Vaccination with nanoparticles displaying gH/gL from Epstein-Barr virus elicits limited cross-protection against rhesus lymphocryptovirus.

Epstein-Barr virus (EBV) is associated with infectious mononucleosis, cancer, and multiple sclerosis. A vaccine that prevents infection and/or EBV-associated morbidity is an unmet need. The viral gH/gL glycoprotein complex is essential for infectivity, making it an attractive vaccine target. Here, we evaluate the immunogenicity of a gH/gL nanoparticle vaccine adjuvanted with the Sigma Adjuvant System (SAS) or a saponin/monophosphoryl lipid A nanoparticle (SMNP) in rhesus macaques. Formulation with SMNP elicits higher titers of neutralizing antibodies and more vaccine-specific CD4+ T cells. All but one animal in the SMNP group were infected after oral challenge with the EBV ortholog rhesus lymphocryptovirus (rhLCV). Their immune plasma had a 10- to 100-fold lower reactivity against rhLCV gH/gL compared to EBV gH/gL. Anti-EBV neutralizing monoclonal antibodies showed reduced binding to rhLCV gH/gL, demonstrating that EBV gH/gL neutralizing epitopes are poorly conserved on rhLCV gH/gL. Prevention of rhLCV infection despite antigenic disparity supports clinical development of gH/gL nanoparticle vaccines against EBV.

Vaccine targeting to mucosal lymphoid tissues promotes humoral immunity in the gastrointestinal tract.

Viruses, bacteria, and parasites frequently cause infections in the gastrointestinal tract, but traditional vaccination strategies typically elicit little or no mucosal antibody responses. Here, we report a strategy to effectively concentrate immunogens and adjuvants in gut-draining lymph nodes (LNs) to induce gut-associated mucosal immunity. We prepared nanoemulsions (NEs) based on biodegradable oils commonly used as vaccine adjuvants, which encapsulated a potent Toll-like receptor agonist and displayed antigen conjugated to their surface. Following intraperitoneal administration, these NEs accumulated in gut-draining mesenteric LNs, priming strong germinal center responses and promoting B cell class switching to immunoglobulin A (IgA). Optimized NEs elicited 10- to 1000-fold higher antigen-specific IgG and IgA titers in the serum and feces, respectively, compared to free antigen mixed with NE, and strong neutralizing antibody titers against severe acute respiratory syndrome coronavirus 2. Thus, robust gut humoral immunity can be elicited by exploiting the unique lymphatic collection pathways of the gut with a lymph-targeting vaccine formulation.

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 of both murine CD19 CAR-T cells in a syngeneic model and human CAR-T cells in a humanized mouse model of B cell acute lymphoblastic leukemia (B-ALL), and enhanced control of leukemia progression. This approach thus enables vaccine boosting to be applied to any clinically-relevant CAR-T cell product.

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

Antigen processing is critical for therapeutic vaccines to generate epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often limits the quantity of epitopes released. We address this challenge using machine learning to ascribe a proteasomal degradation score to epitope sequences. Epitopes with varying scores were translocated into cells using nontoxic anthrax proteins. Epitopes with a low score show pronounced immunogenicity due to antigen processing, but epitopes with a high score show limited immunogenicity. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by these vaccines in clinical settings.

LNP-RNA-engineered adipose stem cells for accelerated diabetic wound healing.

Adipose stem cells (ASCs) have attracted considerable attention as potential therapeutic agents due to their ability to promote tissue regeneration. However, their limited tissue repair capability has posed a challenge in achieving optimal therapeutic outcomes. Herein, we conceive a series of lipid nanoparticles to reprogram ASCs with durable protein secretion capacity for enhanced tissue engineering and regeneration. In vitro studies identify that the isomannide-derived lipid nanoparticles (DIM1T LNP) efficiently deliver RNAs to ASCs. Co-delivery of self-amplifying RNA (saRNA) and E3 mRNA complex (the combination of saRNA and E3 mRNA is named SEC) using DIM1T LNP modulates host immune responses against saRNAs and facilitates the durable production of proteins of interest in ASCs. The DIM1T LNP-SEC engineered ASCs (DS-ASCs) prolong expression of hepatocyte growth factor (HGF) and C-X-C motif chemokine ligand 12 (CXCL12), which show superior wound healing efficacy over their wild-type and DIM1T LNP-mRNA counterparts in the diabetic cutaneous wound model. Overall, this work suggests LNPs as an effective platform to engineer ASCs with enhanced protein generation ability, expediting the development of ASCs-based cell therapies.

Modulating Antigen Availability in Lymphoid Organs to Shape the Humoral Immune Response to Vaccines.

Primary immune responses following vaccination are initiated in draining lymph nodes, where naive T and B cells encounter Ag and undergo coordinated steps of activation. For humoral immunity, the amount of Ag present over time, its localization to follicles and follicular dendritic cells, and the Ag's structural state all play important roles in determining the subsequent immune response. Recent studies have shown that multiple elements of vaccine design can impact Ag availability in lymphoid tissues, including the choice of adjuvant, physical form of the immunogen, and dosing kinetics. These vaccine design elements affect the transport of Ag to lymph nodes, Ag's localization in the tissue, the duration of Ag availability, and the structural integrity of the Ag. In this review, we discuss these findings and their implications for engineering more effective vaccines, particularly for difficult to neutralize pathogens.

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.

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 (αCD45-Cyt) that, upon injection, decorated the surface of leukocytes in the tumor and tumor-draining lymph node (TDLN) without systemic exposure. αCD45-Cyt therapy eradicated both directly treated tumors and untreated distal lesions in multiple syngeneic mouse tumor models. Mechanistically, αCD45-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.

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.

ALK inhibitors increase ALK expression and sensitize neuroblastoma cells to ALK.CAR-T cells.

Selection of the best tumor antigen is critical for the therapeutic success of chimeric antigen receptor (CAR) T cells in hematologic malignancies and solid tumors. The anaplastic lymphoma kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. Here, we describe the development of ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy.

A combined adjuvant approach primes robust germinal center responses and humoral immunity in non-human primates.

Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.

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 CTL's 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.

Enhanced Vaccine Immunogenicity Enabled by Targeted Cytosolic Delivery of Tumor Antigens into Dendritic Cells.

Molecular vaccines comprising antigen peptides and inflammatory cues make up a class of therapeutics that promote immunity against cancer and pathogenic diseases but often exhibit limited efficacy. Here, we engineered an antigen peptide delivery system to enhance vaccine efficacy by targeting dendritic cells and mediating cytosolic delivery. The delivery system consists of the nontoxic anthrax protein, protective antigen (PA), and a single-chain variable fragment (scFv) that recognizes the XCR1 receptor on dendritic cells (DCs). Combining these proteins enabled selective delivery of the N-terminus of lethal factor (LFN) into XCR1-positive cross-presenting DCs. Incorporating immunogenic epitope sequences into LFN showed selective protein translocation in vitro and enhanced the priming of antigen-specific T cells in vivo. Administering DC-targeted constructs with tumor antigens (Trp1/gp100) into mice bearing aggressive B16-F10 melanomas improved mouse outcomes when compared to free antigen, including suppressed tumor growth up to 58% at 16 days post tumor induction (P < 0.0001) and increased survival (P = 0.03). These studies demonstrate that harnessing DC-targeting anthrax proteins for cytosolic antigen delivery significantly enhances the immunogenicity and antitumor efficacy of cancer vaccines.

Noninvasive In Vivo Imaging of T-Cells during Cancer Immunotherapy Using Rare-Earth Nanoparticles.

Only a minority of patients respond positively to cancer immunotherapy, and addressing this variability is an active area of immunotherapy research. Infiltration of tumors by immune cells is one of the most significant prognostic indicators of response and disease-free survival. However, the ability to noninvasively sample the tumor microenvironment for immune cells remains limited. Imaging in the near-infrared-II region using rare-earth nanocrystals is emerging as a powerful imaging tool for high-resolution deep-tissue imaging. In this paper, we demonstrate that these nanoparticles can be used for noninvasive in vivo imaging of tumor-infiltrating T-cells in a highly aggressive melanoma tumor model. We present nanoparticle synthesis and surface modification strategies for the generation of small, ultrabright, and biocompatible rare-earth nanocrystals necessary for deep tissue imaging of rare cell types. The ability to noninvasively monitor the immune contexture of a tumor during immunotherapy could lead to early identification of nonresponding patients in real time, leading to earlier interventions and better outcomes.

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.

Targeted modulation of immune cells and tissues using engineered biomaterials.

Therapies modulating the immune system offer the prospect of treating a wide range of conditions including infectious diseases, cancer and autoimmunity. Biomaterials can promote specific targeting of immune cell subsets in peripheral or lymphoid tissues and modulate the dosage, timing and location of stimulation, thereby improving safety and efficacy of vaccines and immunotherapies. Here we review recent advances in biomaterials-based strategies, focusing on targeting of lymphoid tissues, circulating leukocytes, tissue-resident immune cells and immune cells at disease sites. These approaches can improve the potency and efficacy of immunotherapies by promoting immunity or tolerance against different diseases.

Decoupled neoantigen cross-presentation by dendritic cells limits anti-tumor immunity against tumors with heterogeneous neoantigen expression.

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.

Optimization of an alum-anchored clinical HIV vaccine candidate.

In the ongoing effort to develop a vaccine against HIV, vaccine approaches that promote strong germinal center (GC) responses may be critical to enable the selection and affinity maturation of rare B cell clones capable of evolving to produce broadly neutralizing antibodies. We previously demonstrated an approach for enhancing GC responses and overall humoral immunity elicited by alum-adjuvanted protein immunization via the use of phosphoserine (pSer) peptide-tagged immunogens that stably anchor to alum particles via ligand exchange with the alum particle surface. Here, using a clinically relevant stabilized HIV Env trimer termed MD39, we systematically evaluated the impact of several parameters relevant to pSer tag composition and trimer immunogen design to optimize this approach, including phosphate valency, amino acid sequence of the trimer C-terminus used for pSer tag conjugation, and structure of the pSer tag. We also tested the impact of co-administering a potent saponin/monophosphoryl lipid A (MPLA) nanoparticle co-adjuvant with alum-bound trimers. We identified MD39 trimer sequences bearing an optimized positively-charged C-terminal amino acid sequence, which, when conjugated to a pSer tag with four phosphates and a polypeptide spacer, bound very tightly to alum particles while retaining a native Env-like antigenicity profile. This optimized pSer-trimer design elicited robust antigen-specific GC B cell and serum IgG responses in mice. Through this optimization, we present a favorable MD39-pSer immunogen construct for clinical translation.