Peptide-MHC-targeted retroviruses enable in vivo expansion and gene delivery to tumor-specific T cells.

Tumor-infiltrating-lymphocyte (TIL) therapy has demonstrated that endogenous T cells can be harnessed to initiate an effective anti-tumor response. Despite clinical promise, current TIL production protocols involve weeks-long ex vivo expansions which can affect treatment efficacy. Therefore, additional tools are needed to engineer endogenous tumor-specific T cells to have increased potency while mitigating challenges of manufacturing. Here, we present a strategy for pseudotyping retroviral vectors with peptide-major histocompatibility complexes (pMHC) for antigen-specific gene delivery to CD8 T cells and examine the efficacy of these transduced cells in immunocompetent mouse models. We demonstrate that pMHC-targeted viruses are able to specifically deliver function-enhancing cargoes while simultaneously activating and expanding anti-tumor T cells. The specificity of these viral vectors enables in vivo engineering of tumor-specific T cells, circumventing ex vivo manufacturing processes and improving overall survival in B16F10-bearing mice. Altogether, we have established that pMHC-targeted viruses are efficient vectors for reprogramming and expanding tumor-specific populations of T cells directly in vivo , with the potential to substantially streamline engineered cell therapy production for a variety of applications.

SARS-CoV-2 spike does not interact with the T cell receptor or directly activate T cells.

SARS-CoV-2infection can induce multisystem inflammatory syndrome in children, which resembles superantigen-induced toxic shock syndrome. Recent work has suggested that the SARS-CoV-2 spike (S) protein could act as a superantigen by binding T cell receptors (TCRs) and inducing broad antigen-independent T cell responses. Structure-based computational modeling identified potential TCR-binding sites near the S receptor-binding domain, in addition to a site with homology to known neurotoxins. We experimentally examined the mechanism underpinning this theory-the direct interaction between the TCR and S protein. Surface plasmon resonance of recombinantly expressed S protein and TCR revealed no detectable binding. Orthogonally, we pseudotyped lentiviruses with SARS-CoV-2 S in both wild-type and prefusion-stabilized forms, demonstrated their functionality in a cell line assay, and observed no transduction, activation, or stimulation of proliferation of CD8+ T cells. We conclude that it is unlikely that the SARS-CoV-2 spike protein engages nonspecifically with TCRs or has superantigenic character.

Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells.

To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we design and characterize Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND), a single-transcript, microRNA-based incoherent feedforward loop. We experimentally tune the ComMAND output profile, and we model the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we highlight the precise control afforded by the single-transcript architecture, particularly at relatively low copy numbers. We show that ComMAND tightly regulates transgene expression from lentiviruses and precisely controls expression in primary human T cells, primary rat neurons, primary mouse embryonic fibroblasts, and human induced pluripotent stem cells. Finally, ComMAND effectively sets levels of the clinically relevant transgenes FMRP1 and FXN within a narrow window. Together, ComMAND is a compact tool well-suited to precisely specify expression of therapeutic cargoes.

Antigen identification and high-throughput interaction mapping by reprogramming viral entry.

Deciphering immune recognition is critical for understanding a broad range of diseases and for the development of effective vaccines and immunotherapies. Efforts to do so are limited by a lack of technologies capable of simultaneously capturing the complexity of adaptive immunoreceptor repertoires and the landscape of potential antigens. To address this, we present receptor-antigen pairing by targeted retroviruses, which combines viral pseudotyping and molecular engineering approaches to enable one-pot library-on-library interaction screens by displaying antigens on the surface of lentiviruses and encoding their identity in the viral genome. Antigen-specific viral infection of cell lines expressing human T or B cell receptors allows readout of both antigen and receptor identities via single-cell sequencing. The resulting system is modular, scalable and compatible with any cell type. These techniques provide a suite of tools for targeted viral entry, molecular engineering and interaction screens with broad potential applications.

Perfect adaptation of CD8+ T cell responses to constant antigen input over a wide range of affinities is overcome by costimulation.

Reduced T cell responses by contrast antigen stimulation can be rescued by signals from costimulatory receptors.

Challenges to sustainable immunization systems in Gavi transitioning countries.

The Global Vaccine Action Plan 2011-2020 (GVAP) aims to extend the full benefit of vaccination against vaccine-preventable diseases to all individuals. More than halfway through the Decade of Vaccines, countries classified as Middle-Income by the World Bank struggle to achieve several GVAP targets. Countries transitioning from Gavi, the Vaccine Alliance, represent a key sub-group of Middle Income Countries. Through a review of available literature on the subject, this study documents the lack of comparative analyses on immunization system performance in countries transitioning from Gavi support. Despite increased emphasis on the importance of programmatic sustainability beyond financing through the Gavi 2016-2020 Strategy and availability of data, existing literature has predominantly documented challenges related to domestic financing of immunization. This study complements a review of current literature with an analysis of country assessments conducted by immunization partners since 2011, in an effort to document programmatic challenges related to decision-making for immunization policy, delivery of services, and access to affordable and timely supply in Gavi transitioning countries. In light of the findings, we suggest continued systematic compilation of country performance data beyond financing to inform policy-making, in particular for: (i) development of a more nuanced theory of change towards sustainable immunization programmes and (ii) measurement of progress and key areas for attention and investment.

Nanoparticles for Immune Cytokine TRAIL-Based Cancer Therapy.

The immune cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received significant attention as a cancer therapeutic due to its ability to selectively trigger cancer cell apoptosis without causing toxicity in vivo. While TRAIL has demonstrated significant promise in preclinical studies in mice as a cancer therapeutic, challenges including poor circulation half-life, inefficient delivery to target sites, and TRAIL resistance have hindered clinical translation. Recent advances in drug delivery, materials science, and nanotechnology are now being exploited to develop next-generation nanoparticle platforms to overcome barriers to TRAIL therapeutic delivery. Here, we review the design and implementation of nanoparticles to enhance TRAIL-based cancer therapy. The platforms we discuss are diverse in their approaches to the delivery problem and provide valuable insight into guiding the design of future nanoparticle-based TRAIL cancer therapeutics to potentially enable future translation into the clinic.

Nanomaterial Interactions with Human Neutrophils.

Neutrophils are the most abundant circulating leukocyte and the first point of contact between many drug delivery formulations and human cells. Despite their prevalence and implication in a range of immune functions, little is known about how human neutrophils respond to synthetic particulates. Here, we describe how ex vivo human neutrophils respond to particles which vary in both size (5 nm to 2 µm) and chemistry (lipids, poly(styrene), poly(lactic-co-glycolic acid), and gold). In particular, we show that (i) particle uptake is rapid, typically plateauing within 15 min; (ii) for a given particle chemistry, neutrophils preferentially take up larger particles at the nanoscale, up to 200 nm in size; (iii) uptake of nanoscale poly(styrene) and liposomal particles at concentrations of up to 5 µg/mL does not enhance apoptosis, activation, or cell death; (iv) particle-laden neutrophils retain the ability to degranulate normally in response to chemical stimulation; and (v) ingested particles reside in intracellular compartments that are retained during activation and degranulation. Aside from the implications for design of intravenously delivered particulate formulations in general, we expect these observations to be of particular use for targeting nanoparticles to circulating neutrophils, their clearance site (bone marrow), or distal sites of active inflammation.

Advances in Pediatric Reference Intervals for Biochemical Markers: Establishment of the Caliper Database in Healthy Children and Adolescents.

Clinical laboratory reference intervals provide valuable information to medical practitioners in their interpretation of quantitative laboratory test results, and therefore are critical in the assessment of patient health and in clinical decision-making. The reference interval serves as a health-associated benchmark with which to compare an individual test result. Unfortunately, critical gaps currently exist in accurate and up-to-date pediatric reference intervals for accurate interpretation of laboratory tests performed in children and adolescents. These critical gaps in the available laboratory reference intervals have the clear potential of contributing to erroneous diagnosis or misdiagnosis of many diseases. To address these important gaps, several initiatives have begun internationally by a number of bodies including the KiGGS initiative in Germany, the Aussie Normals in Australia, the AACC-National Children Study in USA, the NORICHILD Initiative in Scandinavia, and the CALIPER study in Canada. In the present article, we will review the gaps in pediatric reference intervals, challenges in establishing pediatric norms in healthy children and adolescents, and the major contributions of the CALIPER program to closing the gaps in this crucial area of pediatric laboratory medicine. We will also discuss the recently published CALIPER reference interval database (www.caliperdatabase.com) developed to provide comprehensive age and gender specific pediatric reference intervals for a larger number of biochemical markers, based on a large and diverse healthy children cohort. The CALIPER database is based on a multiethnic population examining the influence of ethnicity on laboratory reference intervals. Thus the database has proved to be of global benefit and is being adopted by hospital laboratories worldwide.