Darrell J. Irvine

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.

Nanoscale objects are typically internalized by cells into membrane-bounded endosomes and fail to access the cytosolic cell machinery. Whereas some biomacromolecules may penetrate or fuse with cell membranes without overt membrane disruption, no synthetic material of comparable size has shown this property yet. Cationic nano-objects pass through cell membranes by generating transient holes, a process associated with cytotoxicity. Studies aimed at generating cell-penetrating nanomaterials have focused on the effect of size, shape and composition. Here, we compare membrane penetration by two nanoparticle 'isomers' with similar composition (same hydrophobic content), one coated with subnanometre striations of alternating anionic and hydrophobic groups, and the other coated with the same moieties but in a random distribution. We show that the former particles penetrate the plasma membrane without bilayer disruption, whereas the latter are mostly trapped in endosomes. Our results offer a paradigm for analysing the fundamental problem of cell-membrane-penetrating bio- and macro-molecules.

An amphiphile vaccine consisting of a peptide antigen or adjuvant cargo linked to a lipophilic tail is shown to have improved potency and safety in mice by targeting the lymph nodes. Vaccines based on purified peptides, proteins or polysaccharides, together with molecular adjuvants designed to boost the immune response, are potentially safer and easier to manufacture than the alternatives. Generally however, such 'subunit' vaccines elicit weaker immune responses than those using live attenuated pathogens. Darrell Irvine and colleagues demonstrate a simple chemical strategy for the molecular targeting of subunit vaccines to lymphoid organs following parenteral injection. They designed an amphiphilic vaccine consisting of a peptide antigen or adjuvant cargo linked to a lipophilic tail. The resulting CpG-DNA/peptide amph-vaccine showed substantially increased anti-tumour efficacy with reduced toxicity compared to the parent compounds. In cancer patients, visual identification of sentinel lymph nodes (LNs) is achieved by the injection of dyes that bind avidly to endogenous albumin, targeting these compounds to LNs, where they are efficiently filtered by resident phagocytes1,2. Here we translate this ‘albumin hitchhiking’ approach to molecular vaccines, through the synthesis of amphiphiles (amph-vaccines) comprising an antigen or adjuvant cargo linked to a lipophilic albumin-binding tail by a solubility-promoting polar polymer chain. Administration of structurally optimized CpG-DNA/peptide amph-vaccines in mice resulted in marked increases in LN accumulation and decreased systemic dissemination relative to their parent compounds, leading to 30-fold increases in T-cell priming and enhanced anti-tumour efficacy while greatly reducing systemic toxicity. Amph-vaccines provide a simple, broadly applicable strategy to simultaneously increase the potency and safety of subunit vaccines.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTSynthetic Nanoparticles for Vaccines and ImmunotherapyDarrell J. Irvine*†‡§∥⊥, Melissa C. Hanson†‡, Kavya Rakhra‡, and Talar Tokatlian‡View Author Information‡ †Department of Biological Engineering, ‡David H. Koch Institute for Integrative Cancer Research, and ∥Department of Materials Science & Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States§ The Ragon Institute of MGH, Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, Massachusetts 02139, United States⊥ Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, United States*Phone: (617) 452-4174. E-mail: [email protected]Cite this: Chem. Rev. 2015, 115, 19, 11109–11146Publication Date (Web):July 8, 2015Publication History Received19 February 2015Published online8 July 2015Published inissue 14 October 2015https://doi.org/10.1021/acs.chemrev.5b00109Copyright © 2015 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views14483Altmetric-Citations542LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (18 MB) Get e-AlertsSUBJECTS:Antigens,Cells,Immunology,Nanoparticles,Vaccination Get e-Alerts

A major limitation of cell therapies is the rapid decline in viability and function of the transplanted cells. Here we describe a strategy to enhance cell therapy via the conjugation of adjuvant drug-loaded nanoparticles to the surfaces of therapeutic cells. With this method of providing sustained pseudoautocrine stimulation to donor cells, we elicited marked enhancements in tumor elimination in a model of adoptive T cell therapy for cancer. We also increased the in vivo repopulation rate of hematopoietic stem cell grafts with very low doses of adjuvant drugs that were ineffective when given systemically. This approach is a simple and generalizable strategy to augment cytoreagents while minimizing the systemic side effects of adjuvant drugs. In addition, these results suggest therapeutic cells are promising vectors for actively targeted drug delivery.

Therapeutic targeting of the immune system in cancer is now a clinical reality and marked successes have been achieved, most notably through the use of checkpoint blockade antibodies and chimeric antigen receptor T cell therapy. However, efforts to develop new immunotherapy agents or combination treatments to increase the proportion of patients who benefit have met with challenges of limited efficacy and/or significant toxicity. Nanomedicines — therapeutics composed of or formulated in carrier materials typically smaller than 100 nm — were originally developed to increase the uptake of chemotherapy agents by tumours and to reduce their off-target toxicity. Here, we discuss how nanomedicine-based treatment strategies are well suited to immunotherapy on the basis of nanomaterials’ ability to direct immunomodulators to tumours and lymphoid organs, to alter the way biologics engage with target immune cells and to accumulate in myeloid cells in tumours and systemic compartments. We also discuss early efforts towards clinical translation of nanomedicine-based immunotherapy. Getting medicines to the right place at the right time is key to their success. Nanomedicines — drugs formulated in carrier materials that are smaller than 100 nm — show promise in enhancing tumour immunotherapy, owing to the ability to focus their action on target tissues and control their immunomodulatory properties, as reviewed here.

Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8(+) T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8(+) T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.

Vaccines aim to protect against or treat diseases through manipulation of the immune response, promoting either immunity or tolerance. In the former case, vaccines generate antibodies and T cells poised to protect against future pathogen encounter or attack diseased cells such as tumours; in the latter case, which is far less developed, vaccines block pathogenic autoreactive T cells and autoantibodies that target self tissue. Enormous challenges remain, however, as a consequence of our incomplete understanding of human immunity. A rapidly growing field of research is the design of vaccines based on synthetic materials to target organs, tissues, cells or intracellular compartments; to co-deliver immunomodulatory signals that control the quality of the immune response; or to act directly as immune regulators. There exists great potential for well-defined materials to further our understanding of immunity. Here we describe recent advances in the design of synthetic materials to direct immune responses, highlighting successes and challenges in prophylactic, therapeutic and tolerance-inducing vaccines.

Adoptive cell therapy (ACT) with antigen-specific T cells has shown remarkable clinical success; however, approaches to safely and effectively augment T cell function, especially in solid tumors, remain of great interest. Here we describe a strategy to 'backpack' large quantities of supporting protein drugs on T cells by using protein nanogels (NGs) that selectively release these cargos in response to T cell receptor activation. We designed cell surface-conjugated NGs that responded to an increase in T cell surface reduction potential after antigen recognition and limited drug release to sites of antigen encounter, such as the tumor microenvironment. By using NGs that carried an interleukin-15 super-agonist complex, we demonstrated that, relative to systemic administration of free cytokines, NG delivery selectively expanded T cells 16-fold in tumors and allowed at least eightfold higher doses of cytokine to be administered without toxicity. The improved therapeutic window enabled substantially increased tumor clearance by mouse T cell and human chimeric antigen receptor (CAR)-T cell therapy in vivo.

An immunotherapy consisting of a tumor-antigen targeting antibody, PD-1 blocking antibody, extended half-life recombinant IL-2 and a lymph-node-targeted T cell vaccine mobilized innate and adaptive immunity and eradicated large established tumors in a variety of mouse models. Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte–associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8+ T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.

Integrin-mediated cell adhesion is central to cell survival,differentiation and motility. Many cell responses induced by integrins require both receptor occupancy and receptor aggregation, and appear to be regulated by both biochemical and biophysical means. Multidomain extracellular matrix molecules may serve to foster integrin aggregation by presenting local clusters of adhesion ligands, a hypothesis supported by studies with synthetic substrates showing that cell adhesion and migration are enhanced when adhesion ligands are presented in nanoscale clusters. Here, we used a novel synthetic polymer system to present the adhesion ligand GRGDSPK in nanoscale clusters with 1.7, 3.6 or 5.4 peptides per cluster against a non-adhesive background,where the peptide is mobile on a 2 nm polyethylene oxide tether. Average ligand density ranged from 190 to 5270 RGD/μm2. We used these substrates to study the effects of ligand density and clustering on adhesion of wild-type NR6 fibroblasts, which expressα vβ3 andα 5β1, integrins known to bind to linear RGD peptides. The strength of cell-substratum adhesion was quantified using a centrifugal detachment assay to assess the relative number of cells remaining adherent after a 10 minute application of defined distraction force. An unusual relationship between cell detachment and distraction force at relatively low values of applied force was found on substrates presenting the clustered ligand. Although a monotonic decrease in the number of cells remaining attached would be expected with increasing force on all substrates,we instead observed a peak (adhesion reinforcement) in this profile for certain ligand conditions. On substrates presenting clustered ligands, the fraction of cells remaining attached increased as the distraction force was increased to between 70 and 150 pN/cell, then decreased for higher forces. This phenomenon was only observed on substrates presenting higher ligand cluster sizes (n=3.6 or n=5.4) and was more pronounced at higher ligand densities. Adhesion reinforcement was not observed on fibronectin-coated surfaces. These results support previous studies showing that biophysical cues such as ligand spatial arrangement and extracellular matrix rigidity are central to the governance of cell responses to the external environment.

Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptors--allowing complex phenotypes to be studied. We demonstrate the adaptability of this high-throughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function.

The immune system can be a cure or cause of disease, fulfilling a protective role in attacking cancer or pathogenic microbes but also causing tissue destruction in autoimmune disorders. Thus, therapies aimed to amplify or suppress immune reactions are of great interest. However, the complex regulation of the immune system, coupled with the potential systemic side effects associated with traditional systemic drug therapies, has presented a major hurdle for the development of successful immunotherapies. Recent progress in the design of synthetic micro- and nano-particles that can target drugs, deliver imaging agents, or stimulate immune cells directly through their physical and chemical properties is leading to new approaches to deliver vaccines, promote immune responses against tumors, and suppress autoimmunity. In addition, novel strategies, such as the use of particle-laden immune cells as living targeting agents for drugs, are providing exciting new approaches for immunotherapy. This progress report describes recent advances in the design of micro- and nano-particles for immunotherapies and diagnostics.

Abstract Targeted delivery of compounds to particular cell subsets can enhance therapeutic index by concentrating their action on the cells of interest. Because attempts to target tumors directly have yielded limited benefit, we instead target endogenous immune cell subsets in the circulation that can migrate actively into tumors. We describe antibody-targeted nanoparticles that bind to CD8 + T cells in the blood, lymphoid tissues, and tumors of mice. PD-1 + T cells are successfully targeted in the circulation and tumor. The delivery of an inhibitor of TGFβ signaling to PD-1-expressing cells extends the survival of tumor-bearing mice, whereas free drugs have no effect at such doses. This modular platform also enables PD-1-targeted delivery of a TLR7/8 agonist to the tumor microenvironment, increasing the proportion of tumor-infiltrating CD8 + T cells and sensitizing tumors to subsequent anti-PD-1. Targeted delivery of immunotherapy to defined subsets of endogenous leukocytes may be superior to administration of free drugs.

Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.

Cyclic dinucleotides (CDNs) are agonists of stimulator of IFN genes (STING) and have potential as vaccine adjuvants. However, cyclic di-GMP (cdGMP) injected s.c. shows minimal uptake into lymphatics/draining lymph nodes (dLNs) and instead is rapidly distributed to the bloodstream, leading to systemic inflammation. Here, we encapsulated cdGMP within PEGylated lipid nanoparticles (NP-cdGMP) to redirect this adjuvant to dLNs. Compared with unformulated CDNs, encapsulation blocked systemic dissemination and markedly enhanced dLN accumulation in murine models. Delivery of NP-cdGMP increased CD8+ T cell responses primed by peptide vaccines and enhanced therapeutic antitumor immunity. A combination of a poorly immunogenic liposomal HIV gp41 peptide antigen and NP-cdGMP robustly induced type I IFN in dLNs, induced a greater expansion of vaccine-specific CD4+ T cells, and greatly increased germinal center B cell differentiation in dLNs compared with a combination of liposomal HIV gp41 and soluble CDN. Further, NP-cdGMP promoted durable antibody titers that were substantially higher than those promoted by the well-studied TLR agonist monophosphoryl lipid A and comparable to a much larger dose of unformulated cdGMP, without the systemic toxicity of the latter. These results demonstrate that nanoparticulate delivery safely targets CDNs to the dLNs and enhances the efficacy of this adjuvant. Moreover, this approach can be broadly applied to other small-molecule immunomodulators of interest for vaccines and immunotherapy.

The need to optimize vaccine potency while minimizing toxicity in healthy recipients has motivated studies of the formulation of vaccines to control how, when, and where antigens and adjuvants encounter immune cells and other cells/tissues following administration. An effective subunit vaccine must traffic to lymph nodes (LNs), activate both the innate and adaptive arms of the immune system, and persist for a sufficient time to promote a mature immune response. Here, we review approaches to tailor these three aspects of vaccine function through optimized formulations. Traditional vaccine adjuvants activate innate immune cells, promote cell-mediated transport of antigen to lymphoid tissues, and promote antigen retention in LNs. Recent studies using nanoparticles and other lymphatic-targeting strategies suggest that direct targeting of antigens and adjuvant compounds to LNs can also enhance vaccine potency without sacrificing safety. The use of formulations to regulate biodistribution and promote antigen and inflammatory cue co-uptake in immune cells may be important for next-generation molecular adjuvants. Finally, strategies to program vaccine kinetics through novel formulation and delivery strategies provide another means to enhance immune responses independent of the choice of adjuvant. These technologies offer the prospect of enhanced efficacy while maintaining high safety profiles necessary for successful vaccines.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.

Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment. Amph-ligand boosting triggered massive CAR-T expansion, increased donor cell polyfunctionality, and enhanced antitumor efficacy in multiple immunocompetent mouse tumor models. We demonstrate two approaches to generalizing this strategy to any chimeric antigen receptor, enabling this simple non-human leukocyte antigen-restricted approach to enhanced CAR-T functionality to be applied to existing CAR-T designs.

For subunit vaccines, adjuvants play a key role in shaping immunological memory. Nanoparticle (NP) delivery systems for antigens and/or molecular danger signals are promising adjuvants capable of promoting both cellular and humoral immune responses, but in most cases the mechanisms of action of these materials are poorly understood. Here, we studied the immune response elicited by NPs composed of multilamellar "stapled" lipid vesicles carrying a recombinant Plasmodium vivax circumsporozoite antigen, VMP001, both entrapped in the aqueous core and anchored to the lipid bilayer surfaces. Immunization with these particles and monophosphoryl lipid A (MPLA), a US Food and Drug Administration-approved immunostimulatory agonist for Toll-like receptor-4, promoted high-titer, high-avidity antibody responses against VMP001, lasting more than 1 y in mice at 10-fold lower doses than conventional adjuvants. Compared to soluble VMP001 mixed with MPLA, VMP001-NPs promoted broader humoral responses, targeting multiple epitopes of the protein and a more balanced Th1/Th2 cytokine profile from antigen-specific T cells. To begin to understand the underlying mechanisms, we examined components of the B-cell response and found that NPs promoted robust germinal center (GC) formation at low doses of antigen where no GC induction occurred with soluble protein immunization, and that GCs nucleated near depots of NPs accumulating in the draining lymph nodes over time. In parallel, NP vaccination enhanced the expansion of antigen-specific follicular helper T cells (T(fh)), compared to vaccinations with soluble VMP001 or alum. Thus, NP vaccines may be a promising strategy to enhance the durability, breadth, and potency of humoral immunity by enhancing key elements of the B-cell response.

Significance We explored the effect of nontraditional vaccine dosing profiles on antibody titers of vaccines and discovered that certain dosing profiles demonstrate >10-fold higher antibody production than the traditional single-dose prime–boost method. We also present a computational model that captures the experimental results and provides a mechanistic understanding of the biology behind the effectiveness of our strategy. This work has clinical significance in vaccine design because it is a simple method to increase the efficacy of subunit vaccines, which may lead to the development of efficacious vaccines for diseases such as HIV.

<h2>Summary</h2> The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation.

Polycations that absorb protons in response to the acidification of endosomes can theoretically disrupt these vesicles via the "proton sponge" effect. To exploit this mechanism, we created nanoparticles with a segregated core-shell structure for efficient, noncytotoxic intracellular drug delivery. Cross-linked polymer nanoparticles were synthesized with a pH-responsive core and hydrophilic charged shell designed to disrupt endosomes and mediate drug/cell binding, respectively. By sequestering the relatively hydrophobic pH-responsive core component within a more hydrophilic pH-insensitive shell, nontoxic delivery of small molecules and proteins to the cytosol was achieved in dendritic cells, a key cell type of interest in the context of vaccines and immunotherapy.

Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B-cell activation. However, the effects of antigen copy number, spacing and affinity, as well as the dimensionality and rigidity of scaffold presentation on B-cell activation remain poorly understood. Here, we display the clinical vaccine immunogen eOD-GT8, an engineered outer domain of the HIV-1 glycoprotein-120, on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B-cell activation in vitro. We find that B-cell signalling is maximized by as few as five antigens maximally spaced on the surface of a 40-nm viral-like nanoparticle. Increasing antigen spacing up to ~25-30 nm monotonically increases B-cell receptor activation. Moreover, scaffold rigidity is essential for robust B-cell triggering. These results reveal molecular vaccine design principles that may be used to drive functional B-cell responses.

Subunit vaccines offer a safer alternative to traditional organism-based vaccines, but their immunogenicity is impaired. This hurdle might be overcome by the use of micro- and nanodelivery systems carrying the antigen(s). This review discusses the rationale for the use of particulate vaccines and provides an overview of antigen-delivery vehicles currently under investigation. It further highlights the cellular uptake, antigen processing and the presentation by antigen-presenting cells because these processes are partially governed by particle characteristics and eventually determine the immunological outcome. Finally, we address the attractive concept of concomitant delivery of antigens and immunopotentiators. The condensed knowledge could be an asset for rationally designing antigen-delivery vehicles to obtain safe and efficacious vaccines.

DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These 'multilayer tattoo' DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination.

Tumor cells disseminate into compartments that are poorly accessible from circulation, which necessitates high doses of systemic chemotherapy. However, the effectiveness of many drugs, such as the potent topoisomerase I poison SN-38, is hampered by poor pharmacokinetics. To deliver SN-38 to lymphoma tumors in vivo, we took advantage of the fact that healthy lymphocytes can be programmed to phenocopy the biodistribution of the tumor cells. In a murine model of disseminated lymphoma, we expanded autologous polyclonal T cells ex vivo under conditions that retained homing receptors mirroring lymphoma cells, and functionalized these T cells to carry SN-38-loaded nanocapsules on their surfaces. Nanocapsule-functionalized T cells were resistant to SN-38 but mediated efficient killing of lymphoma cells in vitro. Upon adoptive transfer into tumor-bearing mice, these T cells served as active vectors to deliver the chemotherapeutic into tumor-bearing lymphoid organs. Cell-mediated delivery concentrated SN-38 in lymph nodes at levels 90-fold greater than free drug systemically administered at 10-fold higher doses. The live T cell delivery approach reduced tumor burden significantly after 2 weeks of treatment and enhanced survival under conditions where free SN-38 and SN-38-loaded nanocapsules alone were ineffective. These results suggest that tissue-homing lymphocytes can serve as specific targeting agents to deliver nanoparticles into sites difficult to access from the circulation, and thus improve the therapeutic index of chemotherapeutic drugs with unfavorable pharmacokinetics.

Anionic, monolayer-protected gold nanoparticles (AuNPs) have been shown to nondisruptively penetrate cellular membranes. Here, we show that a critical first step in the penetration process is potentially the fusion of such AuNPs with lipid bilayers. Free energy calculations, experiments on unilamellar and multilamellar vesicles, and cell studies all support this hypothesis. Furthermore, we show that fusion is only favorable for AuNPs with core diameters below a critical size that depends on the monolayer composition.

HIV glycans and nanoparticle vaccines Synthetic nanoparticles have attracted widespread interest for vaccine design, but how the immune system generates a response to multimeric nanoparticles remains unclear. Tokatlian et al. studied immunity generated by HIV envelope antigens arranged in either multivalent nanoparticle forms or as single monomers (see the Perspective by Wilson). The nanoparticle HIV immunogens triggered greater antibody responses compared with the monomeric forms. Glycosylation appeared key for enhanced humoral immunity because it spurred binding to mannose-binding lectin, complement fixation, and antigen trafficking to follicular dendritic cells. The findings highlight how the innate immune system recognizes HIV nanoparticles and the importance of antigen glycosylation in the design of next-generation nano-based vaccines. Science , this issue p. 649 ; see also p. 584

Cancer immunotherapy is now a powerful clinical reality, with a steady progression of new drug approvals and a massive pipeline of additional treatments in clinical and preclinical development. However, modulation of the immune system can be a double-edged sword: Drugs that activate immune effectors are prone to serious non-specific systemic inflammation and autoimmune side effects. Drug delivery technologies have an important role to play in harnessing the power of immune therapeutics while avoiding on-target/off-tumor toxicities. Here we review mechanisms of toxicity for clinically-relevant immunotherapeutics, and discuss approaches based in drug delivery technology to enhance the safety and potency of these treatments. These include strategies to merge drug delivery with adoptive cellular therapies, targeting immunotherapies to tumors or select immune cells, and localizing therapeutics intratumorally. Rational design employing lessons learned from the drug delivery and nanomedicine fields has the potential to facilitate immunotherapy reaching its full potential.

Biodegradable core--shell structured nanoparticles with a poly(β-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell were developed for in vivo mRNA delivery with a view toward delivery of mRNA-based vaccines. The pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. Messenger RNA was efficiently adsorbed via electrostatic interactions onto the surface of these net positively charged nanoparticles. In vitro, mRNA-loaded particle uptake by dendritic cells led to mRNA delivery into the cytosol with low cytotoxicity, followed by translation of the encoded protein in these difficult-to-transfect cells at a frequency of ~30%. Particles loaded with mRNA administered intranasally (i.n.) in mice led to the expression of the reporter protein luciferase in vivo as soon as 6 h after administration, a time point when naked mRNA given i.n. showed no expression. At later time points, luciferase expression was detected in naked mRNA-treated mice, but this group showed a wide variation in levels of transfection, compared to particle-treated mice. This system may thus be promising for noninvasive delivery of mRNA-based vaccines.

Therapeutic treatments based on the injection of living cells are in clinical use and preclinical development for diseases ranging from cancer to cardiovascular disease to diabetes. To enhance the function of therapeutic cells, a variety of chemical and materials science strategies are being developed that engineer the surface of therapeutic cells with new molecules, artificial receptors, and multifunctional nanomaterials, synthetically endowing donor cells with new properties and functions. These approaches offer a powerful complement to traditional genetic engineering strategies for enhancing the function of living cells.

Recent studies have demonstrated a simple, potentially universal strategy to enhance vaccine potency, via intralymph node (i.LN) injection. To date, intranodal immunization studies have focused on the delivery of unadjuvanted vaccines (e.g., naked DNA, peptide, or protein). We hypothesized that combining i.LN vaccination with controlled release biomaterials permitting sustained dosing of molecular adjuvants to the local tissue microenvironment would further enhance this promising vaccination strategy. To test this idea, we encapsulated the Toll-like receptor-3 ligand poly(inosinic:cytidylic acid) (polyIC) in biodegradable poly(lactide- co -glycolide) microparticles (MPs) designed to remain extracellular and release polyIC in the LN over several days. Intranodal injection of MPs increased persistence of polyIC in LNs compared to the same dose of soluble polyIC or polyIC formulated in nanoparticles, leading to increased accumulation of Toll-like receptor agonist in LN-resident antigen presenting cells and more enduring dendritic cell activation. Intralymph node injection of ovalbumin mixed with polyIC-releasing MPs enhanced the humoral response and expanded ovalbumin-specific T cells to frequencies as high as 18% among all CD8 + cells following a single injection (8.2-fold greater than the same vaccine given i.m.), a response that could not be matched by antigen mixed with polyIC-loaded nanoparticles or a 10-fold greater dose of soluble polyIC. Thus, i.LN immunization with slow release-formulated adjuvants may be a broadly applicable strategy to enhance therapeutic or prophylactic vaccines.

Despite their potential, conventional whole-cell cancer vaccines prepared by freeze–thawing or irradiation have shown limited therapeutic efficacy in clinical trials. Recent studies have indicated that cancer cells treated with certain chemotherapeutics, such as mitoxantrone, can undergo immunogenic cell death (ICD) and initiate antitumor immune responses. However, it remains unclear how to exploit ICD for cancer immunotherapy. Here, we present a new material-based strategy for converting immunogenically dying tumor cells into a powerful platform for cancer vaccination and demonstrate their therapeutic potential in murine models of melanoma and colon carcinoma. We have generated immunogenically dying tumor cells surface-modified with adjuvant-loaded nanoparticles. Dying tumor cells laden with adjuvant nanodepots efficiently promote activation and antigen cross-presentation by dendritic cells in vitro and elicit robust antigen-specific CD8α+ T-cells in vivo. Furthermore, whole tumor-cell vaccination combined with immune checkpoint blockade leads to complete tumor regression in ∼78% of CT26 tumor-bearing mice and establishes long-term immunity against tumor recurrence. Our strategy presented here may open new doors to “personalized” cancer immunotherapy tailored to individual patient’s tumor cells.

Resting memory CD4+ T-cells harboring latent HIV proviruses represent a critical barrier to viral eradication. Histone deacetylase inhibitors (HDACis), such as suberanilohydroxamic acid (SAHA), romidepsin, and panobinostat have been shown to induce HIV expression in these resting cells. Recently, it has been demonstrated that the low levels of viral gene expression induced by a candidate HDACi may be insufficient to cause the death of infected cells by viral cytopathic effects, necessitating their elimination by immune effectors, such as cytotoxic T-lymphocytes (CTL). Here, we study the impact of three HDACis in clinical development on T-cell effector functions. We report two modes of HDACi-induced functional impairment: i) the rapid suppression of cytokine production from viable T-cells induced by all three HDACis ii) the selective death of activated T-cells occurring at later time-points following transient exposures to romidepsin or, to a lesser extent, panobinostat. As a net result of these factors, HDACis impaired CTL-mediated IFN-γ production, as well as the elimination of HIV-infected or peptide-pulsed target cells, both in liquid culture and in collagen matrices. Romidepsin exerted greater inhibition of antiviral function than SAHA or panobinostat over the dose ranges tested. These data suggest that treatment with HDACis to mobilize the latent reservoir could have unintended negative impacts on the effector functions of CTL. This could influence the effectiveness of HDACi-based eradication strategies, by impairing elimination of infected cells, and is a critical consideration for trials where therapeutic interruptions are being contemplated, given the importance of CTL in containing rebound viremia.

Immunostimulatory agents such as agonistic anti-CD137 and interleukin (IL)−2 generate effective anti-tumor immunity but also elicit serious toxicities, hampering their clinical application. Here we show that combination therapy with anti-CD137 and an IL-2-Fc fusion achieves significant initial anti-tumor activity, but also lethal immunotoxicity deriving from stimulation of circulating leukocytes. To overcome this toxicity, we demonstrate that anchoring IL-2 and anti-CD137 on the surface of liposomes allows these immune agonists to rapidly accumulate in tumors while lowering systemic exposure. In multiple tumor models, immunoliposome delivery achieves anti-tumor activity equivalent to free IL-2/anti-CD137 but with the complete absence of systemic toxicity. Immunoliposomes stimulated tumor infiltration by cytotoxic lymphocytes, cytokine production, and granzyme expression, demonstrating equivalent immunostimulatory effects to the free drugs in the local tumor microenvironment. Thus, surface-anchored particle delivery may provide a general approach to exploit the potent stimulatory activity of immune agonists without debilitating systemic toxicities. Immunostimulatory agents used in cancer treatment often elicit serious toxicities, limiting their clinical application. Here, the authors show that the use of liposomes to intravenously deliver surface-anchored IL-2 and anti-CD137 proteins enables anti-cancer immunity and reduces the toxic side effects.

Adjuvants are central to the efficacy of subunit vaccines. Aluminum hydroxide (alum) is the most commonly used vaccine adjuvant, yet its adjuvanticity is often weak and mechanisms of triggering antibody responses remain poorly understood. We demonstrate that site-specific modification of immunogens with short peptides composed of repeating phosphoserine (pSer) residues enhances binding to alum and prolongs immunogen bioavailability. The pSer-modified immunogens formulated in alum elicited greatly increased germinal center, antibody, neutralizing antibody, memory and long-lived plasma cell responses compared to conventional alum-adsorbed immunogens. Mechanistically, pSer-immunogen:alum complexes form nanoparticles that traffic to lymph nodes and trigger B cell activation through multivalent and oriented antigen display. Direct uptake of antigen-decorated alum particles by B cells upregulated antigen processing and presentation pathways, further enhancing B cell activation. These data provide insights into mechanisms of action of alum and introduce a readily translatable approach to significantly improve humoral immunity to subunit vaccines using a clinical adjuvant.

Generation of potent antibodies by a mutation-selection process called affinity maturation is a key component of effective immune responses. Antibodies that protect against highly mutable pathogens must neutralize diverse strains. Developing effective immunization strategies to drive their evolution requires understanding how affinity maturation happens in an environment where variants of the same antigen are present. We present an in silico model of affinity maturation driven by antigen variants which reveals that induction of cross-reactive antibodies often occurs with low probability because conflicting selection forces, imposed by different antigen variants, can frustrate affinity maturation. We describe how variables such as temporal pattern of antigen administration influence the outcome of this frustrated evolutionary process. Our calculations predict, and experiments in mice with variant gp120 constructs of the HIV envelope protein confirm, that sequential immunization with antigen variants is preferred over a cocktail for induction of cross-reactive antibodies focused on the shared CD4 binding site epitope.

Immunostimulatory agonists such as anti-CD137 and interleukin (IL)-2 have elicited potent antitumor immune responses in preclinical studies, but their clinical use is limited by inflammatory toxicities that result upon systemic administration. We hypothesized that by rigorously restricting the biodistribution of immunotherapeutic agents to a locally accessible lesion and draining lymph node(s), effective local and systemic antitumor immunity could be achieved in the absence of systemic toxicity. We anchored anti-CD137 and an engineered IL-2Fc fusion protein to the surfaces of PEGylated liposomes, whose physical size permitted dissemination in the tumor parenchyma and tumor-draining lymph nodes but blocked entry into the systemic circulation following intratumoral injection. In the B16F10 melanoma model, intratumoral liposome-coupled anti-CD137 + IL-2Fc therapy cured a majority of established primary tumors while avoiding the lethal inflammatory toxicities caused by equivalent intratumoral doses of soluble immunotherapy. Immunoliposome therapy induced protective antitumor memory and elicited systemic antitumor immunity that significantly inhibited the growth of simultaneously established distal tumors. Tumor inhibition was CD8(+) T-cell-dependent and was associated with increased CD8(+) T-cell infiltration in both treated and distal tumors, enhanced activation of tumor antigen-specific T cells in draining lymph nodes, and a reduction in regulatory T cells in treated tumors. These data suggest that local nanoparticle-anchored delivery of immuno-agonists represents a promising strategy to improve the therapeutic window and clinical applicability of highly potent but otherwise intolerable regimens of cancer immunotherapy. Cancer Res; 73(5); 1547-58. ©2012 AACR.

Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin 'foci'. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response.

<h2>Summary</h2> How antigen valency affects B cells <i>in vivo</i> during immune responses is not well understood. Here, using HIV immunogens with defined valencies ranging from 1 to 60, we investigated the role of antigen valency during different phases of B cell responses <i>in vivo</i>. Highly multimerized immunogens preferentially rapidly activated cognate B cells, with little affinity discrimination. This led to strong early induction of the transcription factors IRF4 (interferon regulatory factor 4) and Bcl6, driving both early extrafollicular plasma cell and germinal center responses, in a CD4⁺ T-cell-dependent manner, involving B cells with a broad range of affinities. Low-valency antigens induced smaller effector B cell responses, with preferential recruitment of high-affinity B cells. Thus, antigen valency has multifaceted effects on B cell responses and can dictate affinity thresholds and competitive landscapes for B cells <i>in vivo</i>, with implications for vaccine design.

Sustained exposure of lymphoid tissues to vaccine antigens promotes humoral immunity, but traditional bolus immunizations lead to rapid antigen clearance. We describe a technology to tailor vaccine kinetics in a needle-free platform translatable to human immunization. Solid pyramidal microneedle (MN) arrays were fabricated with silk fibroin protein tips encapsulating a stabilized HIV envelope trimer immunogen and adjuvant, supported on a dissolving polymer base. Upon brief skin application, vaccine-loaded silk tips are implanted in the epidermis/upper dermis where they release vaccine over a time period determined by the crystallinity of the silk matrix. Following MN immunization in mice, Env trimer was released over 2 wk in the skin, correlating with increased germinal center (GC) B cell responses, a ∼1,300-fold increase in serum IgG titers and a 16-fold increase in bone marrow (BM) plasma cells compared with bolus immunization. Thus, implantable MNs provide a practical means to substantially enhance humoral immunity to subunit vaccines.

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody–envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires. The immunogen RC1 facilitates recognition of the V3-glycan patch on the envelope of HIV-1 and elicits specific serological responses in mice and macaques, making it a possible priming immunogen for sequential vaccination strategies in humans.

Vaccines are one of the most powerful technologies supporting public health. The adaptive immune response induced by immunization arises following appropriate activation and differentiation of T and B cells in lymph nodes. Among many parameters impacting the resulting immune response, the presence of antigen and inflammatory cues for an appropriate temporal duration within the lymph nodes, and further within appropriate subcompartments of the lymph nodes- the right timing and location- play a critical role in shaping cellular and humoral immunity. Here we review recent advances in our understanding of how vaccine kinetics and biodistribution impact adaptive immunity, and the underlying immunological mechanisms that govern these responses. We discuss emerging approaches to engineer these properties for future vaccines, with a focus on subunit vaccines.

Collagen-localized cytokines potentiate disparate systemic cancer immunotherapies while minimizing toxicity in many tumor models.

Minimally invasive technologies that can sample and detect cell-free nucleic acid biomarkers from liquid biopsies have recently emerged as clinically useful for early diagnosis of a broad range of pathologies, including cancer. Although blood has so far been the most commonly interrogated bodily fluid, skin interstitial fluid has been mostly overlooked despite containing the same broad variety of molecular biomarkers originating from cells and surrounding blood capillaries. Emerging technologies to sample this fluid in a pain-free and minimally-invasive manner often take the form of microneedle patches. Herein, we developed microneedles that are coated with an alginate-peptide nucleic acid hybrid material for sequence-specific sampling, isolation, and detection of nucleic acid biomarkers from skin interstitial fluid. Characterized by fast sampling kinetics and large sampling capacity (∼6.5 μL in 2 min), this platform technology also enables the detection of specific nucleic acid biomarkers either on the patch itself or in solution after light-triggered release from the hydrogel. Considering the emergence of cell-free nucleic acids in bodily fluids as clinically informative biomarkers, platform technologies that can detect them in an automated and minimally invasive fashion have great potential for personalized diagnosis and longitudinal monitoring of patient-specific disease progression.

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ∼1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained.

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist 'ALT-803', an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies.

Activation of the innate immune STimulator of INterferon Genes (STING) pathway potentiates antitumour immunity, but systemic delivery of STING agonists to tumours is challenging. We conjugated STING-activating cyclic dinucleotides (CDNs) to PEGylated lipids (CDN-PEG-lipids; PEG, polyethylene glycol) via a cleavable linker and incorporated them into lipid nanodiscs (LNDs), which are discoid nanoparticles formed by self-assembly. Compared to state-of-the-art liposomes, intravenously administered LNDs carrying CDN-PEG-lipid (LND-CDNs) exhibited more efficient penetration of tumours, exposing the majority of tumour cells to STING agonist. A single dose of LND-CDNs induced rejection of established tumours, coincident with immune memory against tumour rechallenge. Although CDNs were not directly tumoricidal, LND-CDN uptake by cancer cells correlated with robust T-cell activation by promoting CDN and tumour antigen co-localization in dendritic cells. LNDs thus appear promising as a vehicle for robust delivery of compounds throughout solid tumours, which can be exploited for enhanced immunotherapy.

Therapies that synergistically stimulate immunogenic cancer cell death (ICD), inflammation, and immune priming are of great interest for cancer immunotherapy. However, even multi-agent therapies often fail to trigger all of the steps necessary for self-sustaining anti-tumor immunity. Here we describe self-replicating RNAs encapsulated in lipid nanoparticles (LNP-replicons), which combine three key elements: (1) an LNP composition that potently promotes ICD, (2) RNA that stimulates danger sensors in transfected cells, and (3) RNA-encoded IL-12 for modulation of immune cells. Intratumoral administration of LNP-replicons led to high-level expression of IL-12, stimulation of a type I interferon response, and cancer cell ICD, resulting in a highly inflamed tumor microenvironment and priming of systemic anti-tumor immunity. In several mouse models of cancer, a single intratumoral injection of replicon-LNPs eradicated large established tumors, induced protective immune memory, and enabled regression of distal uninjected tumors. LNP-replicons are thus a promising multifunctional single-agent immunotherapeutic.

Imidazoquinoline derivatives (IMDs) and related compounds function as synthetic agonists of Toll-like receptors 7 and 8 (TLR7/8) and one is FDA approved for topical antiviral and skin cancer treatments. Nevertheless, these innate immune system-activating drugs have potentially much broader therapeutic utility; they have been pursued as antitumor immunomodulatory agents and more recently as candidate vaccine adjuvants for cancer and infectious disease. The broad expression profiles of TLR7/8, poor pharmacokinetic properties of IMDs, and toxicities associated with systemic administration, however, are formidable barriers to successful clinical translation. Herein, we review IMD formulations that have advanced to the clinic and discuss issues related to biodistribution and toxicity that have hampered the further development of these compounds. Recent strategies aimed at enhancing safety and efficacy, particularly through the use of bioconjugates and nanoparticle formulations that alter pharmacokinetics, biodistribution, and cellular targeting, are described. Finally, key aspects of the biology of TLR7 signaling, such as TLR7 tolerance, that may need to be considered in the development of new IMD therapeutics are discussed.

Significance Most of the global population resides in low- and middle-income countries, where current vaccines for COVID-19 remain largely unavailable. For the COVID-19 pandemic, the world will need access to &gt;10 billion doses of vaccines, or more than double the annual volume of vaccines for all other diseases. Many vaccine candidates use the SARS-CoV-2 receptor-binding domain (RBD) antigen. Here, we present an engineered RBD with improved production titers in Pichia pastoris , a yeast commonly used for large-scale, low-cost manufacturing by vaccine manufacturers. The modified RBD also raises an enhanced immune response in mice relative to the Wuhan-Hu-1 sequence used in current candidates. These combined traits make it a promising candidate for next-generation vaccines addressing emerging variants of the virus.

Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses. Using HIV Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B cells lasting at least 6 months, showing promise in regard to difficult vaccine targets.

Anti-tumour inflammatory cytokines are highly toxic when administered systemically. Here, in multiple syngeneic mouse models, we show that the intratumoural injection of recombinantly expressed cytokines bound tightly to the common vaccine adjuvant aluminium hydroxide (alum) (via ligand exchange between hydroxyls on the surface of alum and phosphoserine residues tagged to the cytokine by an alum-binding peptide) leads to weeks-long retention of the cytokines in the tumours, with minimal side effects. Specifically, a single dose of alum-tethered interleukin-12 induced substantial interferon-γ-mediated T-cell and natural-killer-cell activities in murine melanoma tumours, increased tumour antigen accumulation in draining lymph nodes and elicited robust tumour-specific T-cell priming. Moreover, intratumoural injection of alum-anchored cytokines enhanced responses to checkpoint blockade, promoting cures in distinct poorly immunogenic syngeneic tumour models and eliciting control over metastases and distant untreated lesions. Intratumoural treatment with alum-anchored cytokines represents a safer and tumour-agnostic strategy to improving local and systemic anticancer immunity.

Immune cells are being engineered to recognize and respond to disease states, acting as a "living drug" when transferred into patients. Therapies based on engineered immune cells are now a clinical reality, with multiple engineered T cell therapies approved for treatment of hematologic malignancies. Ongoing preclinical and clinical studies are testing diverse strategies to modify the fate and function of immune cells for applications in cancer, infectious disease, and beyond. Here, we discuss current progress in treating human disease with immune cell therapeutics, emerging strategies for immune cell engineering, and challenges facing the field, with a particular emphasis on the treatment of cancer, where the most effort has been applied to date.

Proton leakage from organelles is a common signal for noncanonical light chain 3B (LC3B) lipidation and inflammasome activation, processes induced upon stimulator of interferon genes (STING) activation. On the basis of structural analysis, we hypothesized that human STING is a proton channel. Indeed, we found that STING activation induced a pH increase in the Golgi and that STING reconstituted in liposomes enabled transmembrane proton transport. Compound 53 (C53), a STING agonist that binds the putative channel interface, blocked STING-induced proton flux in the Golgi and in liposomes. STING-induced LC3B lipidation and inflammasome activation were also inhibited by C53, suggesting that STING’s channel activity is critical for these two processes. Thus, STING’s interferon-induction function can be decoupled from its roles in LC3B lipidation and inflammasome activation.

The structural integrity of vaccine antigens is critical to the generation of protective antibody responses, but the impact of protease activity on vaccination in vivo is poorly understood. We characterized protease activity in lymph nodes and found that antigens were rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions, whereas low protease activity and antigen degradation rates were detected in the vicinity of follicular dendritic cells (FDCs). Correlated with these findings, immunization regimens designed to target antigen to FDCs led to germinal centers dominantly targeting intact antigen, whereas traditional immunizations led to much weaker responses that equally targeted the intact immunogen and antigen breakdown products. Thus, spatially compartmentalized antigen proteolysis affects humoral immunity and can be exploited.

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.

The recent clinical success of multiple mRNA-based SARS-CoV-2 vaccines has proven the potential of RNA formulated in lipid nanoparticles (LNPs) in humans, and products based on base-modified RNA, sequence-optimized RNA, and self-replicating RNAs formulated in LNPs are all in various stages of clinical development. However, much remains to be learned about critical parameters governing the manufacturing and use of LNP-RNA formulations. One important issue that has received limited attention in the literature to date is the identification of optimal storage conditions for LNP-RNA that preserve long-term activity of the formulations. Here, we analyzed the physical structure, in vivo expression characteristics, and functional activity of alphavirus-derived self-replicating RNA (repRNA)-loaded LNPs encoding HIV vaccine antigens following storage in varying temperatures, buffers, and in the presence or absence of cryoprotectants. We found that for lipid nanoparticles with compositions similar to clinically-used LNPs, storage in RNAse-free PBS containing 10% (w/v) sucrose at −20 °C was able to maintain vaccine stability and in vivo potency at a level equivalent to freshly prepared vaccines following 30 days of storage. LNPs loaded with repRNA could also be lyophilized with retention of bioactivity.

Chimeric antigen receptor (CAR) T cell therapy effectively treats human cancer, but the loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T cells triggers the engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited the priming of endogenous anti-tumor T cells. This process was accompanied by shifts in CAR T metabolism toward oxidative phosphorylation (OXPHOS) and was critically dependent on CAR-T-derived IFN-γ. Antigen spreading (AS) induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR antigen negative, and heterogeneous tumor control was further enhanced by the genetic amplification of CAR T IFN-γ expression. Thus, CAR-T-cell-derived IFN-γ plays a critical role in promoting AS, and vaccine boosting provides a clinically translatable strategy to drive such responses against solid tumors.

Cellular immune control of HIV is mediated, in part, by induction of single amino acid mutations that reduce viral fitness, but compensatory mutations limit this effect. Here, we sought to determine if higher order constraints on viral evolution exist, because some coordinately linked combinations of mutations may hurt viability. Immune targeting of multiple sites in such a multidimensionally conserved region might render the virus particularly vulnerable, because viable escape pathways would be greatly restricted. We analyzed available HIV sequences using a method from physics to reveal distinct groups of amino acids whose mutations are collectively coordinated ("HIV sectors"). From the standpoint of mutations at individual sites, one such group in Gag is as conserved as other collectively coevolving groups of sites in Gag. However, it exhibits higher order conservation indicating constraints on the viability of viral strains with multiple mutations. Mapping amino acids from this group onto protein structures shows that combined mutations likely destabilize multiprotein structural interactions critical for viral function. Persons who durably control HIV without medications preferentially target the sector in Gag predicted to be most vulnerable. By sequencing circulating viruses from these individuals, we find that individual mutations occur with similar frequency in this sector as in other targeted Gag sectors. However, multiple mutations within this sector are very rare, indicating previously unrecognized multidimensional constraints on HIV evolution. Targeting such regions with higher order evolutionary constraints provides a novel approach to immunogen design for a vaccine against HIV and other rapidly mutating viruses.

We demonstrate that functional polyelectrolyte multilayer (PEM) patches can be attached to a fraction of the surface area of living, individual lymphocytes. Surface-modified cells remain viable at least 48 h following attachment of the functional patch, and patches carrying magnetic nanoparticles allow the cells to be spatially manipulated using a magnetic field. The patch does not completely occlude the cellular surface from the surrounding environment; this approach allows a functional payload to be attached to a cell that is still free to perform its native functions, as suggested by preliminary studies on patch-modified T-cell migration. This approach has potential for broad applications in bioimaging, cellular functionalization, immune system and tissue engineering, and cell-based therapeutics where cell-environment interactions are critical.

Here we introduce a new approach for transcutaneous drug delivery, using microneedles coated with stabilized lipid nanocapsules, for delivery of a model vaccine formulation. Poly(lactide-co-glycolide) microneedle arrays were coated with multilayer films via layer-by-layer assembly of a biodegradable cationic poly(β-amino ester) (PBAE) and negatively charged interbilayer-cross-linked multilamellar lipid vesicles (ICMVs). To test the potential of these nanocapsule-coated microneedles for vaccine delivery, we loaded ICMVs with a protein antigen and the molecular adjuvant monophosphoryl lipid A. Following application of microneedle arrays to the skin of mice for 5 min, (PBAE/ICMV) films were rapidly transferred from microneedle surfaces into the cutaneous tissue and remained in the skin following removal of the microneedle arrays. Multilayer films implanted in the skin dispersed ICMV cargos in the treated tissue over the course of 24 h in vivo, allowing for uptake of the lipid nanocapsules by antigen presenting cells in the local tissue and triggering their activation in situ. Microneedle-mediated transcutaneous vaccination with ICMV-carrying multilayers promoted robust antigen-specific humoral immune responses with a balanced generation of multiple IgG isotypes, whereas bolus delivery of soluble or vesicle-loaded antigen via intradermal injection or transcutaneous vaccination with microneedles encapsulating soluble protein elicited weak, IgG(1)-biased humoral immune responses. These results highlight the potential of lipid nanocapsules delivered by microneedles as a promising platform for noninvasive vaccine delivery applications.

Regulating molecular interactions in the T-cell synapse to prevent autoimmunity or, conversely, to boost anti-tumor immunity has long been a goal in immunotherapy. However, delivering therapeutically meaningful doses of immune-modulating compounds into the synapse represents a major challenge. Here, we report that covalent coupling of maleimide-functionlized nanoparticles (NPs) to free thiol groups on T-cell membrane proteins enables efficient delivery of compounds into the T-cell synapse. We demonstrate that surface-linked NPs are rapidly polarized toward the nascent immunological synapse (IS) at the T-cell/APC contact zone during antigen recognition. To translate these findings into a therapeutic application we tested the NP delivery of NSC-87877, a dual inhibitor of Shp1 and Shp2, key phosphatases that downregulate T-cell receptor activation in the synapse, in the context of adoptive T cell therapy of cancer. Conjugating NSC-87877-loaded NPs to the surface of tumor-specific T cells just prior to adoptive transfer into mice with advanced prostate cancer promoted a much greater T-cell expansion at the tumor site, relative to co-infusing the same drug dose systemically, leading to enhanced survival of treated animals. In summary, our studies support the application of T-cell-linked synthetic NPs as efficient drug delivery vehicles into the IS, as well as the broad applicability of this new paradigm for therapeutically modulating signaling events at the T-cell/APC interface.

T cells are activated by recognition of foreign peptides displayed on the surface of antigen presenting cells (APCs), an event that triggers assembly of a complex microscale structure at the T cell-APC interface known as the immunological synapse (IS). It remains unresolved whether the unique physical structure of the synapse itself impacts the functional response of T cells, independent of the quantity and quality of ligands encountered by the T cell. As a first step toward addressing this question, we created multicomponent protein surfaces presenting lithographically defined patterns of tethered T cell receptor (TCR) ligands (anti-CD3 "activation sites") surrounded by a field of tethered intercellular adhesion molecule-1 (ICAM-1), as a model substrate on which T cells could be seeded to mimic T cell-APC interactions. CD4(+) T cells seeded on these surfaces polarized and migrated; on contact with activation sites, T cells assembled an IS with a structure modulated by the physical pattern of ligand encountered. On surfaces patterned with focal spots of TCR ligand, T cells stably interacted with activation sites, proliferated, and secreted cytokines. In contrast, T cells interacting with activation sites patterned to preclude centralized clustering of TCR ligand failed to form stable contacts with activation sites, exhibited aberrant PKC- clustering in a fraction of cells, and had significantly reduced production of IFN-gamma. These results suggest that focal clustering of TCR ligand characteristic of the "mature" IS may be required under some conditions for full T cell activation.

We describe protein- and oligonucleotide-loaded layer-by-layer (LbL)-assembled multilayer films incorporating a hydrolytically degradable polymer for transcutaneous drug or vaccine delivery. Films were constructed based on electrostatic interactions between a cationic poly(β-amino ester) (denoted Poly-1) with a model protein antigen, ovalbumin (ova), and/or immunostimulatory CpG (cytosine−phosphate diester−guanine-rich) DNA oligonucleotide adjuvant molecules. Linear growth of nanoscale Poly-1/ova bilayers was observed. Dried ova protein-loaded films rapidly deconstructed when rehydrated in saline solutions, releasing ova as nonaggregated/nondegraded protein, suggesting that the structure of biomolecules integrated into these multilayer films is preserved during release. Using confocal fluorescence microscopy and an in vivo murine ear skin model, we demonstrated delivery of ova from LbL films into barrier-disrupted skin, uptake of the protein by skin-resident antigen-presenting cells (Langerhans cells), and transport of the antigen to the skin-draining lymph nodes. Dual incorporation of ova and CpG oligonucleotides into the nanolayers of LbL films enabled dual release of the antigen and adjuvant with distinct kinetics for each component; ova was rapidly released, while CpG was released in a relatively sustained manner. Applied as skin patches, these films delivered ova and CpG to Langerhans cells in the skin. To our knowledge, this is the first demonstration of LbL films applied for the delivery of biomolecules into skin. This approach provides a new route for storage of vaccines and other immunotherapeutics in a solid-state thin film for subsequent delivery into the immunologically rich milieu of the skin.

Dendritic cell vaccines, in which antigen-loaded dendritic cells (DCs) are injected directly into patients to trigger immune responses, are in development as a treatment for cancer and some infectious diseases. In this study, we tested the concept of delivering DCs in an injectable hydrogel matrix, with the aim of harboring dendritic cells for prolonged time periods at a defined site and trapping/concentrating factors secreted by DCs to establish an inflammatory milieu in situ. To achieve these goals, a self-gelling formulation of alginate was developed, obtained by mixing calcium-loaded alginate microspheres with soluble alginate solution and dendritic cells, a formulation that rapidly gelled in vivo. When injected subcutaneously in mice, these alginate ‘vaccination nodes’ containing activated DCs attracted both host dendritic cells and a large number of T cells to the injection sites over a week in vivo, while some of the inoculated DCs trafficked to the draining lymph nodes. Using an adoptive transfer model to track a defined population of T cells responding to immunization with antigen-loaded DCs, we show that DC/alginate immunization led to recruitment of activated antigen-specific T cells to the alginate matrix, in a manner dependent on the presence of the DCs. This gel/DC immunization system may thus be of interest for immunotherapy to direct the accumulation of immune cells at solid tumors or infection sites in the presence of supporting factors co-delivered by the hydrogel matrix.

Immunostimulatory therapies that activate immune response pathways are of great interest for overcoming the immunosuppression present in advanced tumors. Agonistic anti-CD40 antibodies and CpG oligonucleotides have previously demonstrated potent, synergistic anti-tumor effects, but their clinical use even as monotherapies is hampered by dose-limiting inflammatory toxicity provoked upon systemic exposure. We hypothesized that by anchoring immuno-agonist compounds to lipid nanoparticles we could retain the bioactivity of therapeutics in the local tumor tissue and tumor-draining lymph node, but limit systemic exposure to these potent molecules. We prepared PEGylated liposomes bearing surface-conjugated anti-CD40 and CpG and assessed their therapeutic efficacy and systemic toxicity compared to soluble versions of the same immuno-agonists, injected intratumorally in the B16F10 murine model of melanoma. Anti-CD40/CpG-liposomes significantly inhibited tumor growth and induced a survival benefit similar to locally injected soluble anti-CD40 + CpG. Biodistribution analyses following local delivery showed that the liposomal carriers successfully sequestered anti-CD40 and CpG in vivo, reducing leakage into systemic circulation while allowing draining to the tumor-proximal lymph node. Contrary to locally-administered soluble immunotherapy, anti-CD40/CpG-liposomes did not elicit significant increases in serum levels of ALT enzyme, systemic inflammatory cytokines, or overall weight loss, confirming that off-target inflammatory effects had been minimized. The development of a delivery strategy capable of inducing robust anti-tumor responses concurrent with minimal systemic side effects is crucial for the continued progress of potent immunotherapies toward widespread clinical translation.

The immunological synapse (IS) serves a dual role for sustained T cell receptor (TCR) signaling and for TCR downregulation. TC21 (Rras2) is a RRas subfamily GTPase that constitutively associates with the TCR and is implicated in tonic TCR signaling by activating phosphatidylinositol 3-kinase. In this study, we demonstrate that TC21 both cotranslocates with the TCR to the IS and is necessary for TCR internalization from the IS through a mechanism dependent on RhoG, a small GTPase previously associated with phagocytosis. Indeed, we found that the TCR triggers T cells to phagocytose 1–6 μm beads through a TC21- and RhoG-dependent pathway. We further show that TC21 and RhoG are necessary for the TCR-promoted uptake of major histocompatibility complex (MHC) from antigen-presenting cells. Therefore, TC21 and RhoG dependence underlie the existence of a common phagocytic mechanism that drives TCR internalization from the IS together with its peptide-MHC ligand.

A lipid nanocapsule vaccine promotes cross-presentation of antigen with enhanced draining lymph node delivery to elicit an effector memory CD8 + T cell response.

Transcutaneous administration has the potential to improve therapeutics delivery, providing an approach that is safer and more convenient than traditional alternatives, while offering the opportunity for improved therapeutic efficacy through sustained/controlled drug release. To this end, we demonstrate a microneedle materials platform for rapid implantation of controlled-release polymer depots into the cutaneous tissue. Arrays of microneedles comprised of drug-loaded poly(lactide-co-glycolide) (PLGA) microparticles or solid PLGA tips were prepared with a supporting and rapidly water-soluble poly(acrylic acid) (PAA) matrix. Upon application of microneedle patches to the skin of mice, the microneedles perforated the stratum corneum and epidermis. Penetration of the outer skin layers was followed by rapid dissolution of the PAA binder on contact with the interstitial fluid of the epidermis, implanting the microparticles or solid polymer microneedles in the tissue, which were retained following patch removal. These polymer depots remained in the skin for weeks following application and sustained the release of encapsulated cargos for systemic delivery. To show the utility of this approach we demonstrated the ability of these composite microneedle arrays to deliver a subunit vaccine formulation. In comparison to traditional needle-based vaccination, microneedle delivery gave improved cellular immunity and equivalent generation of serum antibodies, suggesting the potential of this approach for vaccine delivery. However, the flexibility of this system should allow for improved therapeutic delivery in a variety of diverse contexts.

Advanced MaterialsVolume 22, Issue 43 p. 4851-4856 Communication Nano-Layered Microneedles for Transcutaneous Delivery of Polymer Nanoparticles and Plasmid DNA Peter C. DeMuth, Peter C. DeMuth Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA)Search for more papers by this authorXingfang Su, Xingfang Su Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA)Search for more papers by this authorRaymond E. Samuel, Raymond E. Samuel Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA)Search for more papers by this authorPaula T. Hammond, Corresponding Author Paula T. Hammond hammond@mit.edu Department of Chemical Engineering and Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA) Paula T. Hammond, Department of Chemical Engineering and Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA). Darrell J. Irvine, Department of Materials Science and Engineering, Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA), Ragon Institute of MIT, MGH, and Harvard Boston, MA 02139, Howard Hughes Medical Institute, 4000 Jones Bridge Rd., Chevy Chase, MDSearch for more papers by this authorDarrell J. Irvine, Corresponding Author Darrell J. Irvine djirvine@mit.edu Department of Materials Science and Engineering, Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA), Ragon Institute of MIT, MGH, and Harvard Boston, MA 02139, Howard Hughes Medical Institute, 4000 Jones Bridge Rd., Chevy Chase, MD Paula T. Hammond, Department of Chemical Engineering and Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA). Darrell J. Irvine, Department of Materials Science and Engineering, Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA), Ragon Institute of MIT, MGH, and Harvard Boston, MA 02139, Howard Hughes Medical Institute, 4000 Jones Bridge Rd., Chevy Chase, MDSearch for more papers by this author Peter C. DeMuth, Peter C. DeMuth Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA)Search for more papers by this authorXingfang Su, Xingfang Su Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA)Search for more papers by this authorRaymond E. Samuel, Raymond E. Samuel Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA)Search for more papers by this authorPaula T. Hammond, Corresponding Author Paula T. Hammond hammond@mit.edu Department of Chemical Engineering and Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA) Paula T. Hammond, Department of Chemical Engineering and Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA). Darrell J. Irvine, Department of Materials Science and Engineering, Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA), Ragon Institute of MIT, MGH, and Harvard Boston, MA 02139, Howard Hughes Medical Institute, 4000 Jones Bridge Rd., Chevy Chase, MDSearch for more papers by this authorDarrell J. Irvine, Corresponding Author Darrell J. Irvine djirvine@mit.edu Department of Materials Science and Engineering, Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA), Ragon Institute of MIT, MGH, and Harvard Boston, MA 02139, Howard Hughes Medical Institute, 4000 Jones Bridge Rd., Chevy Chase, MD Paula T. Hammond, Department of Chemical Engineering and Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA). Darrell J. Irvine, Department of Materials Science and Engineering, Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139 (USA), Ragon Institute of MIT, MGH, and Harvard Boston, MA 02139, Howard Hughes Medical Institute, 4000 Jones Bridge Rd., Chevy Chase, MDSearch for more papers by this author First published: 21 September 2010 https://doi.org/10.1002/adma.201001525Citations: 126Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Multilayer-coated microneedles achieve transcutaneous delivery of plasmid DNA to the viable epidermis. Cy3-labeled plasmid DNA encoding luciferase (yellow) is deposited on biodegradable microneedle arrays through multilayer self-assembly and then delivered to the skin by microneedle application to achieve colocalization with Langerhans dendritic cells (MHC II-GFP–green). Citing Literature Supporting Information Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Filename Description adma_201001525_sm_suppl.pdf1.1 MB suppl Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume22, Issue43November 16, 2010Pages 4851-4856 RelatedInformation

Microneedle vaccines mimic several aspects of cutaneous pathogen invasion by targeting antigen to skin-resident dendritic cells and triggering local inflammatory responses in the skin, which are correlated with enhanced immune responses. Here, we tested whether control over vaccine delivery kinetics can enhance immunity through further mimicry of kinetic profiles present during natural acute infections. An approach for the fabrication of silk/poly(acrylic acid) (PAA) composite microneedles composed of a silk tip supported on a PAA base is reported. On brief application of microneedle patches to skin, the PAA bases rapidly dissolved to deliver a protein subunit vaccine bolus, while also implanting persistent silk hydrogel depots into the skin for a low-level sustained cutaneous vaccine release over 1-2 weeks. Use of this platform to deliver a model whole-protein vaccine with optimized release kinetics resulted in >10-fold increases in antigen-specific T-cell and humoral immune responses relative to traditional parenteral needle-based immunization.

The parasite Plasmodium vivax is the most frequent cause of malaria outside of sub-Saharan Africa, but efforts to develop viable vaccines against P. vivax so far have been inadequate. We recently developed pathogen-mimicking polymeric vaccine nanoparticles composed of the FDA-approved biodegradable polymer poly(lactide-co-glycolide) acid (PLGA) "enveloped" by a lipid membrane. In this study, we sought to determine whether this vaccine delivery platform could be applied to enhance the immune response against P. vivax sporozoites. A candidate malaria antigen, VMP001, was conjugated to the lipid membrane of the particles, and an immunostimulatory molecule, monophosphoryl lipid A (MPLA), was incorporated into the lipid membranes, creating pathogen-mimicking nanoparticle vaccines (VMP001-NPs). Vaccination with VMP001-NPs promoted germinal center formation and elicited durable antigen-specific antibodies with significantly higher titers and more balanced Th1/Th2 responses in vivo, compared with vaccines composed of soluble protein mixed with MPLA. Antibodies raised by NP vaccinations also exhibited enhanced avidity and affinity toward the domains within the circumsporozoite protein implicated in protection and were able to agglutinate live P. vivax sporozoites. These results demonstrate that these VMP001-NPs are promising vaccines candidates that may elicit protective immunity against P. vivax sporozoites.

Strategies to enhance, suppress, or qualitatively shape the immune response are of importance for diverse biomedical applications, such as the development of new vaccines, treatments for autoimmune diseases and allergies, strategies for regenerative medicine, and immunotherapies for cancer. However, the intricate cellular and molecular signals regulating the immune system are major hurdles to predictably manipulating the immune response and developing safe and effective therapies. To meet this challenge, biomaterials are being developed that control how, where, and when immune cells are stimulated in vivo, and that can finely control their differentiation in vitro. We review recent advances in the field of biomaterials for immunomodulation, focusing particularly on designing biomaterials to provide controlled immunostimulation, targeting drugs and vaccines to lymphoid organs, and serving as scaffolds to organize immune cells and emulate lymphoid tissues. These ongoing efforts highlight the many ways in which biomaterials can be brought to bear to engineer the immune system.

Abstract We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function—including Granzyme B—are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology.

By wrapping a ligand-functionalized lipid membrane around a silica core, nanoparticles with a fluid surface are created. These combine unprecedented specificity in binding to cancer cells with the combinatorial delivery of drug cocktails.

In adoptive cell therapy (ACT), autologous tumor-specific T-cells isolated from cancer patients are activated and expanded ex vivo, then infused back into the individual to eliminate metastatic tumors. A major limitation of this promising approach is the rapid loss of ACT T-cell effector function in vivo due to the highly immunosuppressive environment in tumors. Protection of T-cells from immunosuppressive signals can be achieved by systemic administration of supporting adjuvant drugs such as interleukins, chemotherapy, and other immunomodulators, but these adjuvant treatments are often accompanied by serious toxicities and may still fail to optimally stimulate lymphocytes in all tumor and lymphoid compartments. Here we propose a novel strategy to repeatedly stimulate or track ACT T-cells, using cytokines or ACT-cell-specific antibodies as ligands to target PEGylated liposomes to transferred T-cells in vivo. Using F(ab')2 fragments against a unique cell surface antigen on ACT cells (Thy1.1) or an engineered interleukin-2 (IL-2) molecule on an Fc framework as targeting ligands, we demonstrate that >95% of ACT cells can be conjugated with liposomes following a single injection in vivo. Further, we show that IL-2-conjugated liposomes both target ACT cells and are capable of inducing repeated waves of ACT T-cell proliferation in tumor-bearing mice. These results demonstrate the feasibility of repeated functional targeting of T-cells in vivo, which will enable delivery of imaging contrast agents, immunomodulators, or chemotherapy agents in adoptive cell therapy regimens.

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ∼3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or noninternalizing receptor (CD45). When lymphocytes were preloaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell antitumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors.

Amphiphilic oligonucleotides synthesized by covalent conjugation between a hydrophobic diacyllipid tail and chemically stabilized RNA or DNA oligonucleotides can directly label tumor cells on injection into solid tumors. In a murine melanoma tumor model, cell membrane-anchored CpG ODNs with a nuclease-resistant phophorothioate backbone (row a) exhibited significantly enhanced immunostimulatory activity compared to soluble CpG (row b).

Important cell populations reside within tissues and are not accessed by traditional blood draws used to monitor the immune system. To address this issue at an essential barrier tissue, the skin, we created a microneedle-based technology for longitudinal sampling of cells and interstitial fluid, enabling minimally invasive parallel monitoring of immune responses. Solid microneedle projections were coated by a cross-linked biocompatible polymer, which swells upon skin insertion, forming a porous matrix for local leukocyte infiltration. By embedding molecular adjuvants and specific antigens encapsulated in nanocapsules within the hydrogel coating, antigen-specific lymphocytes can be enriched in the recovered cell population, allowing for subsequent detailed phenotypic and functional analysis. We demonstrate this approach in mice immunized with a model protein antigen or infected in the skin with vaccinia virus. After vaccination or infection, sampling microneedles allowed tissue-resident memory T cells (TRMs) to be longitudinally monitored in the skin for many months, during which time the antigen-specific T cell population in systemic circulation contracted to low or undetectable counts. Sampling microneedles did not change the immune status of naïve or antigen-exposed animals. We also validated the ability of cell sampling using human skin samples. This approach may be useful in vaccines and immunotherapies to temporally query TRM populations or as a diagnostic platform to sample for biomarkers in chronic inflammatory and autoimmune disorders, allowing information previously accessible only via invasive biopsies to be obtained in a minimally invasive manner from the skin or other mucosal tissues.

Biocompatible polymer solutions that can crosslink in situ following injection to form stable hydrogels are of interest as depots for sustained delivery of therapeutic factors or cells, and as scaffolds for regenerative medicine. Here, injectable self-gelling alginate formulations obtained by mixing alginate microspheres (as calcium reservoirs) with soluble alginate solutions were characterized for potential use in immunotherapy. Rapid redistribution of calcium ions from microspheres into the surrounding alginate solution led to crosslinking and formation of stable hydrogels. The mechanical properties of the resulting gels correlated with the concentration of calcium-reservoir microspheres added to the solution. Soluble factors such as the cytokine interleukin-2 were readily incorporated into self-gelling alginate matrices by simply mixing them with the formulation prior to gelation. Using alginate microspheres as modular components, strategies for binding immunostimulatory CpG oligonucleotides onto the surface of microspheres were also demonstrated. When injected subcutaneously in the flanks of mice, self-gelling alginate formed soft macroporous gels supporting cellular infiltration and allowing ready access to microspheres carrying therapeutic factors embedded in the matrix. This in situ gelling formulation may thus be useful for stimulating immune cells at desired locales, such as solid tumors or infection sites, as well as for other soft tissue regeneration applications.

Inorganic nanoparticles (NPs) are studied as drug carriers, radiosensitizers and imaging agents, and characterizing nanoparticle biodistribution is essential for evaluating their efficacy and safety. Tracking NPs at the single-cell level with current technologies is complicated by the lack of reliable methods to stably label particles over extended durations in vivo. Here we demonstrate that mass cytometry by time-of-flight provides a label-free approach for inorganic nanoparticle quantitation in cells. Furthermore, mass cytometry can enumerate AuNPs with a lower detection limit of ∼10 AuNPs (3 nm core size) in a single cell with tandem multiparameter cellular phenotyping. Using the cellular distribution insights, we selected an amphiphilic surface ligand-coated AuNP that targeted myeloid dendritic cells in lymph nodes as a peptide antigen carrier, substantially increasing the efficacy of a model vaccine in a B16-OVA melanoma mouse model. This technology provides a powerful new level of insight into nanoparticle fate in vivo.

Amphiphilic gold nanoparticles (amph-NPs), composed of gold cores surrounded by an amphiphilic mixed organic ligand shell, are capable of embedding within and traversing lipid membranes. Here we describe a strategy using crosslink-stabilized lipid nanocapsules (NCs) as carriers to transport such membrane-penetrating particles into tumor cells and promote their transfer to intracellular membranes for enhanced radiotherapy of cancer. We synthesized and characterized interbilayer-crosslinked multilamellar lipid vesicles (ICMVs) carrying amph-NPs embedded in the capsule walls, forming Au-NCs. Confocal and electron microscopies revealed that the intracellular distribution of amph-NPs within melanoma and breast tumor cells following uptake of free particles vs Au-NCs was quite distinct and that amph-NPs initially delivered into endosomes by Au-NCs transferred over a period of hours to intracellular membranes through tumor cells, with greater intracellular spread in melanoma cells than breast carcinoma cells. Clonogenic assays revealed that Au-NCs enhanced radiotherapeutic killing of melanoma cells. Thus, multilamellar lipid capsules may serve as an effective carrier to deliver amphiphilic gold nanoparticles to tumors, where the membrane-penetrating properties of these materials can significantly enhance the efficacy of frontline radiotherapy treatments.

New subunit vaccine formulations with increased potency are of interest to improve immune responses against poorly immunogenic antigens, to avoid vaccine shortages in pandemic situations, and to promote dose-sparing of potent adjuvant molecules that can cause unacceptable side effects in prophylactic vaccination. Here we report strong class-switched, high avidity humoral immune responses elicited by a vaccine system based on poly(lactide-co-glycolide) micro- or nano-particles enveloped by PEGylated phospholipid bilayers, with protein antigens covalently anchored to the lipid surface and lipophilic adjuvants inserted in the bilayer coating. Strikingly, these particles elicited high endpoint antigen-specific IgG titers (>10(6)) sustained for over 100 days after two immunizations with as little as 2.5 ng of antigen. At such low doses, the conventional adjuvant alum or the molecular adjuvants monophosphoryl lipid A (MPLA) or α-galactosylceramide (αGC) failed to elicit responses. Co-delivery of antigen with MPLA or αGC incorporated into the particle bilayers in a pathogen-mimetic fashion further enhanced antibody titers by ~12-fold. MPLA provided the highest sustained IgG titers at these ultra-low antigen doses, while αGC promoted a rapid rise in serum IgG after one immunization, which may be valuable in emergencies such as disease pandemics. The dose of αGC required to boost the antibody response was also spared by particulate delivery. Lipid-enveloped biodegradable micro- and nano-particles thus provide a potent dose-sparing platform for vaccine delivery.

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.

The development of a tuberculosis (TB) vaccine that induces sterilizing immunity to Mycobacterium tuberculosis infection has been elusive. Absence of sterilizing immunity induced by TB vaccines may be due to delayed activation of mucosal dendritic cells (DCs), and subsequent delay in antigen presentation and activation of vaccine-induced CD4+ T-cell responses. Here we show that pulmonary delivery of activated M. tuberculosis antigen-primed DCs into vaccinated mice, at the time of M. tuberculosis exposure, can overcome the delay in accumulation of vaccine-induced CD4+ T-cell responses. In addition, activating endogenous host CD103+ DCs and the CD40-CD40L pathway can similarly induce rapid accumulation of vaccine-induced lung CD4+ T-cell responses and limit early M. tuberculosis growth. Thus, our study provides proof of concept that targeting mucosal DCs can accelerate vaccine-induced T-cell responses on M. tuberculosis infection, and provide insights to overcome bottlenecks in TB vaccine efficacy.

ABSTRACT While development of an HIV vaccine that can induce neutralizing antibodies remains a priority, decades of research have proven that this is a daunting task. However, accumulating evidence suggests that antibodies with the capacity to harness innate immunity may provide some protection. While significant research has focused on the cytolytic properties of antibodies in acquisition and control, less is known about the role of additional effector functions. In this study, we investigated antibody-dependent phagocytosis of HIV immune complexes, and we observed significant differences in the ability of antibodies from infected subjects to mediate this critical effector function. We observed both quantitative differences in the capacity of antibodies to drive phagocytosis and qualitative differences in their FcγR usage profile. We demonstrate that antibodies from controllers and untreated progressors exhibit increased phagocytic activity, altered Fc domain glycosylation, and skewed interactions with FcγR2a and FcγR2b in both bulk plasma and HIV-specific IgG. While increased phagocytic activity may directly influence immune activation via clearance of inflammatory immune complexes, it is also plausible that Fc receptor usage patterns may regulate the immune response by modulating downstream signals following phagocytosis—driving passive degradation of internalized virus, release of immune modulating cytokines and chemokines, or priming of a more effective adaptive immune response.

B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale "cell squeezing" process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8(+)T-cells, and not CD4(+)T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8(+)T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation.

Strategies to qualitatively and quantitatively enhance the humoral response to immunizations with protein and polysaccharide antigens are of broad interest for development of new and more effective vaccines. A strategy of increasing importance is the formulation of antigens into a particulate format, mimicking the physical form of viruses. The potential benefits of enhanced B cell receptor engagement by nanoparticles have been long been appreciated, but recent studies are defining additional important factors governing how nanoparticle immunogens interact with the immune system in the context of lymphoid organs. This review will discuss findings about how nanoparticles enhance humoral immunity in vivo and factors governing the fate of nanoparticle immunogens in lymph nodes.

Antigen- and adjuvant-based bioconjugates that can stimulate the immune system play an important role in vaccine applications. Bioconjugates have demonstrated unique physicochemical and biological properties, enabling vaccines to be delivered to key immune cells, to target specific intracellular pathways, or to mimic immunogenic properties of natural pathogens. In this Review we highlight recent advances in such molecular immunomodulators, with an emphasis on the structure-function relationships that provide the foundation for rational design of safe and effective vaccines and immunotherapies.

An HIV vaccine capable of inducing high and durable levels of broadly neutralizing antibodies has thus far proven elusive. A promising antigen is the membrane-proximal external region (MPER) from gp41, a segment of the viral envelope recognized by a number of broadly neutralizing antibodies. Though an attractive vaccine target due to the linear nature of the epitope and its highly conserved sequence, MPER peptides are poorly immunogenic and may require display on membranes to achieve a physiological conformation matching the native virus. Here we systematically explored how the structure and composition of liposomes displaying MPER peptides impacts the strength and durability of humoral responses to this antigen as well as helper T-cell responses in mice. Administration of MPER peptides anchored to the surface of liposomes induced MPER-specific antibodies whereas MPER administered in oil-based emulsion adjuvants or alum did not, even when combined with Toll-like receptor agonists. High-titer IgG responses to liposomal MPER required the inclusion of molecular adjuvants such as monophosphoryl lipid A. Anti-MPER humoral responses were further enhanced by incorporating high-Tm lipids in the vesicle bilayer and optimizing the MPER density to a mean distance of ∼10-15 nm between peptides on the liposomes' surfaces. Encapsulation of helper epitopes within the vesicles allowed efficient "intrastructural" T-cell help, which promoted IgG responses to MPER while minimizing competing B-cell responses against the helper sequence. These results define several key properties of liposome formulations that promote durable, high-titer antibody responses against MPER peptides, which will be a prerequisite for a successful MPER-targeting vaccine.

Messenger RNA (mRNA) represents a promising class of nucleic-acid-based therapeutics. While numerous nanocarriers have been developed for mRNA delivery, the inherent labile nature of mRNA results in a very low transfection efficiency and poor expression of desired protein. Here we preassemble the mRNA translation initiation structure through an inherent molecular recognition between 7-methylguanosine (m7G)-capped mRNA and eukaryotic initiation factor 4E (eIF4E) protein to form ribonucleoproteins (RNPs), thereby mimicking the first step of protein synthesis inside cells. Subsequent electrostatic stabilization of RNPs with structurally tunable cationic carriers leads to nanosized complexes (nanoplexes), which elicit high levels of mRNA transfection in different cell types by enhancing intracellular mRNA stability and protein synthesis. By investigating a family of synthetic polypeptides bearing different side group arrangements of cationic charge, we find that the molecular structure modulates the nanoscale distance between the mRNA strand and the eIF4E protein inside the nanoplex, which directly impacts the enhancement of mRNA transfection. To demonstrate the biomedical potential of this approach, we use this approach to introduce mRNA/eIF4E nanoplexes to murine dendritic cells, resulting in increased activation of cytotoxic CD8 T cells ex vivo. More importantly, eIF4E enhances gene expression in lungs following a systemic delivery of luciferase mRNA/eIF4E in mice. Collectively, this bioinspired molecular assembly method could lead to a new paradigm of gene delivery.

The utility of layer-by-layer (LbL) coated microneedle (MN) skin patches for transdermal drug delivery has proven to be a promising approach, with advantages over hypodermal injection due to painless and easy self-administration. However, the long epidermal application time required for drug implantation by existing LbL MN strategies (15-90 min) can lead to potential medication noncompliance. Here, we developed a MN platform to shorten the application time in MN therapies based on a synthetic pH-induced charge-invertible polymer poly(2-(diisopropylamino) ethyl methacrylate- b-methacrylic acid) (PDM), requiring only 1 min skin insertion time to implant LbL films in vivo. Following MN-mediated delivery of 0.5 μg model antigen chicken ovalbumin (OVA) in the skin of mice, this system achieved sustained release over 3 days and led to an elevated immune response as demonstrated by significantly higher humoral immunity compared with OVA administration via conventional routes (subcutaneously and intramuscularly). Moreover, in an ex vivo experiment on human skin, we achieved efficient immune activation through MN-delivered LbL films, demonstrated by a rapid uptake of vaccine adjuvants by the antigen presenting cells. These features, rapid administration and the ability to elicit a robust immune response, can potentially enable a broad application of microneedle-based vaccination technologies.

Abstract An HIV vaccine capable of eliciting durable neutralizing antibody responses continues to be an important unmet need. Multivalent nanoparticles displaying a high density of envelope trimers may be promising immunogen forms to elicit strong and durable humoral responses to HIV, but critical particle design criteria remain to be fully defined. To this end, we developed strategies to covalently anchor a stabilized gp140 trimer, BG505 MD39, on the surfaces of synthetic liposomes to study the effects of trimer density and vesicle stability on vaccine-elicited humoral responses in mice. CryoEM imaging revealed homogeneously distributed and oriented MD39 on the surface of liposomes irrespective of particle size, lipid composition, and conjugation strategy. Immunization with covalent MD39-coupled liposomes led to increased germinal center and antigen-specific T follicular helper cell responses and significantly higher avidity serum MD39-specific IgG responses compared to immunization with soluble MD39 trimers. A priming immunization with liposomal-MD39 was important for elicitation of high avidity antibody responses, regardless of whether booster immunizations were administered with either soluble or particulate trimers. The stability of trimer anchoring to liposomes was critical for these effects, as germinal center and output antibody responses were further increased by liposome compositions incorporating sphingomyelin that exhibited high in vitro stability in the presence of serum. Together these data highlight key liposome design features for optimizing humoral immunity to lipid nanoparticle immunogens.