Summary of Data from the Sixth AIAA CFD Drag Prediction Workshop: Case 1 Code Verification.

Results from the Sixth AIAA CFD Drag Prediction Workshop (DPW-VI), Case 1 Code Verification are presented. This test case is for the turbulent flow over a 2D NACA 0012 airfoil using Reynolds-Averaged Navier-Stokes (RANS) turbulence models. A numerical benchmark solution is available for the standard Spalart-Allmaras (SA) turbulence model that can be used for code verification purposes, i.e., to verify that the numerical algorithms employed are consistent and that there are no programming mistakes in the software. For the Case 1 code verification study, there were 31 data submissions from 16 teams: 23 with the SA model (using various versions), 4 with the k-omega SST model (two variants), and one each with k-kl, k-epsilon, an explicit algebraic Reynolds stress model, and the lattice Boltzmann method (LBM) with very large eddy simulation (VLES). Various grid types were employed including structured, unstructured, Cartesian, and adapted grids. The benchmark numerical solution was deemed to be the correct solution for the 21 submissions with the standard SA model, the SA-noft2 variant (without the f t2 term), and the SA-neg variant (designed to avoid nonphysical transient states in discrete settings). While many of these 21 submissions did demonstrate first-order convergence on the finer meshes, others showed either nonconvergent solutions in terms of the aerodynamic forces and moments or converged to the wrong answer. Results for this case highlight the continuing need for rigorous code verification to be conducted as a prerequisite for design, model validation, and analysis studies.

Enhancement of the Tolerogenic Phenotype in the Liver by ImmTOR Nanoparticles.

ImmTOR biodegradable nanoparticles encapsulating rapamycin have been shown to induce a durable tolerogenic immune response to co-administered biologics and gene therapy vectors. Prior mechanism of action studies have demonstrated selective biodistribution of ImmTOR to the spleen and liver following intravenous (IV) administration. In the spleen, ImmTOR has been shown to induce tolerogenic dendritic cells and antigen-specific regulatory T cells and inhibit antigen-specific B cell activation. Splenectomy of mice resulted in partial but incomplete abrogation of the tolerogenic immune response induced by ImmTOR. Here we investigated the ability of ImmTOR to enhance the tolerogenic environment in the liver. All the major resident populations of liver cells, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KC), stellate cells (SC), and hepatocytes, actively took up fluorescent-labeled ImmTOR particles, which resulted in downregulation of MHC class II and co-stimulatory molecules and upregulation of the PD-L1 checkpoint molecule. The LSEC, known to play an important role in hepatic tolerance induction, emerged as a key target cell for ImmTOR. LSEC isolated from ImmTOR treated mice inhibited antigen-specific activation of ovalbumin-specific OT-II T cells. The tolerogenic environment led to a multi-pronged modulation of hepatic T cell populations, resulting in an increase in T cells with a regulatory phenotype, upregulation of PD-1 on CD4+ and CD8+ T cells, and the emergence of a large population of CD4-CD8- (double negative) T cells. ImmTOR treatment protected mice in a concanavalin A-induced model of acute hepatitis, as evidenced by reduced production of inflammatory cytokines, infiltrate of activated leukocytes, and tissue necrosis. Modulation of T cell phenotype was seen to a lesser extent after administration by empty nanoparticles, but not free rapamycin. The upregulation of PD-1, but not the appearance of double negative T cells, was inhibited by antibodies against PD-L1 or CTLA-4. These results suggest that the liver may contribute to the tolerogenic properties of ImmTOR treatment.

Enhancement of liver-directed transgene expression at initial and repeat doses of AAV vectors admixed with ImmTOR nanoparticles.

Systemic AAV (adeno-associated virus) gene therapy is a promising approach for the treatment of inborn errors of metabolism, but questions remain regarding its potency and durability. Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been shown to block the formation of neutralizing anti-capsid antibodies, thereby enabling vector re-administration. Here, we further demonstrate that ImmTOR admixed with AAV vectors also enhances hepatic transgene expression at the initial dose of AAV vector, independent of its effects on adaptive immunity. ImmTOR enhances AAV trafficking to the liver, resulting in increased hepatic vector copy numbers and transgene mRNA expression. Enhanced transgene expression occurs through a mechanism independent of the AAV receptor and cannot be replicated in vivo with free rapamycin or empty nanoparticles. The multipronged mechanism of ImmTOR action makes it an attractive candidate to enable more efficient transgene expression at first dose while simultaneously inhibiting adaptive responses against AAV to enable repeat dosing.

ImmTOR nanoparticles enhance AAV transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia.

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.

Synthetic vaccine particles for durable cytolytic T lymphocyte responses and anti-tumor immunotherapy.

We previously reported that synthetic vaccine particles (SVP) encapsulating antigens and TLR agonists resulted in augmentation of immune responses with minimal production of systemic inflammatory cytokines. Here we evaluated two different polymer formulations of SVP-encapsulated antigens and tested their ability to induce cytolytic T lymphocytes (CTL) in combination with SVP-encapsulated adjuvants. One formulation led to efficient antigen processing and cross-presentation, rapid and sustained CTL activity, and expansion of CD8+ T cell effector memory cells locally and centrally, which persisted for at least 1-2 years after a single immunization. SVP therapeutic dosing resulted in suppression of tumor growth and a substantial delay in mortality in several syngeneic mouse cancer models. Treatment with checkpoint inhibitors and/or cytotoxic drugs, while suboptimal on their own, showed considerable synergy with SVP immunization. SVP encapsulation of endosomal TLR agonists provided superior CTL induction, therapeutic benefit and/or improved safety profile compared to free adjuvants. SVP vaccines encapsulating mutated HPV-16 E7 and E6/E7 recombinant proteins led to induction of broad CTL activity and strong inhibition of TC-1 tumor growth, even when administered therapeutically 13-14 days after tumor inoculation in animals bearing palpable tumors. A pilot study in non-human primates showed that SVP-encapsulated E7/E6 adjuvanted with SVP-encapsulated poly(I:C) led to robust induction of antigen-specific T and B cell responses.

Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release.

Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions in vivo, leading to potential adverse events and safety concerns. We have developed a synthetic vaccine particle (SVP) technology that enables co-encapsulation of antigen with potent adjuvants. We demonstrate that co-delivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles results in a strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. In contrast, antigen encapsulated into nanoparticles and admixed with free TLR7/8 agonist leads to lower immunogenicity and rapid induction of high levels of inflammatory cytokines in the serum (e.g., TNF-a and IL-6 levels are 50- to 200-fold higher upon injection of free resiquimod (R848) than of nanoparticle-encapsulated R848). Conversely, local immune stimulation as evidenced by cellular infiltration of draining lymph nodes and by intranodal cytokine production was more pronounced and persisted longer when SVP-encapsulated TLR agonists were used. The strong local immune activation achieved using a modular self-assembling nanoparticle platform markedly enhanced immunogenicity and was equally effective whether antigen and adjuvant were co-encapsulated in a single nanoparticle formulation or co-delivered in two separate nanoparticles. Moreover, particle encapsulation enabled the utilization of CpG oligonucleotides with the natural phosphodiester backbone, which are otherwise rapidly hydrolyzed by nucleases in vivo. The use of SVP may enable clinical use of potent TLR agonists as vaccine adjuvants for indications where cellular immunity or robust humoral responses are required.

Conservation of CD1 intracellular trafficking patterns between mammalian species.

Dendritic cells (DC) are potent APCs that sample Ags from the surrounding environment and present them to naive T cells using cell surface Ag-presenting molecules. The DC in both lymphoid and nonlymphoid tissues express high levels of CD1, a cell surface glycoprotein capable of presenting lipids and glycolipids to T cells. Distinct group 1 CD1 isoforms (CD1a, -b, -c) in man are known to traffic to different parts of the endocytic system where microbial Ags may be sampled. Guinea pigs are the only known rodent species that express the group 1 CD1 proteins. Therefore, we examined the expression and trafficking of guinea pig CD1 (gpCD1) isoforms on isolated DC. Confocal microscopy using mAbs specific for individual gpCD1 isoforms revealed differential trafficking of two distinct CD1b isoforms within DC. Colocalization of MHC class II was observed with the gpCD1b1 isoform, consistent with localization in the late endosomes of DC. In contrast, the gpCD1b3 isoform lacks an endosomal sorting motif and remains on the cell surface. Following incubation with Mycobacterium tuberculosis lipoarabinomannan, colocalization of endocytosed lipoarabinomannan with the gpCD1b1 isoform was observed but not with the gpCD1b3 isoform, which remained primarily on the cell surface. These data demonstrate that guinea pig DC express CD1 isoforms with unique trafficking patterns that recapitulate the patterns seen for human CD1 isoforms. This suggests evolutionary pressure for a conserved mechanism in mammals that allows CD1 to sample lipid Ags from various subcompartments of the endocytic system.