Toll-like receptor 4 stimulation with the detoxified ligand monophosphoryl lipid A improves Alzheimer's disease-related pathology.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. The pathogenesis of this neurodegenerative disease, currently without curative treatment, is associated with the accumulation of amyloid ß (Aß) in brain parenchyma and cerebral vasculature. AD patients are unable to clear this toxic peptide, leading to Aß accumulation in their brains and, presumably, the pathology associated with this devastating disease. Compounds that stimulate the immune system to clear Aß may therefore have great therapeutic potential in AD patients. Monophosphoryl lipid A (MPL) is an LPS-derived Toll-like receptor 4 agonist that exhibits unique immunomodulatory properties at doses that are nonpyrogenic. We show here that repeated systemic injections of MPL, but not LPS, significantly improved AD-related pathology in APP(swe)/PS1 mice. MPL treatment led to a significant reduction in Aß load in the brain of these mice, as well as enhanced cognitive function. MPL induced a potent phagocytic response by microglia while triggering a moderate inflammatory reaction. Our data suggest that the Toll-like receptor 4 agonist MPL may be a treatment for AD.

Exogenous fms-like tyrosine kinase 3 ligand overrides brain immune privilege and facilitates recognition of a neo-antigen without causing autoimmune neuropathology.

Soluble antigens diffuse out of the brain and can thus stimulate a systemic immune response, whereas particulate antigens (from infectious agents or tumor cells) remain within brain tissue, thus failing to stimulate a systemic immune response. Immune privilege describes how the immune system responds to particulate antigens localized selectively within the brain parenchyma. We believe this immune privilege is caused by the absence of antigen presenting dendritic cells from the brain. We tested the prediction that expression of fms-like tyrosine kinase ligand 3 (Flt3L) in the brain will recruit dendritic cells and induce a systemic immune response against exogenous influenza hemagglutinin in BALB/c mice. Coexpression of Flt3L with HA in the brain parenchyma induced a robust systemic anti-HA immune response, and a small response against myelin basic protein and proteolipid protein epitopes. Depletion of CD4(+)CD25+ regulatory T cells (Tregs) enhanced both responses. To investigate the autoimmune impact of these immune responses, we characterized the neuropathological and behavioral consequences of intraparenchymal injections of Flt3L and HA in BALB/c and C57BL/6 mice. T cell infiltration in the forebrain was time and strain dependent, and increased in animals treated with Flt3L and depleted of Tregs; however, we failed to detect widespread defects in myelination throughout the forebrain or spinal cord. Results of behavioral tests were all normal. These results demonstrate that Flt3L overcomes the brain's immune privilege, and supports the clinical development of Flt3L as an adjuvant to stimulate clinically effective immune responses against brain neo-antigens, for example, those associated with brain tumors.

Combined Flt3L/TK gene therapy induces immunological surveillance which mediates an immune response against a surrogate brain tumor neoantigen.

Glioblastoma multiforme (GBM) is a primary brain tumor with a median survival of 14.6 months postdiagnosis. The infiltrative nature of GBM prevents complete resection and residual brain tumor cells give rise to recurrent GBM, a hallmark of this disease. Recurrent GBMs are known to harbor numerous mutations/gene rearrangements when compared to the primary tumor, which leads to the potential expression of novel proteins that could serve as tumor neoantigens. We have developed a combined immune-based gene therapeutic approach for GBM using adenoviral (Ads) mediated gene delivery of Herpes Simplex Virus Type 1-thymidine kinase (TK) into the tumor mass to induce tumor cells' death combined with an adenovirus expressing fms-like tyrosine kinase 3 ligand (Flt3L) to recruit dendritic cells (DCs) into the tumor microenvironment. This leads to the induction of specific anti-brain tumor immunity and immunological memory. In a model of GBM recurrence, we demonstrate that Flt3L/TK mediated immunological memory is capable of recognizing brain tumor neoantigens absent from the original treated tumor. These data demonstrate that the Flt3L/TK gene therapeutic approach can induce systemic immunological memory capable of recognizing a brain tumor neoantigen in a model of recurrent GBM.

Whole-blood cytokine secretion assay as a high-throughput alternative for assessing the cell-mediated immunity profile after two doses of an adjuvanted SARS-CoV-2 recombinant protein vaccine candidate.

OBJECTIVES: We previously described the Phase I-II evaluation of SARS-CoV-2 recombinant protein candidate vaccine, CoV2-PreS-dTM, with AF03- or AS03-adjuvant systems (ClinicalTrials.gov, NCT04537208). Here, we further characterise the cellular immunogenicity profile of this vaccine candidate using a whole-blood secretion assay in parallel to intracellular cytokine staining (ICS) of cryopreserved peripheral blood mononuclear cells (PBMCs). METHODS: A randomly allocated subset of 90 healthy, SARS-CoV-2-seronegative adults aged ≥ 18 years who had received (random allocation) one or two separate injections (on study day [D]1 and D22) of saline placebo or CoV2-PreS-dTM formulated with AS03 or AF03 were included. Cytokine secretion was assessed using a TruCulture® whole-blood stimulation system in combination with multiplex bead array, and intracellular cytokine profiles were evaluated on thawed PBMCs following ex vivo stimulation with recombinant S protein at pre-vaccination (D1), post-dose 1 (D22) and post-dose 2 (D36). RESULTS: Both methods detected similar vaccine-induced responses after the first and second doses. We observed a Th1 bias (Th1/Th2 ratio > 1.0) for most treatment groups when analysed in whole blood, mainly characterised by increased IFN-γ, IL-2 and TNF-α secretion. Among participants aged ≥ 50 years, the Th1/Th2 ratio was higher for those who received vaccine candidate with AS03 versus AF03 adjuvant. ICS revealed that this higher Th1/Th2 ratio resulted from higher levels of IFN-γ expression and that the vaccine induced polyfunctional CD4+ T cells. CONCLUSIONS: The whole-blood cytokine secretion assay is a high-throughput alternative for assessing the quantity and character of vaccine-induced cellular responses.

AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity.

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-kappaB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4(+) T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

Pulmonary-Resident Memory Lymphocytes: Pivotal Orchestrators of Local Immunity Against Respiratory Infections.

There is increasing evidence that lung-resident memory T and B cells play a critical role in protecting against respiratory reinfection. With a unique transcriptional and phenotypic profile, resident memory lymphocytes are maintained in a quiescent state, constantly surveying the lung for microbial intruders. Upon reactivation with cognate antigen, these cells provide rapid effector function to enhance immunity and prevent infection. Immunization strategies designed to induce their formation, alongside novel techniques enabling their detection, have the potential to accelerate and transform vaccine development. Despite most data originating from murine studies, this review will discuss recent insights into the generation, maintenance and characterisation of pulmonary resident memory lymphocytes in the context of respiratory infection and vaccination using recent findings from human and non-human primate studies.

Endoplasmic reticulum stress response and bile acid signatures associate with multi-strain seroresponsiveness during elderly influenza vaccination.

The elderly are an important target for influenza vaccination, and the determination of factors that underlie immune responsiveness is clinically valuable. We evaluated the immune and metabolic profiles of 205 elderly Singaporeans administered with Vaxigrip. Despite high seroprotection rates, we observed heterogeneity in the response. We stratified the cohort into complete (CR) or incomplete responders (IR), where IR exhibited signs of accelerated T cell aging. We found a higher upregulation of genes associated with the B-cell endoplasmic-reticulum stress response in CR, where XBP-1 acts as a key upstream regulator. B-cells from IR were incapable of matching the level of XBP-1 upregulation observed in CR after inducing ER stress with tunicamycin in vitro. Metabolic signatures also distinguished CR and IR - as CR presented with a greater diversity of bile acids. Our findings suggest that the ER-stress pathway activation could improve influenza vaccination in the elderly.

High-capacity adenovirus vector-mediated anti-glioma gene therapy in the presence of systemic antiadenovirus immunity.

Gene therapy is proposed as a novel therapeutic strategy for treating glioblastoma multiforme (GBM), a devastating brain cancer. In the clinic, antivector immune responses pose formidable challenges. Herein we demonstrate that high-capacity adenovirus vectors (HC-Ads) carrying the conditional cytotoxic gene herpes simplex virus type 1-thymidine kinase (TK) induce tumor regression and long-term survival in an intracranial glioma model, even in the presence of systemic antiadenovirus immunity, as could be encountered in patients. First-generation Ad-TK failed to elicit tumor regression in this model. These results pave the way for implementing HC-Ad-TK-mediated gene therapy as a powerful adjuvant for treating GBM.

Immunization against the transgene but not the TetON switch reduces expression from gutless adenoviral vectors in the brain.

Immune responses against vectors or encoded transgenes can impose limitations on gene therapy. We demonstrated that tetracycline-regulated high-capacity adenoviral vectors (HC-Ads) sustain regulated transgene expression in the brain even in the presence of systemic pre-existing immune responses against adenoviruses. In this study we assessed whether systemic pre-existing immune responses against the transgene products, i.e., beta-Gal or the tetracycline-dependent (TetON) regulatory transcription factors (rtTA2(S)M2 and the tTS(Kid)), affect transgene expression levels and the safety profile of HC-Ads in the brain. We pre-immunized mice with plasmids encoding the TetON switch expressing rtTA2(S)M2 and the tTS(Kid) or beta-Gal. HC-Ads expressing beta-Gal under the control of the TetON switch were then injected into the striatum. We assessed levels and distribution of beta-Gal expression, and evaluated local inflammation and neuropathological changes. We found that systemic immunity against beta-Gal, but not against the TetON switch, led to inflammation and reduction of transgene expression in the striatum. Therefore, the regulatory TetON switch appears to be safe to use, and capable of sustaining transgene expression in the brain even in the presence of an immune response against its components. Systemic immunity against the transgene had the effect of curtailing its expression, thereby affecting the efficacy and safety of gene delivery to the brain. This factor should be considered when developing gene therapies for neurological use.

Protection of p27(Kip1) mRNA by quaking RNA binding proteins promotes oligodendrocyte differentiation.

The quaking (Qk) locus expresses a family of RNA binding proteins, and the expression of several alternatively spliced isoforms coincides with the development of oligodendrocytes and the onset of myelination. Quaking viable (Qk(v)) mice harboring an autosomal recessive mutation in this locus have uncompacted myelin in the central nervous system owing to the inability of oligodendrocytes to properly mature. Here we show that the expression of two QKI isoforms, absent from oligodendrocytes of Qk(v) mice, induces cell cycle arrest of primary rat oligodendrocyte progenitor cells and differentiation into oligodendrocytes. Injection of retroviruses expressing QKI into the telencephalon of mouse embryos induced differentiation and migration of multipotential neural progenitor cells into mature oligodendrocytes localized in the corpus callosum. The mRNA encoding the cyclin-dependent kinase (CDK)-inhibitor p27(Kip1) was bound and stabilized by QKI, leading to an increased accumulation of p27(Kip1) protein in oligodendrocytes. Our findings demonstrate that QKI is upstream of p27(Kip1) during oligodendrocyte differentiation.