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Human immunodeficiency virus type 1 (HIV-1)-specific monoclonal antibodies with extraordinary potency and breadth have recently been described. In humanized mice, combinations of monoclonal antibodies have been shown to suppress viraemia, but the therapeutic potential of these monoclonal antibodies has not yet been evaluated in primates with an intact immune system. Here we show that administration of a cocktail of HIV-1-specific monoclonal antibodies, as well as the single glycan-dependent monoclonal antibody PGT121, resulted in a rapid and precipitous decline of plasma viraemia to undetectable levels in rhesus monkeys chronically infected with the pathogenic simian–human immunodeficiency virus SHIV-SF162P3. A single monoclonal antibody infusion afforded up to a 3.1 log decline of plasma viral RNA in 7 days and also reduced proviral DNA in peripheral blood, gastrointestinal mucosa and lymph nodes without the development of viral resistance. Moreover, after monoclonal antibody administration, host Gag-specific T-lymphocyte responses showed improved functionality. Virus rebounded in most animals after a median of 56 days when serum monoclonal antibody titres had declined to undetectable levels, although, notably, a subset of animals maintained long-term virological control in the absence of further monoclonal antibody infusions. These data demonstrate a profound therapeutic effect of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys as well as an impact on host immune responses. Our findings strongly encourage the investigation of monoclonal antibody therapy for HIV-1 in humans., National Institutes of Health (U.S.) (AI055332), National Institutes of Health (U.S.) (AI060354), National Institutes of Health (U.S.) (AI078526), National Institutes of Health (U.S.) (AI084794), National Institutes of Health (U.S.) (AI095985), National Institutes of Health (U.S.) (AI096040), National Institutes of Health (U.S.) (AI100148), National Institutes of Health (U.S.) (AI10063), Bill & Melinda Gates Foundation (OPP1033091), Bill & Melinda Gates Foundation (OPP1033115), Bill & Melinda Gates Foundation (OPP1040741), Bill & Melinda Gates Foundation (OPP1040753), Ragon Institute of MGH, MIT, and Harvard, Stavros S. Niarchos Foundation, Howard Hughes Medical Institute (Investigator)

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., David H. Koch Institute for Integrative Cancer Research at MIT (Koch Institute Support (core) Grant P30-CA14051), National Cancer Institute (U.S.), National Institutes of Health (U.S.) (grant AI091693), National Institutes of Health (U.S.) (grant AI104715), National Institutes of Health (U.S.) (AI095109), United States. Dept. of Defense (contract W911NF-13-D-0001), United States. Dept. of Defense (contract W911NF-07-D-0004), Ragon Institute of MGH, MIT, and Harvard

Valency requirements for B cell activation upon antigen encounter are poorly understood. OB1 transnuclear B cells express an IgG1 B cell receptor (BCR) specific for ovalbumin (OVA), the epitope of which can be mimicked using short synthetic peptides to allow antigen-specific engagement of the BCR. By altering length and valency of epitope-bearing synthetic peptides, we examined the properties of ligands required for optimal OB1 B cell activation. Monovalent engagement of the BCR with an epitope-bearing 17-mer synthetic peptide readily activated OB1 B cells. Dimers of the minimal peptide epitope oriented in an N to N configuration were more stimulatory than their C to C counterparts. Although shorter length correlated with less activation, a monomeric 8-mer peptide epitope behaved as a weak agonist that blocked responses to cell-bound peptide antigen, a blockade which could not be reversed by CD40 ligation. The 8-mer not only delivered a suboptimal signal, which blocked subsequent responses to OVA, anti-IgG, and anti-kappa, but also competed for binding with OVA. Our results show that fine-tuning of BCR-ligand recognition can lead to B cell nonresponsiveness, activation, or inhibition., National Institutes of Health (U.S.) (grant R01 AI087879), National Institutes of Health (U.S.) (grant R01 GM100518), National Institutes of Health (U.S.) (grant P01 AI091580), Netherlands Organization for Scientific Research

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., National Institutes of Health (U.S.) (AI095109), National Institutes of Health (U.S.) (CA140476), United States. Dept. of Defense (Contract W81XWH-10-1-0290), United States. Dept. of Defense (Contract W911NF-07-D-0004), Ragon Institute of MGH, MIT and Harvard

Chemoattractants regulate diverse immunological, developmental, and pathological processes, but how cell migration patterns are shaped by attractant production in tissues remains incompletely understood. Using computational modeling and chemokine-releasing microspheres (CRMs), cell-sized attractant-releasing beads, we analyzed leukocyte migration in physiologic gradients of CCL21or CCL19 produced by beads embedded in 3D collagen gels. Individual T-cells that migrated into contact with CRMs exhibited characteristic highly directional migration to attractant sources independent of their starting position in the gradient (and thus independent of initial gradient strength experienced) but the fraction of responding cells was highly sensitive to position in the gradient. These responses were consistent with modeling calculations assuming a threshold absolute difference in receptor occupancy across individual cells of [similar]10 receptors required to stimulate chemotaxis. In sustained gradients eliciting low receptor desensitization, attracted T-cells or dendritic cells swarmed around isolated CRMs for hours. With increasing CRM density, overlapping gradients and high attractant concentrations caused a transition from local swarming to transient “hopping” of cells bead to bead. Thus, diverse migration responses observed in vivo may be determined by chemoattractant source density and secretion rate, which govern receptor occupancy patterns in nearby cells., National Institutes of Health (U.S.) (NIH EB007280), Howard Hughes Medical Institute (Investigator)

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′)[subscript 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., National Institutes of Health (U.S.) (CA140476), National Institutes of Health (U.S.) (CA172164), United States. Dept. of Defense (Contract W81XWH-10-1-0290), National Cancer Institute (U.S.) (Koch Institute Support (core) Grant P30-CA14051)

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., United States. Dept. of Defense (contract W911NF-13-D-0001), United States. Dept. of Defense (contract W911NF-07-D-0004), National Institutes of Health (U.S.) (AI095109), Bill & Melinda Gates Foundation, Ragon Institute of MGH, MIT, and Harvard, National Institutes of Health (U.S.) (AI091693), Howard Hughes Medical Institute (Investigator), Carigest SA

Viral immune evasion by sequence variation is a major hindrance to HIV-1 vaccine design. To address this challenge, our group has developed a computational model, rooted in physics, that aims to predict the fitness landscape of HIV-1 proteins in order to design vaccine immunogens that lead to impaired viral fitness, thus blocking viable escape routes. Here, we advance the computational models to address previous limitations, and directly test model predictions against in vitro fitness measurements of HIV-1 strains containing multiple Gag mutations. We incorporated regularization into the model fitting procedure to address finite sampling. Further, we developed a model that accounts for the specific identity of mutant amino acids (Potts model), generalizing our previous approach (Ising model) that is unable to distinguish between different mutant amino acids. Gag mutation combinations (17 pairs, 1 triple and 25 single mutations within these) predicted to be either harmful to HIV-1 viability or fitness-neutral were introduced into HIV-1 NL4-3 by site-directed mutagenesis and replication capacities of these mutants were assayed in vitro. The predicted and measured fitness of the corresponding mutants for the original Ising model (r = −0.74, p = 3.6×10[superscript −6]) are strongly correlated, and this was further strengthened in the regularized Ising model (r = −0.83, p = 3.7×10[superscript −12]). Performance of the Potts model (r = −0.73, p = 9.7×10[superscript −9]) was similar to that of the Ising model, indicating that the binary approximation is sufficient for capturing fitness effects of common mutants at sites of low amino acid diversity. However, we show that the Potts model is expected to improve predictive power for more variable proteins. Overall, our results support the ability of the computational models to robustly predict the relative fitness of mutant viral strains, and indicate the potential value of this approach for understanding viral immune evasion, Ragon Institute of MGH, MIT and Harvard, National Institutes of Health (U.S.) (Director's Pioneer Award)

T cells orchestrate pathogen-specific adaptive immune responses by identifying peptides derived from pathogenic proteins that are displayed on the surface of infected cells. Host cells also display peptide fragments from the host’s own proteins. Incorrectly identifying peptides derived from the body’s own proteome as pathogenic can result in autoimmune disease. To minimize autoreactivity, immature T cells that respond to self-peptides are deleted in the thymus by a process called negative selection. However, negative selection is imperfect, and autoreactive T cells exist in healthy individuals. To understand how autoimmunity is yet avoided, without loss of responsiveness to pathogens, we have developed a model of T-cell training and response. Our model shows that T cells reliably respond to infection and avoid autoimmunity because collective decisions made by the T-cell population, rather than the responses of individual T cells, determine biological outcomes. The theory is qualitatively consistent with experimental data and yields a criterion for thymic selection to be adequate for suppressing autoimmunity., National Institutes of Health (U.S.). Pioneer Award, National Science Foundation (U.S.) (Grant 12-06323)

Mass cytometry enables an unprecedented number of parameters to be measured in individual cells at a high throughput, but the large dimensionality of the resulting data severely limits approaches relying on manual “gating.” Clustering cells based on phenotypic similarity comes at a loss of single-cell resolution and often the number of subpopulations is unknown a priori. Here we describe ACCENSE, a tool that combines nonlinear dimensionality reduction with density-based partitioning, and displays multivariate cellular phenotypes on a 2D plot. We apply ACCENSE to 35-parameter mass cytometry data from CD8+ T cells derived from specific pathogen-free and germ-free mice, and stratify cells into phenotypic subpopulations. Our results show significant heterogeneity within the known CD8+ T-cell subpopulations, and of particular note is that we find a large novel subpopulation in both specific pathogen-free and germ-free mice that has not been described previously. This subpopulation possesses a phenotypic signature that is distinct from conventional naive and memory subpopulations when analyzed by ACCENSE, but is not distinguishable on a biaxial plot of standard markers. We are able to automatically identify cellular subpopulations based on all proteins analyzed, thus aiding the full utilization of powerful new single-cell technologies such as mass cytometry., Poitras Foundation (Predoctoral Fellowship), Massachusetts Institute of Technology. Ragon Institute of MGH, MIT and Harvard, National Institutes of Health (U.S.) (PO1 AI091580)

Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide–MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4–MHCII interaction is generally weaker than CD8–MHCI., National Institutes of Health (U.S.). Pioneer Award

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., Howard Hughes Medical Institute (Investigator), Ragon Institute of MGH, MIT, and Harvard, National Institutes of Health (U.S.) (NIH AI095109), United States. Dept. of Defense. Institute for Soldier Nanotechnologies (contract W911NF-07-D-0004), United States. Dept. of Defense. Institute for Soldier Nanotechnologies (contract W911NF-07-0004)

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[superscript +] T-cell–dependent and was associated with increased CD8[superscript +] 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., Dana-Farber/Harvard Cancer Center-MIT Bridge Project Fund

Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors., Janssen Pharmaceutical Ltd., National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051), National Science Foundation (U.S.). Graduate Research Fellowship, Singapore. Agency for Science, Technology and Research, Swiss National Science Foundation (Fellowship for Advanced Researchers PA00P3 139659)

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic., National Science Foundation (U.S.). Graduate Research Fellowship, National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051), Janssen Pharmaceutical Ltd., Klarman Family Foundation

Natural killer (NK) cells are a subset of innate immune lymphocytes that interrogate potential target cells and rapidly respond by lysing them or secreting inflammatory immunomodulators. Productive interactions between NK cells and targets such as tumor cells or virally infected cells are critical for immunological control of malignancies and infections. For individual NK cells, however, the relationship between the characteristics of these cell–cell interactions, cytolysis, and secretory activity is not well understood. Here, we used arrays of subnanoliter wells (nanowells) to monitor individual NK cell–target cell interactions and quantify the resulting cytolytic and secretory responses. We show that NK cells operate independently when lysing a single target cell and that lysis is most probable during an NK cell's first encounter with a target. Furthermore, we demonstrate that the secretion of interferon-γ (IFN-γ) occurs most often among NK cells that become the least motile upon contacting a target cell but is largely independent of cytolysis. Our findings demonstrate that integrated analysis of the cell–cell interaction parameters, cytolytic activity, and secretory activity of single NK cells can reveal new insights into how these complex functions are related within individual cells., Charles A. Dana Foundation, W. M. Keck Foundation, Ragon Institute of MGH, MIT and Harvard, National Science Foundation (U.S.) (Fellowship), Massachusetts Institute of Technology (Collamore-Rogers Fellowship)

The Abs made by B lymphocytes on first encountering an antigen bind it with low intrinsic affinity, and, over time, the average affinity of the Abs made against that antigen gradually increases. These changes, known as affinity maturation, were found initially for serum antibodies that recognized small, chemically well-defined epitopes (e.g., 2,4-dinitrophenyl). That similar affinity increases occur in responses to protein antigens, which elicit most immune responses to infections or vaccination, has been generally assumed but difficult to prove (1) largely because of the complexity and multiplicity of epitopes (antigenic determinants) on even small, single-chain proteins (2). Though some protein epitopes are linear stretches of amino acids, they usually are configurational clusters of noncontiguous residues best delineated crystallographically in antibody–antigen complexes (3); and more often than not, the diverse antibodies elicited to a protein antigen bind to different epitopes on that protein (4), confounding efforts to convincingly demonstrate affinity maturation. There are at least five epitopes on the influenza virus hemagglutinin (HA) that binds the virions to host cells (5). By comparing the binding properties and structures of affinity matured Abs to one of the HA epitopes with those of their progenitor Abs, the elegant study by Schmidt et al. reported in PNAS (6) provides clear evidence for affinity maturation of Abs. The study is notable, moreover, for its focus on the human immune response to influenza virus vaccination with a conventional influenza seasonal vaccine (FLUZON flu shot). By bringing together diverse approaches (crystallography, molecular dynamics simulations, and kinetic studies), the authors shed intriguing light on how the affinity enhancement of affinity matured antibodies is achieved.

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)., United States. Dept. of Defense (DAAD19- 02-D-0002), United States. Dept. of Defense (W911NF-07-D-0004), Ragon Institute of MGH, MIT and Harvard, National Institutes of Health (U.S.) (NIH Biotechnology Training Program at MIT), Howard Hughes Medical Institute (Investigator)

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, a microneedle materials platform is demonstrated for rapid implantation of controlled-release polymer depots into the cutaneous tissue. Arrays of microneedles composed of drug-loaded poly(lactide-co-glycolide) (PLGA) microparticles or solid PLGA tips are 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 perforate the stratum corneum and epidermis. Penetration of the outer skin layers is 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 are retained following patch removal. These polymer depots remain in the skin for weeks following application and sustain the release of encapsulated cargos for systemic delivery. To show the utility of this approach the ability of these composite microneedle arrays to deliver a subunit vaccine formulation is demonstrated. In comparison to traditional needle-based vaccination, microneedle delivery gives 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., Massachusetts Institute of Technology. Ragon Institute of MGH, MIT and Harvard, National Institutes of Health (U.S.) (Award AI095109), United States. Army Research Office (Contract W911NF-07-D-0004)

We present a method that uses fluorescent cellular barcodes to increase the number of unique samples that can be analyzed simultaneously by microengraving, a nanowell array-based technique for quantifying the secretory responses of thousands of single cells in parallel. Using n different fluorescent dyes to generate 2n unique cellular barcodes, we achieved a 2n-fold reduction in the number of arrays and quantity of reagents required per sample. The utility of this approach was demonstrated in three applications of interest in clinical and experimental immunology. Using barcoded human peripheral blood mononuclear cells and T cells, we constructed dose–response curves, profiled the secretory behavior of cells treated with mechanistically distinct stimuli, and tracked the secretory behaviors of different lineages of CD4+ T helper cells. In addition to increasing the number of samples analyzed by generating secretory profiles of single cells from multiple populations in a time- and reagent-efficient manner, we expect that cellular barcoding in combination with microengraving will facilitate unique experimental opportunities for quantitatively analyzing interactions among heterogeneous cells isolated in small groups (2–5 cells)., Howard Hughes Medical Institute (Investigator), National Institute of Allergy and Infectious Diseases (U.S.) (Grant 1R56AI104274), National Institute of Allergy and Infectious Diseases (U.S.) (Grant 1U19AI089992), National Institute of Allergy and Infectious Diseases (U.S.) (Grant 5U01AI068618), National Science Foundation (U.S.) (Fellowship), Massachusetts Institute of Technology (Collamore-Rogers Fellowship), Ragon Institute of MGH, MIT and Harvard, W. M. Keck Foundation

available in PMC 2011 September 1, 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., Ragon Institute of MGH, MIT and Harvard, Bill & Melinda Gates Foundation, United States. Dept. of Defense (contract W911NF-07-D-0004), National Institutes of Health (U.S.) (P41RR002250), National Institutes of Health (U.S.) (RC2GM092599)

Plant-derived Type I toxins are candidate anticancer therapeutics requiring cytosolic delivery into tumor cells. We tested a concept for two-stage delivery, whereby tumor cells precoated with an antibody-targeted gelonin toxin were killed by exposure to endosome-disrupting polymer nanoparticles. Co-internalization of particles and tumor cell-bound gelonin led to cytosolic delivery and >50-fold enhancement of toxin efficacy. This approach allows the extreme potency of gelonin to be focused on tumors with significantly reduced potential for off-target toxicity., United States. Dept. of Defense (Institute for Soldier Nanotechnology, contract W911NF-07-D-0004), Howard Hughes Medical Institute (Investigator), Singapore. Agency for Science, Technology and Research

Without therapy, most people infected with human immunodeficiency virus (HIV) ultimately progress to AIDS. Rare individuals (‘elite controllers’) maintain very low levels of HIV RNA without therapy, thereby making disease progression and transmission unlikely. Certain HLA class I alleles are markedly enriched in elite controllers, with the highest association observed for HLA-B57 (ref. 1). Because HLA molecules present viral peptides that activate CD8+ T cells, an immune-mediated mechanism is probably responsible for superior control of HIV. Here we describe how the peptide-binding characteristics of HLA-B57 molecules affect thymic development such that, compared to other HLA-restricted T cells, a larger fraction of the naive repertoire of B57-restricted clones recognizes a viral epitope, and these T cells are more cross-reactive to mutants of targeted epitopes. Our calculations predict that such a T-cell repertoire imposes strong immune pressure on immunodominant HIV epitopes and emergent mutants, thereby promoting efficient control of the virus. Supporting these predictions, in a large cohort of HLA-typed individuals, our experiments show that the relative ability of HLA-B alleles to control HIV correlates with their peptide-binding characteristics that affect thymic development. Our results provide a conceptual framework that unifies diverse empirical observations, and have implications for vaccination strategies., Mark and Lisa Schwartz Foundation, National Institutes of Health (U.S.) (Director’s Pioneer award), Philip T. and Susan M. Ragon Foundation, Jane Coffin Childs Memorial Fund for Medical Research, Bill & Melinda Gates Foundation, National Institute of Allergy and Infectious Diseases (U.S.), National Institutes of Health (U.S.) (contract no. HHSN261200800001E), National Institutes of Health (U.S.). Intramural Research Program, National Cancer Institute (U.S.), Center for Cancer Research (National Cancer Institute (U.S.))

Second-generation sequencing is gradually becoming the method of choice for miRNA detection and expression profiling. Given the relatively small number of miRNAs and improvements in DNA sequencing technology, studying miRNA expression profiles of multiple samples in a single flow cell lane becomes feasible. Multiplexing strategies require marking each miRNA library with a DNA barcode. Here we report that barcodes introduced through adapter ligation confer significant bias on miRNA expression profiles. This bias is much higher than the expected Poisson noise and masks significant expression differences between miRNA libraries. This bias can be eliminated by adding barcodes during PCR amplification of libraries. The accuracy of miRNA expression measurement in multiplexed experiments becomes a function of sample number., National Human Genome Research Institute (U.S.) (Center for Excellence in Genome Sciences grant), Canadian Institutes of Health Research, National Heart, Lung, and Blood Institute, National Institutes of Health (U.S.) (SysCODE Consortium), United States-Israel Binational Science Foundation (grant no. 2009290), Ragon Institute of MGH, MIT and Harvard (Fellowship), Leducq Foundation

From AIDS Vaccine 2012, Boston, MA, USA. 9-12 September 2012., Background: In BLT (bone marrow-liver-thymus) humanized mice, human thymocytes are educated by autologous human thymic tissue, resulting in functional human T cells capable of rapidly selecting for CTL escape mutations in HIV. In contrast, limitations to B cell maturation have been noted. But despite this, we show for the first time that HIV infected BLT mice can produce class-switched anti-HIV antibodies with neutralizing activities. Methods: Humanized BLT mice were generated by transplanting irradiated NOD-scid/IL2rgnull (NSG) mice with fetal thymus and liver fragments and then injecting them with autologous human CD34+ stem cells. BLT mice were then infected with HIVJRCSF and bled at various time-points. HIV neutralizing activity was measured using Tat-induced luciferase reporter TZM-bl cells. Results: Human transitional B cells were present in greater frequencies in BLT mice than adult humans. Most of these cells had a T1 phenotype in the blood and spleen. But despite this B cell maturation defect, class-switched IgG Abs against various HIV proteins were detected by Western Blot in HIV-infected BLT mice. Using ELISA to determine anti-p24 IgG Ab titers, Abs were present as early as 8 weeks post infection (p.i.), with peak Ab titers seen after 15 weeks. One infected mouse demonstrated a peak titer similar to that seen in a chronically infected human. Finally, plasma samples from infected BLT mice after 22 weeks p.i. demonstrated neutralizing activities against the challenge virus. Average IC50 neutralizing titers in these mice were similar to those from infected human samples. Conclusion: The ability of humanized BLT mice to generate functional humoral immune responses may be further improved by strategies to improve their B cell maturation, which will further improve the potential of these mice to become a model system to study candidate HIV vaccines and therapies.

Adenosine-to-inosine (A-to-I) editing modifies RNA transcripts from their genomic blueprint. A prerequisite for this process is a double-stranded RNA (dsRNA) structure. Such dsRNAs are formed as part of the microRNA (miRNA) maturation process, and it is therefore expected that miRNAs are affected by A-to-I editing. Editing of miRNAs has the potential to add another layer of complexity to gene regulation pathways, especially if editing occurs within the miRNA–mRNA recognition site. Thus, it is of interest to study the extent of this phenomenon. Current reports in the literature disagree on its extent; while some reports claim that it may be widespread, others deem the reported events as rare. Utilizing a next-generation sequencing (NGS) approach supplemented by an extensive bioinformatic analysis, we were able to systematically identify A-to-I editing events in mature miRNAs derived from human brain tissues. Our algorithm successfully identified many of the known editing sites in mature miRNAs and revealed 17 novel human sites, 12 of which are in the recognition sites of the miRNAs. We confirmed most of the editing events using in vitro ADAR overexpression assays. The editing efficiency of most sites identified is very low. Similar results are obtained for publicly available data sets of mouse brain-regions tissues. Thus, we find that A-to-I editing does alter several miRNAs, but it is not widespread.

Blood plasma viral loads and the time to progress to AIDS differ widely among untreated HIV-infected humans. Although people with certain HLA (HLA-I) alleles are more likely to control HIV infections without therapy, the majority of such untreated individuals exhibit high viral loads and progress to AIDS. Stochastic effects are considered unimportant for evolutionary dynamics in HIV-infected people when viral load is high or when selective forces strongly drive mutation. We describe a computational study of host–pathogen interaction demonstrating that stochastic effects can have a profound influence on disease dynamics, even in cases of high viral load and strong selective pressure. These stochastic effects are pronounced when the virus must traverse a fitness “barrier” in sequence space to escape the host’s cytotoxic T-lymphocyte (CTL) response, as often occurs when a fitness defect imposed by a CTL-driven mutation must be compensated for by other mutations. These “barrier-crossing” events are infrequent and stochastic, resulting in divergent disease outcomes in genetically identical individuals infected by the same viral strain. Our results reveal how genetic determinants of the CTL response control the probability with which an individual is able to control HIV infection indefinitely, and thus provide clues for vaccine design., Jane Coffins Childs Foundation, Massachusetts Institute of Technology. Ragon Institute of MGH, MIT and Harvard, National Institutes of Health (U.S.) (Director’s Pioneer Award)

Online supplemental material. Table S1 shows the expression of genes related to drug metabolism in XBP1-deficient liver. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20111298/DC1., The mammalian stress sensor IRE1α plays a central role in the unfolded protein, or endoplasmic reticulum (ER), stress response by activating its downstream transcription factor XBP1 via an unconventional splicing mechanism. IRE1α can also induce the degradation of a subset of mRNAs in a process termed regulated IRE1-dependent decay (RIDD). Although diverse mRNA species can be degraded by IRE1α in vitro, the pathophysiological functions of RIDD are only beginning to be explored. Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in young adults in the United States and is primarily caused by CYP1A2-, CYP2E1-, and CYP3A4-driven conversion of APAP into hepatotoxic metabolites. We demonstrate here that genetic ablation of XBP1 results in constitutive IRE1α activation in the liver, leading to RIDD of Cyp1a2 and Cyp2e1 mRNAs, reduced JNK activation, and protection of mice from APAP-induced hepatotoxicity. A pharmacological ER stress inducer that activated IRE1α suppressed the expression of Cyp1a2 and Cyp2e1 in WT, but not IRE1α-deficient mouse liver, indicating the essential role of IRE1α in the down-regulation of these mRNAs upon ER stress. Our study reveals an unexpected function of RIDD in drug metabolism., National Institutes of Health (U.S.) (grant AI32412), National Institutes of Health (U.S.) (grant DK082448), National Institutes of Health (U.S.) (grant DK089211), American Heart Association

In biochemical networks, identifying key proteins and protein-protein reactions that regulate fluctuation-driven transitions leading to pathological cellular function is an important challenge. Using large deviation theory, we develop a semianalytical method to determine how changes in protein expression and rate parameters of protein-protein reactions influence the rate of such transitions. Our formulas agree well with computationally costly direct simulations and are consistent with experiments. Our approach reveals qualitative features of key reactions that regulate stochastic transitions., National Cancer Institute (U.S.). Physical Sciences-Oncology Center, National Institutes of Health (U.S.) (P01 AI09158001)

T cells play an important role in the adaptive immune system, quickly activating effector functions in response to small numbers of antigenic peptides but rarely activating in response to constant interaction with most endogenous peptides. Emerging experimental evidence suggests that key membrane-bound signaling proteins such as the T cell receptor and the adaptor protein Lat are spatially organized into small clusters on the T cell membrane. We use spatially resolved, stochastic computer simulations to study how the inhomogeneous distribution of molecules affects the portion of the T cell signaling network in which the kinase ZAP-70, originating in T cell receptor clusters, phosphorylates Lat. To gain insight into the effects of protein clustering, we compare the signaling response from membranes with clustered proteins to the signaling response from membranes with homogeneously distributed proteins. Given a fixed amount of ZAP-70 (a proxy for degree of TCR stimulation) that must diffuse into contact with Lat molecules, the spatially homogeneous system responds faster and results in higher levels of phosphorylated Lat. Analysis of the spatial distribution of proteins demonstrates that, in the homogeneous system, nearest ZAP-70 and Lat proteins are closer on average and fewer Lat molecules share the same closest ZAP-70 molecule, leading to the faster response time. The results presented here suggest that spatial clustering of proteins on the T cell membrane may suppress the propagation of signal from ZAP-70 to Lat, thus providing a regulatory mechanism by which T cells suppress transient, spurious signals induced by stimulation of T cell receptors by endogenous peptides. Because this suppression of spurious signals may occur at a cost to sensitivity, we discuss recent experimental results suggesting other potential mechanisms by which ZAP-70 and Lat may interact to initiate T cell activation., United States. National Institutes of Health (Grant 1P01AI091580-01)

Vaccination and the application of a vaginal microbicide have traditionally been considered independent methods to prevent the sexual transmission of HIV-1 to women. Both techniques can be effective in macaque models, and limited efficacy has been observed in clinical trials for each. Here, we have addressed whether vaccines and microbicides can be used together to provide reinforced protection against virus challenge of rhesus macaques. In two separate experiments, four groups of animals were vaccinated with a T-cell–based adenovirus (Ad) vectored vaccine aimed at reducing postinfection viral loads and/or a partially effective dose of a vaginal microbicide aimed at blocking infection of a high-dose vaginal challenge with SIVmac251 or SHIV-162P3. In the first study, the only two protected animals were in the group that received Ad26/Ad5HVR48 vaccine vectors combined with the fusion inhibitor T-1249 as the vaginal microbicide before SIVmac251 challenge. In the second study, vaccination with Ad35/Ad26 vectors combined with the CCR5 inhibitor maraviroc as the vaginal microbicide led to significant reductions of both acquisition of infection and postinfection viral loads following SHIV-SF162P3 challenge. As expected, the vaccine by itself reduced viral loads but had no acquisition effect, whereas the microbicide had a partial acquisition effect but minimal impact on viral loads. For both measures of protective efficacy, the vaccine–microbicide combination differed more from controls than did either separate intervention. Overall, the data suggest that vaccines and microbicides are complementary techniques that may protect better when used together than separately., Massachusetts Institute of Technology. Ragon Institute of MGH, MIT and Harvard, National Institutes of Health (U.S.) (Cooperative Agreement U19 AI76982), National Institutes of Health (U.S.) (Grant AI078526), National Institutes of Health (U.S.) (Grant AI066924)

Differential expression of human leukocyte antigen C (HLA-C) allotypes is mediated by the binding of a microRNA, miR-148a, to the 3′ untranslated region of some, but not all, HLA-C alleles. The binding results in lower levels of HLA-C expression, which is associated with higher levels of HIV-1 viral load among infected individuals. The alternative set of HLA-C alleles has several substitutions in the miR-148a binding site that prevent binding and HLA-C downregulation; these high-expression alleles associate with control of HIV-1 viral load. We show that the common ancestor of all extant HLA-C alleles was suppressed by miR-148a. Substitutions that prevent miR-148a binding arose by a sequence exchange event between an HLA-C allele and an HLA-B (MIM 142830) allele of a B∗07-like lineage. The event occurred 3–5 million years ago, resulting in an HLA-C variant that escape from miR-148a downregulation. We present evidence suggesting that selection played a role in the successful spread of the HLA-C escape alleles, giving rise to 7 of the 14 extant HLA-C lineages. Notably, critical peptide and KIR binding residues of the escape variants have selectively converged to resemble the sequence of their inhibited counterparts, such that the inhibited and escape groupings differ primarily by their levels of expression., National Institutes of Health (U.S.) (contract no. HHSN261200800001E), National Cancer Institute (U.S.), Cancer Research Institute (New York, N.Y.), National Institutes of Health (U.S.) (Intramural Research Program)

Background Despite high potential for HIV-1 genetic variation, the emergence of some mutations is constrained by fitness costs, and may be associated with compensatory amino acid (AA) co-variation. To characterize the interplay between Cytotoxic T Lymphocyte (CTL)-mediated pressure and HIV-1 evolutionary pathways, we investigated AA co-variation in Gag sequences obtained from 449 South African individuals chronically infected with HIV-1 subtype C. Methodology/Principal Findings Individuals with CTL responses biased toward Gag presented lower viral loads than individuals with under-represented Gag-specific CTL responses. Using methods that account for founder effects and HLA linkage disequilibrium, we identified 35 AA sites under Human Leukocyte Antigen (HLA)-restricted CTL selection pressure and 534 AA-to-AA interactions. Analysis of two-dimensional distances between co-varying residues revealed local stabilization mechanisms since 40% of associations involved neighboring residues. Key features of our co-variation analysis included sites with a high number of co-varying partners, such as HLA-associated sites, which had on average 55% more connections than other co-varying sites. Conclusions/Significance Clusters of co-varying AA around HLA-associated sites (especially at typically conserved sites) suggested that cooperative interactions act to preserve the local structural stability and protein function when CTL escape mutations occur. These results expose HLA-imprinted HIV-1 polymorphisms and their interlinked mutational paths in Gag that are likely due to opposite selective pressures from host CTL-mediated responses and viral fitness constraints., Bill & Melinda Gates Foundation (#43437), American Foundation for AIDS Research (#107005-43-RFNT), Center for AIDS Research (CFAR), United States. Public Health Service (AI057005)

Background: Despite the extensive genetic diversity of HIV-1, viral evolution in response to immune selective pressures follows broadly predictable mutational patterns. Sites and pathways of Human Leukocyte-Antigen (HLA)-associated polymorphisms in HIV-1 have been identified through the analysis of population-level data, but the full extent of immune escape pathways remains incompletely characterized. Here, in the largest analysis of HIV-1 subtype B sequences undertaken to date, we identify HLA-associated polymorphisms in the three HIV-1 proteins most commonly considered in cellular-based vaccine strategies. Results are organized into protein-wide escape maps illustrating the sites and pathways of HLA-driven viral evolution. Methodology/Principal Findings: HLA-associated polymorphisms were identified in HIV-1 Gag, Pol and Nef in a multicenter cohort of >1500 chronically subtype-B infected, treatment-naïve individuals from established cohorts in Canada, the USA and Western Australia. At q≤0.05, 282 codons commonly mutating under HLA-associated immune pressures were identified in these three proteins. The greatest density of associations was observed in Nef (where close to 40% of codons exhibited a significant HLA association), followed by Gag then Pol (where ~15–20% of codons exhibited HLA associations), confirming the extensive impact of immune selection on HIV evolution and diversity. Analysis of HIV codon covariation patterns identified over 2000 codon-codon interactions at q≤0.05, illustrating the dense and complex networks of linked escape and secondary/compensatory mutations. Conclusions/Significance: The immune escape maps and associated data are intended to serve as a user-friendly guide to the locations of common escape mutations and covarying codons in HIV-1 subtype B, and as a resource facilitating the systematic identification and classification of immune escape mutations. These resources should facilitate research in HIV epitope discovery and host-pathog, National Institute of Allergy and Infectious Diseases (Award Number U01AI068636 and AI068634)