RESUMO
Several FDA-approved adjuvants signal through the NLRP3 inflammasome and IL-1ß release. Identifying small molecules that induce IL-1ß release could allow targeted delivery and structure-function optimization, thereby improving safety and efficacy of next-generation adjuvants. In this work, we leverage our existing high throughput data set to identify small molecules that induce IL-1ß release. We find that ribociclib induces IL-1ß release when coadministered with a TLR4 agonist in an NLRP3- and caspase-dependent fashion. Ribociclib was formulated with a TLR4 agonist into liposomes, which were used as an adjuvant in an ovalbumin prophylactic vaccine model. The liposomes induced antigen-specific immunity in an IL-1 receptor-dependent fashion. Furthermore, the liposomes were coadministered with a tumor antigen and used in a therapeutic cancer vaccine, where they facilitated rejection of E.G7-OVA tumors. While further chemical optimization of the ribociclib scaffold is needed, this study provides proof-of-concept for its use as an IL-1 producing adjuvant in various immunotherapeutic contexts.
RESUMO
The innate immune response is vital for the success of prophylactic vaccines and immunotherapies. Control of signaling in innate immune pathways can improve prophylactic vaccines by inhibiting unfavorable systemic inflammation and immunotherapies by enhancing immune stimulation. In this work, we developed a machine learning-enabled active learning pipeline to guide in vitro experimental screening and discovery of small molecule immunomodulators that improve immune responses by altering the signaling activity of innate immune responses stimulated by traditional pattern recognition receptor agonists. Molecules were tested by in vitro high throughput screening (HTS) where we measured modulation of the nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB) and the interferon regulatory factors (IRF) pathways. These data were used to train data-driven predictive models linking molecular structure to modulation of the NF-κB and IRF responses using deep representational learning, Gaussian process regression, and Bayesian optimization. By interleaving successive rounds of model training and in vitro HTS, we performed an active learning-guided traversal of a 139 998 molecule library. After sampling only â¼2% of the library, we discovered viable molecules with unprecedented immunomodulatory capacity, including those capable of suppressing NF-κB activity by up to 15-fold, elevating NF-κB activity by up to 5-fold, and elevating IRF activity by up to 6-fold. We extracted chemical design rules identifying particular chemical fragments as principal drivers of specific immunomodulation behaviors. We validated the immunomodulatory effect of a subset of our top candidates by measuring cytokine release profiles. Of these, one molecule induced a 3-fold enhancement in IFN-ß production when delivered with a cyclic di-nucleotide stimulator of interferon genes (STING) agonist. In sum, our machine learning-enabled screening approach presents an efficient immunomodulator discovery pipeline that has furnished a library of novel small molecules with a strong capacity to enhance or suppress innate immune signaling pathways to shape and improve prophylactic vaccination and immunotherapies.
RESUMO
Adjuvants are a critical component of vaccines. Adjuvants typically target receptors that activate innate immune signaling pathways. Historically, adjuvant development has been laborious and slow, but has begun to accelerate over the past decade. Current adjuvant development consists of screening for an activating molecule, formulating lead molecules with an antigen, and testing this combination in an animal model. There are very few adjuvants approved for use in vaccines, however, as new candidates often fail due to poor clinical efficacy, intolerable side effects, or formulation limitations. Here, we consider new approaches using tools from engineering to improve next-generation adjuvant discovery and development. These approaches will create new immunological outcomes that will be evaluated with novel diagnostic tools. Potential improved immunological outcomes include reduced vaccine reactogenicity, tunable adaptive responses, and enhanced adjuvant delivery. Evaluations of these outcomes can leverage computational approaches to interpret "big data" obtained from experimentation. Applying engineering concepts and solutions will provide alternative perspectives, further accelerating the field of adjuvant discovery.
RESUMO
Stimulation of the innate immune system is crucial in both effective vaccinations and immunotherapies. This is often achieved through adjuvants, molecules that usually activate pattern recognition receptors (PRRs) and stimulate two innate immune signaling pathways: the nuclear factor kappa-light-chain-enhancer of activated B-cells pathway (NF-κB) and the interferon regulatory factors pathway (IRF). Here, we demonstrate the ability to alter and improve adjuvant activity via the addition of small molecule "immunomodulators". By modulating signaling activity instead of receptor binding, these molecules allow the customization of select innate responses. We demonstrate both inhibition and enhancement of the products of the NF-κB and IRF pathways by several orders of magnitude. Some modulators apply generally across many receptors, while others focus specifically on individual receptors. Modulators boost correlates of a protective immune responses in a commercial flu vaccine model and reduced correlates of reactogenicity in a typhoid vaccine model. These modulators have a range of applications: from adjuvanticity in prophylactics to enhancement of immunotherapy.
RESUMO
Introduction: Tolerogenic Dendritic Cells (tolDCs) have an exceptional promise as a potential therapy for autoimmune disease and transplantation rejection. TolDCs are a unique phenotype of antigen presenting cells (APCs) that can influence naïve T cells into antigen specific T regulatory cells (Tregs), which can re-establish tolerance against auto/allo-antigens in the long term. Despite their promise, tolDCs have not found clinical success. Most strategies seek to generate tolDCs ex vivo by differentiating naïve dendritic cells (DCs) with immunosuppressive agents. Recently, we developed a tolDC generation strategy, which we call Push/Pull Immunomodulation (PPI). In PPI, DCs are treated with combinations of toll-like-receptor (TLR) agonists and immunomodulatory agents, which generate more robust, Treg-inducing tolDCs than previous strategies. Here, we seek to identify more potent and clinically viable PPI formulations using data from a high-throughput screening project. Methods: Over 40,000 combinations of pathogen-associated molecular patterns (PAMPs) and immunomodulatory small molecules were screened using a modified murine macrophage line, RAW dual cells, to observe the effect of these combinations on two major immune regulatory transcription factors, NF-κB and IRF. Combinations were further screened for inflammatory cytokine activity using a human monocyte cell line, THP-1, then on murine DCs. Leading candidates were co-cultured with T cells to assess antigen specific T cell responses. Results: From this data, we identified 355 combinations that showed low or moderate IRF activity, low NF-κB activity, low inflammatory cytokine generation and good viability: all hallmarks of tolerogenic potential. We further screened these 355 combinations using bone marrow derived DCs (BMDCs) and identified 10 combinations that demonstrated high IL-10 (tolerogenic) and low TNF-α (inflammatory) secretion. After further optimizing these combinations, we identified two combinations that generate robust tolDCs from BMDCs ex vivo. We further show that these PPI-tolDCs can also generate antigen specific Tregs but do not increase overall Treg populations. Discussion: These second-generation PPI formulations have significant potential to generate robust tolDCs and strong antigen specific Tregs.
RESUMO
Tandem duplicated genes are common features of genomes, but the phenotypic consequences of their origins are not well understood. It is not known whether a simple doubling of gene expression should be expected, or else some other expression outcome. This study describes an experimental framework using engineered deletions to assess any contribution of locally acting cis- and globally acting trans-regulatory factors to expression interactions of particular tandem duplicated genes. Acsx1L (CG6300) and Acsx1R (CG11659) are tandem duplicates of a putative acyl-CoA synthetase gene found in Drosophila melanogaster. Experimental deletions of the duplicated segments were used to investigate whether the presence of 1 tandem duplicated block influences the expression of its neighbor. Acsx1L, the gene in the left block, shows much higher expression than either its duplicate Acsx1R or the single Acsx1 in Drosophila simulans. Acsx1L expression decreases drastically upon deleting the right-hand duplicated block. Crosses among wildtype and deletion strains show that high tandem expression is primarily due to cis-acting interactions between the duplicated blocks. No effect of these genes on cuticular hydrocarbons was detected. Sequence and phylogenetic analysis suggest that the duplication rose to fixation in D. melanogaster and has been subject to extensive gene conversion. Some strains actually carry 3 tandem copies, yet strains with 3 Acsx1s do not have higher expression levels than strains with 2. Surveys of tandem duplicate expression have typically not found the expected 2-fold increase in expression. This study suggests that cis-regulatory interactions between duplicated blocks could be responsible for this trend.
Assuntos
Drosophila melanogaster , Duplicação Gênica , Animais , Drosophila melanogaster/genética , Evolução Molecular , Dosagem de Genes , Genes Duplicados , FilogeniaRESUMO
The genomic architecture of barriers to gene exchange during the speciation process is poorly understood. The genomic islands model suggests that loci associated with barriers to gene exchange prevent introgression of nearby genomic regions via linkage disequilibrium. But few analyses of the actual genomic location of non-introgressing loci in closely related species exist. In a previous study Maroja et al. showed that in the hybridizing field crickets, Gryllus firmus and G. pennsylvanicus, 50 non-introgressing loci are localized on two autosomal regions and the X chromosome, but they were not able to map the loci along the X chromosome because they used a male informative cross. Here, we localize the introgressing and non-introgressing loci on the X chromosome, and reveal that all X-linked non-introgressing loci are restricted to a 50-cM region with 10 of these loci mapped to a single location. We discuss the implications of this finding to speciation.