime repair exonuclease 1 (TREX1) gene causes a monogenic form of cutaneous lupus in humans and the genetically precise TREX1 D18N mice recapitulate a lupus-like disease. TREX1 degrades single- and double-stranded DNA (dsDNA), and the link between failed DNA degradation by nucleases, including nucleoside-diphosphate kinases (NM23H1/H2) and Deoxyribonuclease II (DNase II), and anaemia prompted our studies to investigate whether TREX1 dysfunction contributes to anaemia. Utilizing the TREX1 D18N mice we demonstrate that (1) TREX1 mutant mice develop normocytic normochromic anaemia and (2) TREX1 exonuclease participates in the degradation of DNA originating from erythroblast nuclei during definitive erythropoiesis. Gene expression, hematocrit, hemoglobin, immunohistochemistry (IHC) and flow cytometry were used to quantify dysfunctional erythropoiesis. An altered response to induced anaemia in the TREX1 D18N mice was determined through IHC, flow cytometry, and interferon-stimulated gene (ISG) expression analysis of the liver, spleen and erythroblastic islands (EBIs). IHC, flow cytometry, and ISG expression studies were performed in vitro to determine the role of TREX1 in the degradation of erythroblast DNA within EBIs. The TREX1 D18N mice exhibit altered erythropoiesis including a 20% reduction in hematocrit, 10-20 fold increased erythropoietic gene expression levels in the spleen and phenotypic signs of normocytic normochromic anaemia. Anaemia in TREX1 D18N mice is accompanied by increased erythropoietin (Epo), normal hepcidin levels and the TREX1 D18N mice display an inappropriate response to anaemic challenge. Enhanced ISG expression results from failed processing and subsequent sensing of undegraded erythroblast DNA in EBIs. TREX1 participates in the degradation of erythroblast DNA in the EBI and TREX1 D18N mice exhibit a normocytic normochromic anaemia.