Rapid Identification of MHCII-Binding Peptides Through Microsphere-Assisted Peptide Screening (MAPS).

CD4+ T cells play a vital role in the immune response, and their function requires T cell receptor (TCR) recognition of peptide epitopes presented in complex with MHC class II (MHCII) molecules. Consequently, rapidly identifying peptides that bind MHCII is critical to understanding and treating infectious disease, cancer, autoimmunity, allergy, and transplant rejection. Computational methods provide a fast, ultrahigh-throughput approach to predict MHCII-binding peptides but lack the accuracy of experimental methods. In contrast, experimental methods offer accurate, quantitative results at the expense of speed. To address the gap between these two approaches, we developed a high-throughput, semiquantitative experimental screening strategy termed microsphere-assisted peptide screening (MAPS). Here, we use the Zika virus envelope protein as an example to demonstrate the rapid identification of MHCII-binding peptides from a single pathogenic protein using MAPS. This process involves several key steps including peptide library design, peptide exchange into MHCII, peptide-MHCII loading onto microspheres, flow cytometry screening, and data analysis to identify peptides that bind to one or more MHCII alleles.

Rapid microsphere-assisted peptide screening (MAPS) of promiscuous MHCII-binding peptides in Zika virus envelope protein.

Despite promising developments in computational tools, peptide-class II MHC (MHCII) binding predictors continue to lag behind their peptide-class I MHC counterparts. Consequently, peptide-MHCII binding is often evaluated experimentally using competitive binding assays, which tend to sacrifice throughput for quantitative binding detail. Here, we developed a high-throughput semiquantitative peptide-MHCII screening strategy termed microsphere-assisted peptide screening (MAPS) that aims to balance the accuracy of competitive binding assays with the throughput of computational tools. Using MAPS, we screened a peptide library from Zika virus envelope (E) protein for binding to four common MHCII alleles (DR1, DR4, DR7, DR15). Interestingly, MAPS revealed a significant overlap between peptides that promiscuously bind multiple MHCII alleles and antibody neutralization sites. This overlap was also observed for rotavirus outer capsid glycoprotein VP7, suggesting a deeper relationship between B cell and CD4+ T cell specificity which can facilitate the design of broadly protective vaccines to Zika and other viruses.

Enhancing the Yield and Quality of Influenza Virus-like Particles (VLPs) Produced in Insect Cells by Inhibiting Cytopathic Effects of Matrix Protein M2.

To provide broader protection and eliminate the need for annual update of influenza vaccines, biomolecular engineering of influenza virus-like particles (VLPs) to display more conserved influenza proteins such as the matrix protein M2 has been explored. However, achieving high surface density of full-length M2 in influenza VLPs has been left unrealized. In this study, we show that the ion channel activity of M2 induces significant cytopathic effects in Spodoptera frugiperda (Sf9) insect cells when expressed using M2-encoding baculovirus. These effects include altered Sf9 cell morphology and reduced baculovirus replication, resulting in impaired influenza protein expression and thus VLP production. On the basis of the function of M2, we hypothesized that blocking its ion channel activity could potentially relieve these cytopathic effects, and thus restore influenza protein expression to improve VLP production. The use of the M2 inhibitor amantadine indeed improves Sf9 cellular expression not only of M2 (∼3-fold), but also of hemagglutinin (HA) (∼7-fold) and of matrix protein M1 (∼3-fold) when coexpressed to produce influenza VLPs. This increased cellular expression of all three influenza proteins further leads to ∼2-fold greater VLP yield. More importantly, the quality of the resulting influenza VLPs is significantly improved, as demonstrated by the ∼2-fold, ∼50-fold, and ∼2-fold increase in the antigen density to approximately 53 HA, 48 M1, and 156 M2 per influenza VLP, respectively. Taken together, this study represents a novel approach to enable the efficient incorporation of full-length M2 while enhancing both the yield and quality of influenza VLPs produced by Sf9 cells.

Construction and Screening of an Antigen-Derived Peptide Library Displayed on Yeast Cell Surface for CD4+ T Cell Epitope Identification.

Antigenic peptides (termed T cell epitopes) are assembled with major histocompatibility complex (MHC) molecules and presented on the surface of antigen-presenting cells (APCs) for T cell recognition. T cells engage these peptide-MHCs using T cell receptors (TCRs). Because T cell epitopes determine the specificity of a T cell immune response, their prediction and identification are important steps in developing peptide-based vaccines and immunotherapies. In recent years, a number of computational methods have been developed to predict T cell epitopes by evaluating peptide-MHC binding; however, the success of these methods has been limited for MHC class II (MHCII) due to the structural complexity of MHCII antigen presentation. Moreover, while peptide-MHC binding is a prerequisite for a T cell epitope, it alone is not sufficient. Therefore, T cell epitope identification requires further functional verification of the MHC-binding peptide using professional APCs, which are difficult to isolate, expand, and maintain. To address these issues, we have developed a facile, accurate, and high-throughput method for T cell epitope mapping by screening antigen-derived peptide libraries in complex with MHC protein displayed on yeast cell surface. Here, we use hemagglutinin and influenza A virus X31/A/Aichi/68 as examples to describe the key steps in identification of CD4+ T cell epitopes from a single antigenic protein and the entire genome of a pathogen, respectively. Methods for single-chain peptide MHC vector design, yeast surface display, peptide library generation in Escherichia coli, and functional screening in Saccharomyces cerevisiae are discussed.

Elucidating structure-performance relationships in whole-cell cooperative enzyme catalysis.

Cooperative enzyme catalysis in nature has long inspired the application of engineered multi-enzyme assemblies for industrial biocatalysis. Despite considerable interest, efforts to harness the activity of cell-surface displayed multi-enzyme assemblies have been based on trial and error rather than rational design due to a lack of quantitative tools. In this study, we developed a quantitative approach to whole-cell biocatalyst characterization enabling a comprehensive study of how yeast-surface displayed multi-enzyme assemblies form. Here we show that the multi-enzyme assembly efficiency is limited by molecular crowding on the yeast cell surface, and that maximizing enzyme density is the most important parameter for enhancing cellulose hydrolytic performance. Interestingly, we also observed that proximity effects are only synergistic when the average inter-enzyme distance is > ~130 nm. The findings and the quantitative approach developed in this work should help to advance the field of biocatalyst engineering from trial and error to rational design.

Protein-Scaffold Directed Nanoscale Assembly of T Cell Ligands: Artificial Antigen Presentation with Defined Valency, Density, and Ratio.

Tuning antigen presentation to T cells is a critical step in investigating key aspects of T cell activation. However, existing technologies have a limited ability to control the spatial and stoichiometric organization of T cell ligands on 3D surfaces. Here, we developed an artificial antigen presentation platform based on protein scaffold-directed assembly that allows fine control over the spatial and stoichiometric organization of T cell ligands on a 3D yeast cell surface. Using this system, we observed that the T cell activation threshold on a 3D surface is independent of peptide-major histocompatibility complex (pMHC) valency but instead is determined by the overall pMHC surface density. When intercellular adhesion molecule 1 (ICAM-1) was coassembled with pMHC, it enhanced antigen recognition sensitivity by 6-fold. Further, T cells responded with different magnitudes to varying ratios of pMHC and ICAM-1 and exhibited a maximum response at a ratio of 15% pMHC and 85% ICAM-1, introducing an additional parameter for tuning T cell activation. This protein scaffold-directed assembly technology is readily transferrable to acellular surfaces for translational research as well as large-scale T-cell manufacturing.

Engineering Novel and Improved Biocatalysts by Cell Surface Display.

Biocatalysts, especially enzymes, have the ability to catalyze reactions with high product selectivity, utilize a broad range of substrates, and maintain activity at low temperature and pressure. Therefore, they represent a renewable, environmentally friendly alternative to conventional catalysts. Most current industrial-scale chemical production processes using biocatalysts employ soluble enzymes or whole cells expressing intracellular enzymes. Cell surface display systems differ by presenting heterologous enzymes extracellularly, overcoming some of the limitations associated with enzyme purification and substrate transport. Additionally, coupled with directed evolution, cell surface display is a powerful platform for engineering enzymes with enhanced properties. In this review, we will introduce the molecular and cellular principles of cell surface display and discuss how it has been applied to engineer enzymes with improved properties as well as to develop surface-engineered microbes as whole-cell biocatalysts.

In search of on-line locality effects in sentence comprehension.

Many comprehension theories assert that increasing the distance between elements participating in a linguistic relation (e.g., a verb and a noun phrase argument) increases the difficulty of establishing that relation during on-line comprehension. Such locality effects are expected to increase reading times and are thought to reveal properties and limitations of the short-term memory system that supports comprehension. Despite their theoretical importance and putative ubiquity, however, evidence for on-line locality effects is quite narrow linguistically and methodologically: It is restricted almost exclusively to self-paced reading of complex structures involving a particular class of syntactic relation. We present 4 experiments (2 self-paced reading and 2 eyetracking experiments) that demonstrate locality effects in the course of establishing subject-verb dependencies; locality effects are seen even in materials that can be read quickly and easily. These locality effects are observable in the earliest possible eye-movement measures and are of much shorter duration than previously reported effects. To account for the observed empirical patterns, we outline a processing model of the adaptive control of button pressing and eye movements. This model makes progress toward the goal of eliminating linking assumptions between memory constructs and empirical measures in favor of explicit theories of the coordinated control of motor responses and parsing.

Attention enhances the neural processing of relevant features and suppresses the processing of irrelevant features in humans: a functional magnetic resonance imaging study of the Stroop task.

We present a functional MRI experiment investigating the neural basis of feature-based attention in humans using the Stroop task. Cortical areas specifically involved in color processing and word reading were first identified in individual participants using independent tests. These areas were then probed during the Stroop task (in which participants must selectively attend to the font color of a word while ignoring the word itself). We found that activation in functionally defined color areas increased during the task relative to a neutral color-naming task while activation in functionally defined word areas decreased. These results are consistent with a biased competition model of feature-based attention in which the processing of attended features is enhanced and the processing of ignored features is suppressed.

Nature versus nurture in ventral visual cortex: a functional magnetic resonance imaging study of twins.

Using functional magnetic resonance imaging, we estimated neural activity in twins to study genetic influences on the cortical response to categories of visual stimuli (faces, places, and pseudowords) that are known to elicit distinct patterns of activity in ventral visual cortex. The neural activity patterns in monozygotic twins were significantly more similar than in dizygotic twins for the face and place stimuli, but there was no effect of zygosity for pseudowords (or chairs, a control category). These results demonstrate that genetics play a significant role in determining the cortical response to faces and places, but play a significantly smaller role (if any) in the response to orthographic stimuli.

Aging reduces neural specialization in ventral visual cortex.

The present study investigated whether neural structures become less functionally differentiated and specialized with age. We studied ventral visual cortex, an area of the brain that responds selectively to visual categories (faces, places, and words) in young adults, and that shows little atrophy with age. Functional MRI was used to estimate neural activity in this cortical area, while young and old adults viewed faces, houses, pseudowords, and chairs. The results demonstrated significantly less neural specialization for these stimulus categories in older adults across a range of analyses.