Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs.

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.

Identification of HPV16 E1 and E2-specific T cells in the oropharyngeal cancer tumor microenvironment.

BACKGROUND: High-risk human papillomavirus (HPV) is a primary cause of an increasing number of oropharyngeal squamous cell carcinomas (OPSCCs). The viral etiology of these cancers provides the opportunity for antigen-directed therapies that are restricted in scope compared with cancers without viral components. However, specific virally-encoded epitopes and their corresponding immune responses are not fully defined. METHODS: To understand the OPSCC immune landscape, we conducted a comprehensive single-cell analysis of HPV16+ and HPV33+ primary tumors and metastatic lymph nodes. We used single-cell analysis with encoded peptide-human leukocyte antigen (HLA) tetramers to analyze HPV16+ and HPV33+ OPSCC tumors, characterizing the ex vivo cellular responses to HPV-derived antigens presented in major Class I and Class II HLA alleles. RESULTS: We identified robust cytotoxic T-cell responses to HPV16 proteins E1 and E2 that were shared across multiple patients, particularly in HLA-A*01:01 and HLA-B*08:01. Responses to E2 were associated with loss of E2 expression in at least one tumor, indicating the functional capacity of these E2-recognizing T cells and many of these interactions validated in a functional assay. Conversely, cellular responses to E6 and E7 were limited in quantity and cytotoxic capacity, and tumor E6 and E7 expression persisted. CONCLUSIONS: These data highlight antigenicity beyond HPV16 E6 and E7 and nominate candidates for antigen-directed therapies.

Allelic variation in class I HLA determines CD8+ T cell repertoire shape and cross-reactive memory responses to SARS-CoV-2.

Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multiomic technology to generate a unified ex vivo characterization of the CD8+ T cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across four major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCRα/ß sequence diversity, and the utilization of pre-existing SARS-CoV-2-reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations notably influence the CD8+ T cell repertoire shape and utilization of immune recall upon SARS-CoV-2 infection.

High-throughput flow alignment of barcoded hydrogel microparticles.

Suspension (particle-based) arrays offer several advantages over conventional planar arrays in the detection and quantification of biomolecules, including the use of smaller sample volumes, more favorable probe-target binding kinetics, and rapid probe-set modification. We present a microfluidic system for the rapid alignment of multifunctional hydrogel microparticles designed to bear one or several biomolecule probe regions, as well as a graphical code to identify the embedded probes. Using high-speed imaging, we have developed and optimized a flow-through system that (1) allows for a high particle throughput, (2) ensures proper particle alignment for decoding and target quantification, and (3) can be reliably operated continuously without clogging. A tapered channel flanked by side focusing streams is used to orient the flexible, tablet-shaped particles into a well-ordered flow in the center of the channel. The effects of channel geometry, particle geometry, particle composition, particle loading density, and barcode design are explored to determine the best combination for eventual use in biological assays. Particles in the optimized system move at velocities of approximately 50 cm s(-1) and with throughputs of approximately 40 particles s(-1). Simple physical models and CFD simulations have been used to investigate flow behavior in the device.

Lock release lithography for 3D and composite microparticles.

We present a method called "Lock Release Lithography (LRL)" that utilizes a combination of channel topography, mask design, and pressure-induced channel deformation to form and release particles in a cycled fashion. This technique provides a means for the high-throughput production of particles with complex 3D morphologies and composite particles with spatially configurable chemistries. In this work, we demonstrate a diverse set of functional particles including those displaying heterogeneous swelling characteristics and containing functional entities such as nucleic acids, proteins and beads.

Soft microflow sensors.

We present a rapid prototyping method for integrating functional components in conventional PDMS microfluidic devices. We take advantage of stop-flow lithography (D. Dendukuri, S. S. Gu, D. C. Pregibon, T. A. Hatton and P. S. Doyle, Lab Chip, 2007, 7, 818)(1) to achieve the in situ fabrication of mobile and deformable elements with a controlled mechanical response. This strategy is applied to the fabrication of microflow sensors based on a deformable spring-like structure. We show that these sensors have a large dynamic range, typically 3 to 4 orders of magnitude, and that they can be scaled down to measure flows in the nl per min range. We prepared sensors with different geometries, and their flow-elongation characteristics were modeled with a simple hydrodynamic model, with good agreement between model and experiments.

Optimization of encoded hydrogel particles for nucleic acid quantification.

The accurate quantification of nucleic acids is of utmost importance for clinical diagnostics, drug discovery, and basic science research. These applications require the concurrent measurement of multiple targets while demanding high-throughput analysis, high sensitivity, specificity between closely related targets, and a wide dynamic range. In attempt to create a technology that can simultaneously meet these demands, we recently developed a method of multiplexed analysis using encoded hydrogel particles. Here, we demonstrate tuning of hydrogel porosity with semi-interpenetrating networks of poly(ethylene glycol), develop a quantitative model to understand hybridization kinetics, and use the findings from these studies to enhance particle design for nucleic acid detection. With an optimized particle design and efficient fluorescent labeling scheme, we demonstrate subattomole sensitivity and single-nucleotide specificity for small RNA targets.

Stop-flow lithography in a microfluidic device.

Polymeric particles in custom designed geometries and with tunable chemical anisotropy are expected to enable a variety of new technologies in diverse areas such as photonics, diagnostics and functional materials. We present a simple, high throughput and high resolution microfluidic method to synthesize such polymeric particles. Building off earlier work that we have done on continuous flow lithography (CFL) (D. Dendukuri, D. C. Pregibon, J. Collins, T. A. Hatton, P. S. Doyle, Nat. Mater., 2006, 5, 365-369; ref. 1), we have devised and implemented a new setup that uses compressed air driven flows in preference to syringe pumps to synthesize particles using a technique that we call stop-flow lithography (SFL). A flowing stream of oligomer is stopped before polymerizing an array of particles into it, providing for much improved resolution over particles synthesized in flow. The formed particles are then flushed out at high flow rates before the cycle of stop-polymerize-flow is repeated. The high flow rates enable orders-of-magnitude improvements in particle throughput over CFL. However, the deformation of the PDMS elastomer due to the imposed pressure restricts how quickly the flow can be stopped before each polymerization event. We have developed a simple model that captures the dependence of the time required to stop the flow on geometric parameters such as the height, length and width of the microchannel, as well as on the externally imposed pressure. Further, we show that SFL proves to be superior to CFL even for the synthesis of chemically anisotropic particles with sharp interfaces between distinct sections.

Correlation of high levels of antibodies to multiple pre-erythrocytic Plasmodium falciparum antigens and protection from infection.

High levels of antibodies to multiple antigens may be more strongly associated with protection from infection than antibodies to a single antigen. Antibody-associated protection against Plasmodium falciparum infection was assessed in a cohort of 68 adults living in an area of holoendemic malaria in Kenya. Antibodies to the pre-erythrocytic antigens circumsporozoite protein (CSP), liver-stage antigen-1 (LSA-1), thrombospondin-related adhesive protein (TRAP), and blood-stage antigens apical membrane antigen-1 (AMA-1), erythrocyte binding antigen-175 (EBA-175), and merozoite surface protein 1 (MSP-1) were tested. Peptides were used for CSP (NANP repeat) and LSA-1 (central repeat), and recombinant antigens were used for TRAP (aa D(48)-K(394)), AMA-1 (ectodomain, non-glycosylated), EBA-175 (non-glycosylated), and MSP-1 (MSP-1(19)). Weekly microscopy testing for P. falciparum infection was performed over a 12-week period after drug-mediated clearance of P. falciparum parasitemia. Individuals with high levels of IgG antibodies (> 2 arbitrary units) to CSP, LSA-1, and TRAP had a 57% decrease in the risk of infection (95% confidence interval = 20-77%, P = 0.016). This decreased risk remained significant after adjustment for age, prior parasitemia, bed net use, sickle cell trait, and village of residence. In contrast, protection against infection did not correlate with high levels of IgG antibodies to blood-stage antigens or IgM antibodies to pre-erythrocytic or blood-stage antigens. High levels of IgG antibodies to CSP, LSA-1, and TRAP may be useful immune correlates of protection against P. falciparum infection in malaria-endemic populations.

Compressed-air flow control system.

We present the construction and operation of a compressed-air driven flow system that can be used for a variety of microfluidic applications that require rapid dynamic response and precise control of multiple inlet streams. With the use of inexpensive and readily available parts, we describe how to assemble this versatile control system and further explore its utility in continuous- and pulsed-flow microfluidic procedures for the synthesis and analysis of microparticles.

Hierarchical assembly of viral nanotemplates with encoded microparticles via nucleic acid hybridization.

We demonstrate hierarchical assembly of tobacco mosaic virus (TMV)-based nanotemplates with hydrogel-based encoded microparticles via nucleic acid hybridization. TMV nanotemplates possess a highly defined structure and a genetically engineered high density thiol functionality. The encoded microparticles are produced in a high throughput microfluidic device via stop-flow lithography (SFL) and consist of spatially discrete regions containing encoded identity information, an internal control, and capture DNAs. For the hybridization-based assembly, partially disassembled TMVs were programmed with linker DNAs that contain sequences complementary to both the virus 5' end and a selected capture DNA. Fluorescence microscopy, atomic force microscopy (AFM), and confocal microscopy results clearly indicate facile assembly of TMV nanotemplates onto microparticles with high spatial and sequence selectivity. We anticipate that our hybridization-based assembly strategy could be employed to create multifunctional viral-synthetic hybrid materials in a rapid and high-throughput manner. Additionally, we believe that these viral-synthetic hybrid microparticles may find broad applications in high capacity, multiplexed target sensing.

Multifunctional encoded particles for high-throughput biomolecule analysis.

High-throughput screening for genetic analysis, combinatorial chemistry, and clinical diagnostics benefits from multiplexing, which allows for the simultaneous assay of several analytes but necessitates an encoding scheme for molecular identification. Current approaches for multiplexed analysis involve complicated or expensive processes for encoding, functionalizing, or decoding active substrates (particles or surfaces) and often yield a very limited number of analyte-specific codes. We present a method based on continuous-flow lithography that combines particle synthesis and encoding and probe incorporation into a single process to generate multifunctional particles bearing over a million unique codes. By using such particles, we demonstrate a multiplexed, single-fluorescence detection of DNA oligomers with encoded particle libraries that can be scanned rapidly in a flow-through microfluidic channel. Furthermore, we demonstrate with high specificity the same multiplexed detection using individual multiprobe particles.

Magnetically and biologically active bead-patterned hydrogels.

We present a new approach to the direct patterning of biologically and magnetically active microbeads in nonbiofouling polymer scaffolds for use in microfluidic devices. Briefly, the process involves treatment of a glass substrate, conformal contact bonding of a PDMS microchannel on the substrate, filling of the channel with beads and prepolymer solution, and UV-initiated photopolymerization of a mask-defined pattern using a standard inverted microscope. This versatile and simple method allows for the rapid fabrication of dispersed or packed bead patterns in poly(ethylene glycol) (PEG) hydrogels that are covalently linked to glass surfaces. By exploiting the relative opacity of the microbeads used, we are able to create both partially exposed and fully encapsulated bead patterns. To demonstrate the utility of this new technology, we separated magnetic bead-bound B lymphocytes from T lymphocytes on a PEG-encapsulated magnetic filtration platform and also captured B cells directly on patterned, protein-decorated beads in a flow-through microfluidic device. Beyond cell sorting, the accurate patterning of industrially standardized, chemically diverse microbeads may have significant implications for microchip-based analyte detection.

Continuous-flow lithography for high-throughput microparticle synthesis.

Precisely shaped polymeric particles and structures are widely used for applications in photonic materials, MEMS, biomaterials and self-assembly. Current approaches for particle synthesis are either batch processes or flow-through microfluidic schemes that are based on two-phase systems, limiting the throughput, shape and functionality of the particles. We report a one-phase method that combines the advantages of microscope projection photolithography and microfluidics to continuously form morphologically complex or multifunctional particles down to the colloidal length scale. Exploiting the inhibition of free-radical polymerization near PDMS surfaces, we are able to repeatedly pattern and flow rows of particles in less than 0.1 s, affording a throughput of near 100 particles per second using the simplest of device designs. Polymerization was also carried out across laminar, co-flowing streams to generate Janus particles containing different chemistries, whose relative proportions could be easily tuned. This new high-throughput technique offers unprecedented control over particle size, shape and anisotropy.

Gamma interferon responses to Plasmodium falciparum liver-stage antigen 1 and thrombospondin-related adhesive protein and their relationship to age, transmission intensity, and protection against malaria.

Gamma interferon (IFN-gamma) responses to the Plasmodium falciparum antigens liver-stage antigen 1 (LSA-1) and thrombospondin-related adhesive protein (TRAP) are thought to be important in protection against malaria. Optimal methods of testing and the effects of age and transmission intensity on these responses are unknown. IFN-gamma responses to LSA-1 and TRAP peptides were assessed by the enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) in children and adults from areas of stable and unstable malaria transmission in Kenya. Adults in the areas of stable and unstable transmission had similar frequencies and levels of IFN-gamma responses to LSA-1 and TRAP as determined by ELISPOT and ELISA. In contrast, IFN-gamma responses to the LSA-1 T3 peptide (assessed by ELISPOT) and to any LSA-1 peptide (assessed by ELISA) were less frequent in children in the area of unstable transmission than in children in the area of stable transmission. IFN-gamma responses to LSA-1 were more frequently detected by ELISA than by ELISPOT in the stable-transmission area. IFN-gamma responses detected by ELISA and ELISPOT did not correlate with each other. In children in the stable-transmission area, IFN-gamma responses to LSA-1 peptides assessed by ELISA, but not by ELISPOT, were associated with protection against clinical malaria and anemia. IFN-gamma responses to LSA-1 appear to require repeated P. falciparum exposure and/or increased age and, as measured by ELISA, are associated with protection against clinical malaria and anemia.