Biology subjects

Irvine, D. J.

Publications and source records attributed to Irvine, D. J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Vaccines combining slow delivery and follicle targeting of antigens increase germinal center B cell clonal diversity and clonal expansion

Vaccines incorporating slow delivery, multivalent antigen display, or immunomodulation through adjuvants have an important role to play in shaping the humoral immune response. Here we analyzed mechanisms of action of a clinically relevant combination adjuvant strategy, where phosphoserine (pSer)-tagged immunogens bound to aluminum hydroxide (alum) adjuvant (promoting prolonged antigen delivery to draining lymph nodes) are combined with a potent saponin nanoparticle adjuvant termed SMNP (which alters lymph flow and antigen entry into lymph nodes). When employed with a stabilized HIV Env trimer antigen in mice, this combined adjuvant approach promoted substantial enhancements in germinal center (GC) and antibody responses relative to either adjuvant alone. Using scRNA-seq and scBCR-seq, we found that the alum-pSer/SMNP combination both increased the diversity of GC B cell clones and increased GC B cell clonal expansion, coincident with increases in the expression of Myc and the proportion of S-phase GC B cells. To gain insight into the source of these changes in the GC response, we analyzed antigen biodistribution and structural integrity in draining lymph nodes and found that the combination adjuvant approach, but not alum-pSer delivery or SMNP alone, promoted accumulation of highly intact antigen on follicular dendritic cells, reflecting an integration of the slow antigen delivery and altered lymph node uptake effects of these two adjuvants. These results demonstrate how adjuvants with complementary mechanisms of action impacting vaccine biodistribution and kinetics can synergize to enhance humoral immunity.

immunology↗

bioRxiv2024

Dose-dependent regulation of immune memory responses against HIV by saponin monophosphoryl lipid A nanoparticle adjuvant

The induction of durable protective immune responses is the main goal of prophylactic vaccines, and adjuvants play an important role as drivers of such responses. Despite advances in vaccine strategies, a safe and effective HIV vaccine remains a significant challenge. The use of an appropriate adjuvant is crucial to the success of HIV vaccines. Here we assessed the saponin/MPLA nanoparticle (SMNP) adjuvant with an HIV envelope (Env) trimer, evaluating the safety and impact of multiple variables including adjuvant dose (16-fold dose range), immunization route, and adjuvant composition on the establishment of Env-specific memory T and B cell responses (TMem and BMem) and long-lived plasma cells in non-human primates. Robust BMem were detected in all groups, but a 6-fold increase was observed in the highest SMNP dose group vs. the lowest dose group. Similarly, stronger vaccine responses were induced in the highest SMNP dose for CD40L+OX40+ CD4 TMem (11-fold), IFN{gamma}+ CD4 TMem (15-fold), IL21+ CD4 TMem (9-fold), circulating TFH (3.6-fold), bone marrow plasma cells (7-fold), and binding IgG (1.3-fold). Substantial tier-2 neutralizing antibodies were only observed in the higher SMNP dose groups. These investigations highlight the dose-dependent potency of SMNP in non-human primates, which are relevant for human use and next-generation vaccines.

immunology↗

bioRxiv2024

Generation of an inflammatory niche in an injectable hydrogel depot through recruitment of key immune cells improves efficacy of mRNA vaccines

Messenger RNA (mRNA) delivered in lipid nanoparticles (LNPs) rose to the forefront of vaccine candidates during the COVID-19 pandemic due in part to scalability, adaptability, and potency. Yet there remain critical areas for improvements of these vaccines in durability and breadth of humoral responses. In this work, we explore a modular strategy to target mRNA/LNPs to antigen presenting cells with an injectable polymer-nanoparticle (PNP) hydrogel depot technology which recruits key immune cells and forms an immunological niche in vivo. We characterize this niche on a single cell level and find it is highly tunable through incorporation of adjuvants like MPLAs and 3M-052. Delivering commercially available SARS-CoV-2 mRNA vaccines in PNP hydrogels improves the durability and quality of germinal center reactions, and the magnitude, breadth, and durability of humoral responses. The tunable immune niche formed within PNP hydrogels effectively skews immune responses based on encapsulated adjuvants, creating opportunities to precisely modulate mRNA/LNP vaccines for various indications from infectious diseases to cancers.

bioengineering↗

bioRxiv2024

'Target-and-release' nanoparticles for effective immunotherapy of metastatic ovarian cancer

Immunotherapies such as checkpoint inhibitors (CPI) are effective in treating several advanced cancers, but these treatments have had limited success in metastatic ovarian cancer (OC). Here, we engineered liposomal nanoparticles (NPs) carrying a layer-by-layer (LbL) polymer coating that promotes their binding to the surface of OC cells. Covalent anchoring of the potent immunostimulatory cytokine interleukin-12 (IL-12) to phospholipid headgroups of the liposome core enabled the LbL particles to concentrate IL-12 in disseminated OC tumors following intraperitoneal administration. Shedding of the LbL coating and serum protein-mediated extraction of IL-12-conjugated lipids from the liposomal core over time enabled IL-12 to disseminate in the tumor bed following rapid NP localization in tumor nodules. Optimized IL-12 LbL-NPs promoted robust T cell accumulation in ascites and tumors in mouse models, extending survival compared to free IL-12 and remarkedly sensitizing tumors to CPI, leading to curative treatments and immune memory.

bioengineering↗

bioRxiv2024

Visualizing lipid nanoparticle trafficking for mRNA vaccine delivery in non-human primates

mRNA delivered using lipid nanoparticles (LNPs) has become an important subunit vaccine modality, but mechanisms of action for mRNA vaccines remain incompletely understood. Here, we synthesized a metal chelator-lipid conjugate enabling positron emission tomography (PET) tracer labeling of LNP/mRNA vaccines for quantitative visualization of vaccine trafficking in live non-human primates (NHPs). Following i.m. injection, we observed LNPs distributing through injected muscle tissue, simultaneous with rapid trafficking to draining lymph nodes (dLNs). Deltoid injection of LNPs mimicking human vaccine administration led to stochastic LNP delivery to 3 different sets of dLNs. LNP uptake in dLNs was confirmed by histology, and cellular analysis of tissues via flow cytometry identified antigen-presenting cells as the primary cell type responsible for early LNP uptake and mRNA translation. These results provide insights into the biodistribution of mRNA vaccines administered at clinically relevant doses, injection volumes, and injection sites in an important large animal model for vaccine development.

bioengineering↗

bioRxiv2024

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR (FMC63) as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion and memory development of CD19 CAR-T cells in both syngeneic and humanized mouse models of B-ALL/Lymphoma, and enhanced control of disease progression. This approach thus enables vaccine boosting to be applied to any clinically relevant CAR-T cell product.

bioengineering↗

bioRxiv2024

Glycan-costumed virus-like particles promote type 1 anti-tumor immunity

Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) shapes immunity by directing T cell function. Strategies to activate specific DC signaling pathways via targeted receptor engagement are crucial to unlocking type 1 cellular immunity. Here, we engineered a glycan-costumed virus-like particle (VLP) vaccine that delivers programmable peptide antigens to induce tumor-specific cellular immunity in vivo. VLPs encapsulating TLR7 agonists and decorated with a selective mannose-derived ligand for the lectin DC-SIGN induced robust DC activation and type 1 cellular immunity, whereas VLPs lacking this key DC-SIGN ligand failed to promote DC-mediated immunity. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, inhibiting tumor growth in a murine melanoma model. Thus, VLPs employing lectin-driven immune reprogramming provide a framework for advancing cancer immunotherapies.

biochemistry↗

bioRxiv2024

Local delivery of cell surface-targeted immunocytokines programs systemic anti-tumor immunity

Cytokine therapies are potent immunotherapy agents but exhibit severe dose-limiting toxicities. One strategy to overcome this involves engineering cytokines for intratumoral retention following local delivery. Here, we develop a localized cytokine therapy that elicits profound anti-tumor immunity by engineered targeting to the ubiquitous leukocyte receptor CD45. We designed CD45-targeted immunocytokines (CD45-Cyt) that, upon injection, decorated the surface of leukocytes in the tumor and tumor-draining lymph node (TDLN) without systemic exposure. CD45-Cyt therapy eradicated both directly treated tumors and untreated distal lesions in multiple syngeneic mouse tumor models. Mechanistically, CD45-Cyt triggered prolonged pSTAT signaling and reprogrammed tumor-specific CD8+ T cells in the TDLN to exhibit an anti-viral transcriptional signature. CD45 anchoring represents a broad platform for protein retention by host immune cells for use in immunotherapy.

immunology↗

bioRxiv2023

Two-dose "extended priming" immunization amplifies humoral immune responses by synchronizing vaccine delivery with the germinal center response

"Extended priming" immunization regimens that prolong exposure of the immune system to vaccines during the primary immune response have shown promise in enhancing humoral immune responses to a variety of subunit vaccines in preclinical models. We previously showed that escalating-dosing immunization (EDI), where a vaccine is dosed every other day in an increasing pattern over 2 weeks dramatically amplifies humoral immune responses. But such a dosing regimen is impractical for prophylactic vaccines. We hypothesized that simpler dosing regimens might replicate key elements of the immune response triggered by EDI. Here we explored "reduced ED" immunization regimens, assessing the impact of varying the number of injections, dose levels, and dosing intervals during EDI. Using a stabilized HIV Env trimer as a model antigen combined with a potent saponin adjuvant, we found that a two-shot extended-prime regimen consisting of immunization with 20% of a given vaccine dose followed by a second shot with the remaining 80% of the dose 7 days later resulted in increased total GC B cells, 5-10-fold increased frequencies of antigen-specific GC B cells, and 10-fold increases in serum antibody titers compared to single bolus immunization. Computational modeling of the GC response suggested that this enhanced response is mediated by antigen delivered in the second dose being captured more efficiently as immune complexes in follicles, predictions we verified experimentally. Our computational and experimental results also highlight how properly designed reduced ED protocols enhance activation and antigen loading of dendritic cells and activation of T helper cells to amplify humoral responses. These results suggest that a two-shot priming approach can be used to substantially enhance responses to subunit vaccines.

immunology↗

bioRxiv2023

WASP facilitates tumor mechanosensitivity in T lymphocytes

Cytotoxic T lymphocytes (CTLs) carry out immunosurveillance by scanning target cells of diverse physical properties for the presence of antigens. While the recognition of cognate antigen by the T cell receptor is the primary signal for CTL activation, it has become increasingly clear that the mechanical stiffness of target cells plays an important role in antigen-triggered T cell responses. However, the molecular machinery within CTLs that transduces the mechanical information of tumor cells remains unclear. We find that CTLs mechanosensitive ability requires the activity of the actin-organizing protein Wiskott-Aldrich Syndrome Protein (WASP). WASP activation is modulated by the mechanical properties of antigen-presenting contexts across a wide range of target cell stiffnesses and activated WASP then mediates mechanosensitive activation of early TCR signaling markers in the CTL. Our results provide a molecular link between antigen mechanosensing and CTL immune response and suggest that CTL-intrinsic cytoskeletal organizing principles enable the processing of mechanical information from diverse target cells.

immunology↗

bioRxiv2023

Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons

Antigen processing is critical for producing epitope peptides that are presented by HLA molecules for T cell recognition. Therapeutic vaccines aim to harness these epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often reduces vaccine efficacy through limiting the quantity of epitopes released. Here, we set out to improve antigen processing by harnessing protein degradation signals called degrons from the ubiquitin-proteasome system. We used machine learning to generate a computational model that ascribes a proteasomal degradation score between 0 and 100. Epitope peptides with varying degron activities were synthesized and translocated into cells using nontoxic anthrax proteins: protective antigen (PA) and the N-terminus of lethal factor (LFN). Immunogenicity studies revealed epitope sequences with a low score (<25) show pronounced T-cell activation but epitope sequences with a higher score (>75) provide limited activation. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity, through conserving the epitope region but varying the flanking sequence. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by vaccine therapeutics in clinical settings.

immunology↗

bioRxiv2023

Focusing antibody responses to the fusion peptide in rhesusmacaques

Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to elicit immune responses against the conserved fusion peptide. Antibody specificities and GC responses were tracked longitudinally using electron microscopy polyclonal epitope mapping (EMPEM) and lymph node fine-needle aspirates, respectively. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.

immunology↗

bioRxiv2022

Decoupled neoantigen cross-presentation in tumors with high intratumor heterogeneity reduces dendritic cell activation to limit anti-tumor immunity

Cancer immunotherapies, in particular checkpoint blockade immunotherapy (CBT), can induce control of cancer growth, with a fraction of patients experiencing durable responses. However, the majority of patients currently do not respond to CBT and the molecular determinants of resistance have not been fully elucidated. Mounting clinical evidence suggests that the clonal status of neoantigens (NeoAg) impacts the anti-tumor T cell response. High intratumor heterogeneity (ITH), where the majority of NeoAgs are expressed subclonally, is correlated with poor clinical response to CBT and poor infiltration with tumor-reactive T cells. However, the mechanism by which ITH blunts tumor-reactive T cells is unclear. We developed a transplantable murine lung cancer model to characterize the immune response against a defined set of NeoAgs expressed either clonally or subclonally to model low or high ITH, respectively. Here we show that clonal expression of a weakly immunogenic NeoAg with a relatively strong NeoAg increased the immunogenicity of tumors with low but not high ITH. Mechanistically we determined that clonal NeoAg expression allowed cross-presenting dendritic cells to acquire and present both NeoAgs. Dual NeoAg presentation by dendritic cells was associated with a more mature DC phenotype and a higher stimulatory capacity. These data suggest that clonal NeoAg expression can induce more potent anti-tumor responses due to more stimulatory dendritic cell: T cell interactions. Therapeutic vaccination targeting subclonally expressed NeoAgs could be used to boost anti-tumor T cell responses.

immunology↗

bioRxiv2022

Eradication of tumors with pre-existing antigenic heterogeneity by vaccine-mediated co-engagement of CAR T and endogenous T-cells

Chimeric Antigen Receptor (CAR) T-cell therapy can be effective in treating human cancer but loss of the antigen recognized by the CAR poses a major obstacle. Here, we report an approach for vaccine boosting CAR T-cells, which triggers engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T-cells promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited priming of endogenous anti-tumor T-cells (antigen spreading). This process was accompanied by a shift in toward oxidative phosphorylation in CAR T-cells and was critically dependent on CAR-T-derived IFN-{gamma}. Antigen spreading induced by vaccine-boosted CAR-T enabled a proportion of complete responses even when the initial tumor was 50% CAR-antigen-negative, and heterogenous tumor control was further enhanced by genetically amplifying CAR-T IFN-{gamma} expression. Thus, CAR T-cell-derived IFN-{gamma} plays a critical role in promoting antigen spreading, and vaccine boosting provides a clinically-translatable strategy to drive such responses against solid tumors.

immunology↗

bioRxiv2022

Reprogramming brain immunosurveillance with engineered cytokines

Immune surveillance of the brain is regulated by resident non-neuronal cells and the blood-brain barrier.1 Dys-regulation of immunosurveillance is a hallmark feature of several diseases2-5 including brain tumors6 that interact with and rely heavily on immune cells,7 suggesting that disrupting the neuroimmunology of tumors could slow their progression. Yet few tools are available to control brain immunology in vivo with local precision, and fewer yet are used for therapeutic intervention. 2 Here, we propose engineered cytokines as a neuroimmune-modulation platform. We demonstrate that the residence time of cytokines in the brain can be tuned by binding them to the extracellular matrix or synthetic scaffolds. We then show that the aluminum hydroxide adjuvant (alum) is retained in the brain >2 weeks. Tethering of inflammatory cytokines such as interleukins (IL) 2 and 12 to alum yields extended neuroinflammation and brain immunosurveillance after intracranial administration, while avoiding systemic toxicity. In mouse models of both immunologically hot and cold brain tumors, the intracranial deposition of alum-tethered cytokines causes significant delay in tumor progression. RNA profiling reveals that engineered cytokines engage both innate and adaptive immunity in the brain. These findings suggest that engineered cytokines can reprogram brain immunosurveillance, informing the development of future therapies for neuroimmune diseases.

bioengineering↗

bioRxiv2022

Identification of highly cross-reactive mimotopes for a public T cell response in murine melanoma

While immune checkpoint blockade results in durable responses for some patients, many others have not experienced such benefits. These treatments rely upon reinvigorating specific T cell-antigen interactions. However, it is often unknown what antigens are being recognized by T cells or how to potently induce antigen-specific responses in a broadly applicable manner. Here, we characterized the CD8+ T cell response to a murine model of melanoma following combination immunotherapy to determine the basis of tumor recognition. Sequencing of tumor-infiltrating T cells revealed a repertoire of highly homologous TCR sequences that were particularly expanded in treated mice and which recognized an antigen from an endogenous retrovirus. While vaccination against this peptide failed to raise a protective T cell response in vivo, engineered antigen mimotopes induced a significant expansion of CD8+ T cells cross-reactive to the original antigen. Vaccination with mimotopes resulted in killing of antigen-loaded cells in vivo yet showed modest survival benefit in a prophylactic vaccine paradigm. Together, this work demonstrates the identification of a dominant tumor-associated antigen and generation of mimotopes which can induce robust functional T cell responses that are cross-reactive to the endogenous antigen across multiple individuals.

immunology↗

bioRxiv2021

Long-lasting germinal center responses to a priming immunization with continuous proliferation and somatic mutation

Germinal centers (GCs) are the engines of antibody evolution. Using HIV Env protein immunogen priming in rhesus monkeys (RM) followed by a long period without further immunization, we demonstrate GC B cells (BGC) lasted at least 6 months (29 weeks), all the while maintaining rapid proliferation. A 186-fold BGC cell increase was present by week 10 compared to a conventional immunization. Single cell transcriptional profiling revealed that both light zone and dark zone GC states were sustained throughout the 6 months. Antibody somatic hypermutation (SHM) of BGC cells continued to accumulate throughout the 29 week priming period, with evidence of selective pressure. Additionally, Env-binding BGC cells were still 49-fold above baseline 29 weeks after immunization, suggesting that they could be active for significantly longer periods of time. High titers of HIV neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing significant immunodominance challenges for B cells, among other difficulties. Memory B cells (BMem) generated under these long priming conditions had higher levels of SHM, and both BMem cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning the >6-month GC period were identified, demonstrating continuous GC activity and selection for at least 191 days, with no additional antigen exposure. A long prime, adjuvanted, slow delivery (12-day) immunization approach holds promise for difficult vaccine targets, and suggests that patience can have great value for tuning GCs to maximize antibody responses.

immunology↗

bioRxiv2021

Spatially regulated protease activity in lymph nodes renders B cell follicles a sanctuary for retention of intact antigens

The structural integrity of vaccine antigens is critical, as antigen degradation in vivo could eliminate neutralizing epitopes and create competing B cell responses against irrelevant breakdown products. Using FRET imaging and imaging zymography, we found that protease activity and antigen breakdown are spatially heterogeneous in lymph nodes. Following protein immunization, antigens are rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions of the tissue. By contrast, the follicles and follicular dendritic cell (FDC) networks exhibit low protease activity and antigen degradation rates. Immunization regimens targeting antigen rapidly to FDCs led to germinal centers (GCs) where responses to intact antigen were highly dominant, while traditional bolus immunizations led to weaker GC responses where more GC B cells bound to breakdown products than intact antigen. Thus, spatially-compartmentalized antigen proteolysis impacts humoral immunity and can be exploited to enhance vaccine-induced production of antibody responses against key pathogen structural epitopes.

immunology↗