Fundamentals of Capillary Electrophoretic Migration and Separation of SDS Proteins in Borate Cross-Linked Dextran Gels.

Recent progress in the development and production of new, innovative protein therapeutics require rapid and adjustable high-resolution bioseparation techniques. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using a borate (B) cross-linked dextran (D) separation matrix is widely employed today for rapid consistency analysis of therapeutic proteins in manufacturing and release testing. Transient borate cross-linking of the semirigid dextran polymer chains leads to a high-resolution separation gel for SDS-protein complexes. To understand the migration and separation basis of the D/B gel, the present work explores various gel formulations of dextran monomer (2, 5, 7.5, and 10%) and borate cross-linker (2 and 4%) concentrations. Ferguson plots were analyzed for a mixture of protein standards with molecular weights ranging from 20 to 225 kDa, and the resulting nonlinear concave curves pointed to nonclassical sieving behavior. While the 2% D/4% B gel resulted in the fastest analysis time, the 10% D/2% B gel was found to produce the greatest separation window, even higher than with the 10% D/4% B gel, due to a significant increase in the electroosmotic flow of the former composition in the direction opposite to SDS-protein complex migration. The study then focused on SDS-CGE separation of a therapeutic monoclonal antibody and its subunits. A combination of molecular weight and shape selectivity as well as, to a lesser extent, surface charge density differences (due to glycosylation on the heavy chain) influenced migration. Greater molecular weight selectivity occurred for the higher monomer concentration gels, while improved glycoselectivity was obtained using a more dilute gel, even as low as 2% D/2% B. This latter gel took advantage of the dextran-borate-glycoprotein complexation. The study revealed that by modulating the dextran (monomer) and borate (cross-linker) concentration ratios of the sieving matrix, one can optimize the separation for specific biopharmaceutical modalities with excellent column-to-column, run-to-run, and gel-to-gel migration time reproducibilities (<0.96% relative standard deviation (RSD)). The widely used 10% dextran/4% borate gel represents a good screening option, which can then be followed by a modified composition, optimized for a specific separation as necessary.

Proteomic Profiling of IgG1 Producing CHO Cells Using LC/LC-SPS-MS3: The Effects of Bioprocessing Conditions on Productivity and Product Quality.

The biopharmaceutical market is dominated by monoclonal antibodies, the majority of which are produced in Chinese hamster ovary (CHO) cell lines. Intense cell engineering, in combination with optimization of various process parameters results in increasing product titers. To enable further improvements in manufacturing processes, detailed information about how certain parameters affect cellular mechanisms in the production cells, and thereby also the expressed drug substance, is required. Therefore, in this study the effects of commonly applied changes in bioprocessing parameters on an anti-IL8 IgG1 producing CHO DP-12 cell line were investigated on the level of host cell proteome expression combined with product quality assessment of the expressed IgG1 monoclonal antibody. Applying shifts in temperature, pH and dissolved oxygen concentration, respectively, resulted in altered productivity and product quality. Furthermore, analysis of the cells using two-dimensional liquid chromatography-mass spectrometry employing tandem mass tag based isotopic quantitation and synchronous precursor selection-MS3 detection revealed substantial changes in the protein expression profiles of CHO cells. Pathway analysis indicated that applied bioprocessing conditions resulted in differential activation of oxidative phosphorylation. Additionally, activation of ERK5 and TNFR1 signaling suggested an affected cell cycle. Moreover, in-depth product characterization by means of charge variant analysis, peptide mapping, as well as structural and functional analysis, revealed posttranslational and structural changes in the expressed drug substance. Taken together, the present study allows the conclusion that, in anti-IL8 IgG1 producing CHO DP-12 cells, an improved energy metabolism achieved by lowering the cell culture pH is favorable when aiming towards high antibody production rates while maintaining product quality.

Multi-Omics Reveals Impact of Cysteine Feed Concentration and Resulting Redox Imbalance on Cellular Energy Metabolism and Specific Productivity in CHO Cell Bioprocessing.

Chinese hamster ovary (CHO) cells are currently the primary host cell lines used in biotherapeutic manufacturing of monoclonal antibodies (mAbs) and other biopharmaceuticals. Cellular energy metabolism and endoplasmic reticulum (ER) stress are known to greatly impact cell growth, viability, and specific productivity of a biotherapeutic; but the molecular mechanisms are not fully understood. The authors previously employed multi-omics profiling to investigate the impact of a reduction in cysteine (Cys) feed concentration in a fed-batch process and found that disruption of the redox balance led to a substantial decline in cell viability and titer. Here, the multi-omics findings are expanded, and the impact redox imbalance has on ER stress, mitochondrial homeostasis, and lipid metabolism is explored. The reduced Cys feed activates the amino acid response (AAR), increases mitochondrial stress, and initiates gluconeogenesis. Multi-omics analysis reveals that together, ER stress and AAR signaling shift the cellular energy metabolism to rely primarily on anaplerotic reactions, consuming amino acids and producing lactate, to maintain energy generation. Furthermore, the pathways are demonstrated in which this shift in metabolism leads to a substantial decline in specific productivity and altered mAb glycosylation. Through this work, meaningful bioprocess markers and targets for genetic engineering are identified.

Multi-Omics Study on the Impact of Cysteine Feed Level on Cell Viability and mAb Production in a CHO Bioprocess.

There is continual demand to maximize CHO cell culture productivity of a biotherapeutic while maintaining product quality. In this study, a comprehensive multi-omics analysis is performed to investigate the cellular response to the level of dosing of the amino acid cysteine (Cys) in the production of a monoclonal antibody (mAb). When Cys feed levels are insufficient, there is a significant decrease in protein titer. Multi-omics (metabolomics and proteomics, with support from RNAseq) is performed over the time course of the CHO bioprocess producing an IgG1 mAb in 5 L bioreactors. Pathway analysis reveals that insufficient levels of Cys in the feed lead to Cys depletion in the cell. This depletion negatively impacts antioxidant molecules, such as glutathione (GSH) and taurine, leading to oxidative stress with multiple deleterious cellular effects. In this paper, the resultant ER stress and subsequent apoptosis that affects cell viability and viable cell density has been considered. Key metabolic enzymes and metabolites are identified that can be potentially monitored as the process progresses and/or increased in the cell either by nutrient feeding or genetic engineering. This work reinforces the centrality of redox balance to cellular health and success of the bioprocess as well as the power of multi-omics to provide an in-depth understanding of the CHO cell biology during biopharmaceutical production.

Complementary middle-down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis - mass spectrometry.

High-resolution capillary zone electrophoresis - mass spectrometry (CZE-MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle-down and intact CZE-MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post-translational modifications (PTMs) and glycosylation structures. Middle-down and intact CZE separations were performed in an acidified methanol-water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle-down analysis of the IdeS-digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X-deamidated, 1X-deamidated, and non-deamidated forms at baseline resolution. In the course of the middle-down CZE-MS analysis, separation of glycoforms of the FC /2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE-MS2 . Incorporation of TCEP-HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE-MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X-glycosylated, 1X-glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE-MS represents a complementary approach to the more conventional liquid-chromatography - mass spectrometry-based approaches.

Quantitative Proteomic Profiling of the Human Ovary from Early to Mid-Gestation Reveals Protein Expression Dynamics of Oogenesis and Folliculogenesis.

The in vivo gene networks involved in coordinating human fetal ovarian development remain obscure. In this study, quantitative mass spectrometry was conducted on ovarian tissue collected at key stages during the first two trimesters of human gestational development, confirming the expression profiling data using immunofluorescence, as well as in vitro modeling with human oogonial stem cells (OSCs) and human embryonic stem cells (ESCs). A total of 3,837 proteins were identified in samples spanning developmental days 47-137. Bioinformatics clustering and Ingenuity Pathway Analysis identified DNA mismatch repair and base excision repair as major pathways upregulated during this time. In addition, MAEL and TEX11, two key meiosis-related proteins, were identified as highly expressed during the developmental window associated with fetal oogenesis. These findings were confirmed and extended using in vitro differentiation of OSCs into in vitro derived oocytes and of ESCs into primordial germ cell-like cells and oocyte-like cells, as models. In conclusion, the global protein expression profiling data generated by this study have provided novel insights into human fetal ovarian development in vivo and will serve as a valuable new resource for future studies of the signaling pathways used to orchestrate human oogenesis and folliculogenesis.

Lipidomics of CHO Cell Bioprocessing: Relation to Cell Growth and Specific Productivity of a Monoclonal Antibody.

As the demand for biological therapeutic proteins rises, there is an increasing need for robust and highly efficient bioprocesses, specifically, maximizing protein production by controlling the cellular nutritional and metabolic needs. A comprehensive lipidomics analysis has been performed, for the first time, over the time course of CHO cells producing an IgG1 monoclonal antibody (mAb) with fed batch 5 L bioreactors. The dynamic nature and importance of the CHO lipidome, especially on cellular growth and specific productivity, is demonstrated. A robust LC-MS method using positive and negative mode ESI was developed for lipid identification and quantitation of 377 unique lipids. The analysis revealed large changes in lipid features between the different days in bioprocessing including accumulation of triacylglycerol (TG) and lysophospholipid species with depletion of diacylglycerol (DG) species. Exploring pathway analysis where the lipid data was combined with polar metabolites and transcriptomics (RNA sequencing) revealed differences in lipid metabolism between the various stages of cellular growth and highlighted the role of key features of lipid metabolism on cell growth and specific productivity. The study demonstrates the importance of lipidomics in the expanding role of 'Omics methodologies in gaining insight into cellular behavior during protein production in a fed batch bioprocess.

Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry.

Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). Graphical Abstract ᅟ.

Host Cell Protein Profiling by Targeted and Untargeted Analysis of Data Independent Acquisition Mass Spectrometry Data with Parallel Reaction Monitoring Verification.

Host cell proteins (HCPs) are process-related impurities of biopharmaceuticals that remain at trace levels despite multiple stages of downstream purification. Currently, there is interest in implementing LC-MS in biopharmaceutical HCP profiling alongside conventional ELISA, because individual species can be identified and quantitated. Conventional data dependent LC-MS is hampered by the low concentration of HCP-derived peptides, which are 5-6 orders of magnitude less abundant than the biopharmaceutical-derived peptides. In this paper, we present a novel data independent acquisition (DIA)-MS workflow to identify HCP peptides using automatically combined targeted and untargeted data processing, followed by verification and quantitation using parallel reaction monitoring (PRM). Untargeted data processing with DIA-Umpire provided a means of identifying HCPs not represented in the assay library used for targeted, peptide-centric, data analysis. An IgG1 monoclonal antibody (mAb) purified by Protein A column elution, cation exchange chromatography, and ultrafiltration was analyzed using the workflow with 1D-LC. Five protein standards added at 0.5 to 100 ppm concentrations were detected in the background of the purified mAb, demonstrating sensitivity to low ppm levels. A calibration curve was constructed on the basis of the summed peak areas of the three highest intensity fragment ions from the highest intensity peptide of each protein standard. Sixteen HCPs were identified and quantitated on the basis of the calibration curve over the range of low ppm to over 100 ppm in the purified mAb sample. The developed approach achieves rapid HCP profiling using 1D-LC and specific identification exploiting the high mass accuracy and resolution of the mass spectrometer.

Binding Site Characterization of AM1336, a Novel Covalent Inverse Agonist at Human Cannabinoid 2 Receptor, Using Mass Spectrometric Analysis.

Cannabinoid 2 receptor (CB2R), a Class-A G-protein coupled receptor (GPCR), is a promising drug target under a wide array of pathological conditions. Rational drug design has been hindered due to our poor understanding of the structural features involved in ligand binding. Binding of a high-affinity biarylpyrazole inverse agonist AM1336 to a library of the human CB2 receptor (hCB2R) cysteine-substituted mutants provided indirect evidence that two cysteines in transmembrane helix-7 (H7) were critical for the covalent attachment. We used proteomics analysis of the hCB2R with bound AM1336 to directly identify peptides with covalently attached ligand and applied in silico modeling for visualization of the ligand-receptor interactions. The hCB2R, with affinity tags (FlaghCB2His6), was produced in a baculovirus-insect cell expression system and purified as a functional receptor using immunoaffinity chromatography. Using mass spectrometry-based bottom-up proteomic analysis of the hCB2R-AM1336, we identified a peptide with AM1336 attached to the cysteine C284(7.38) in H7. The hCB2R homology model in lipid bilayer accommodated covalent attachment of AM1336 to C284(7.38), supporting both biochemical and mass spectrometric data. This work consolidates proteomics data and in silico modeling and integrates with our ligand-assisted protein structure (LAPS) experimental paradigm to assist in structure-based design of cannabinoid antagonist/inverse agonists.

Functional role and therapeutic targeting of p21-activated kinase 4 in multiple myeloma.

Dysregulated oncogenic serine/threonine kinases play a pathological role in diverse forms of malignancies, including multiple myeloma (MM), and thus represent potential therapeutic targets. Here, we evaluated the biological and functional role of p21-activated kinase 4 (PAK4) and its potential as a new target in MM for clinical applications. PAK4 promoted MM cell growth and survival via activation of MM survival signaling pathways, including the MEK-extracellular signal-regulated kinase pathway. Furthermore, treatment with orally bioavailable PAK4 allosteric modulator (KPT-9274) significantly impacted MM cell growth and survival in a large panel of MM cell lines and primary MM cells alone and in the presence of bone marrow microenvironment. Intriguingly, we have identified FGFR3 as a novel binding partner of PAK4 and observed significant activity of KPT-9274 against t(4;14)-positive MM cells. This set of data supports PAK4 as an oncogene in myeloma and provide the rationale for the clinical evaluation of PAK4 modulator in myeloma.

Advanced Precursor Ion Selection Algorithms for Increased Depth of Bottom-Up Proteomic Profiling.

Conventional TopN data-dependent acquisition (DDA) LC-MS/MS analysis identifies only a limited fraction of all detectable precursors because the ion-sampling rate of contemporary mass spectrometers is insufficient to target each precursor in a complex sample. TopN DDA preferentially targets high-abundance precursors with limited sampling of low-abundance precursors and repeated analyses only marginally improve sample coverage due to redundant precursor sampling. In this work, advanced precursor ion selection algorithms were developed and applied in the bottom-up analysis of HeLa cell lysate to overcome the above deficiencies. Precursors fragmented in previous runs were efficiently excluded using an automatically aligned exclusion list, which reduced overlap of identified peptides to ∼10% between replicates. Exclusion of previously fragmented high-abundance peptides allowed deeper probing of the HeLa proteome over replicate LC-MS runs, resulting in the identification of 29% more peptides beyond the saturation level achievable using conventional TopN DDA. The gain in peptide identifications using the developed approach translated to the identification of several hundred low-abundance protein groups, which were not detected by conventional TopN DDA. Exclusion of only identified peptides compared with the exclusion of all previously fragmented precursors resulted in an increase of 1000 (∼10%) additional peptide identifications over four runs, suggesting the potential for further improvement in the depth of proteomic profiling using advanced precursor ion selection algorithms.

Combined metabolomics and proteomics reveals hypoxia as a cause of lower productivity on scale-up to a 5000-liter CHO bioprocess.

Large-scale bioprocessing is key to the successful manufacturing of a biopharmaceutical. However, cell viability and productivity are often lower in the scale-up from laboratory to production. In this study, we analyzed CHO cells, which showed lower percent viabilities and productivity in a 5-KL production scale bioreactor compared to a 20-L bench-top scale under seemingly identical process parameters. An increase in copper concentration in the media from 0.02 µM to 0.4 µM led to a doubling of percent viability in the production scale albeit still at a lower level than the bench-top scale. Combined metabolomics and proteomics revealed the increased copper reduced the presence of reactive oxygen species (ROS) in the 5-KL scale process. The reduction in oxidative stress was supported by the increased level of glutathione peroxidase in the lower copper level condition. The excess ROS was shown to be due to hypoxia (intermittent), as evidenced by the reduction in fibronectin with increased copper. The 20-L scale showed much less hypoxia and thus less excess ROS generation, resulting in little to no impact to productivity with the increased copper in the media. The study illustrates the power of 'Omics in aiding in the understanding of biological processes in biopharmaceutical production.

High Resolution CZE-MS Quantitative Characterization of Intact Biopharmaceutical Proteins: Proteoforms of Interferon-ß1.

New and improved methods are required for the enhanced characterization of complex biopharmaceuticals, especially those with charge and glycan heterogeneity. High resolution separation and mass spectrometry (MS) analysis of intact proteoforms can contribute significantly to the characterization of such proteins, many of which are glycoproteins. Here, we report on capillary zone electrophoresis (CZE) coupled via a commercial CESI sheathless interface to an Orbitrap ELITE MS for the intact analysis of recombinant human interferon-ß1 (Avonex, rhIFN-ß1), a biopharmaceutical with complex glycosylation at a single N-linked site. Using a cross-linked polyethylenimine coating, column efficiencies between 350,000 and 450,000 plates were produced, allowing separation based on charge and subtle hydrodynamic volume differences. A total of 138 proteoforms were found, and 55 were quantitated. Charge species due to deamidation and sialylation were separated by CZE. Given the high column efficiency, isobaric positional isomers of a single sialic acid on biantennary glycan antennae were resolved. Further, triantennary isomers (antenna on α(1-3) or α(1-6) arms) were separated and confirmed by exoglycosidase digestion. Proteoforms of the N-terminal cleavage of methionine were detected by precursor molecular weight and top-down ETD and HCD analysis of the reduced protein. Quantitative analysis suggested potential correlations between the methionine loss with the relative amount of the deamidation, as well as the level of deamidation with glycan structure. We demonstrate that high resolution CZE separation of intact glycoprotein species coupled to MS has significant potential for the in-depth characterization and quantitative analysis of biopharmaceutical proteoforms.

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells.

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up-regulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial- and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production.

Immunological mechanisms of the antitumor effects of supplemental oxygenation.

Antitumor T cells either avoid or are inhibited in hypoxic and extracellular adenosine-rich tumor microenvironments (TMEs) by A2A adenosine receptors. This may limit further advances in cancer immunotherapy. There is a need for readily available and safe treatments that weaken the hypoxia-A2-adenosinergic immunosuppression in the TME. Recently, we reported that respiratory hyperoxia decreases intratumoral hypoxia and concentrations of extracellular adenosine. We show that it also reverses the hypoxia-adenosinergic immunosuppression in the TME. This, in turn, stimulates (i) enhanced intratumoral infiltration and reduced inhibition of endogenously developed or adoptively transfered tumor-reactive CD8 T cells, (ii) increased proinflammatory cytokines and decreased immunosuppressive molecules, such as transforming growth factor-ß (TGF-ß), (iii) weakened immunosuppression by regulatory T cells, and (iv) improved lung tumor regression and long-term survival in mice. Respiratory hyperoxia also promoted the regression of spontaneous metastasis from orthotopically grown breast tumors. These effects are entirely T cell- and natural killer cell-dependent, thereby justifying the testing of supplemental oxygen as an immunological coadjuvant to combine with existing immunotherapies for cancer.

An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood.

Isolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000-2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100-200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers.

Physical detection of influenza A epitopes identifies a stealth subset on human lung epithelium evading natural CD8 immunity.

Vaccines eliciting immunity against influenza A viruses (IAVs) are currently antibody-based with hemagglutinin-directed antibody titer the only universally accepted immune correlate of protection. To investigate the disconnection between observed CD8 T-cell responses and immunity to IAV, we used a Poisson liquid chromatography data-independent acquisition MS method to physically detect PR8/34 (H1N1), X31 (H3N2), and Victoria/75 (H3N2) epitopes bound to HLA-A*02:01 on human epithelial cells following in vitro infection. Among 32 PR8 peptides (8-10mers) with predicted IC50 < 60 nM, 9 were present, whereas 23 were absent. At 18 h postinfection, epitope copies per cell varied from a low of 0.5 for M13-11 to a high of >500 for M1(58-66) with PA, HA, PB1, PB2, and NA epitopes also detected. However, aside from M1(58-66), natural CD8 memory responses against conserved presented epitopes were either absent or only weakly observed by blood Elispot. Moreover, the functional avidities of the immunodominant M1(58-66)/HLA-A*02:01-specific T cells were so poor as to be unable to effectively recognize infected human epithelium. Analysis of T-cell responses to primary PR8 infection in HLA-A*02:01 transgenic B6 mice underscores the poor avidity of T cells recognizing M1(58-66). By maintaining high levels of surface expression of this epitope on epithelial and dendritic cells, the virus exploits the combination of immunodominance and functional inadequacy to evade HLA-A*02:01-restricted T-cell immunity. A rational approach to CD8 vaccines must characterize processing and presentation of pathogen-derived epitopes as well as resultant immune responses. Correspondingly, vaccines may be directed against "stealth" epitopes, overriding viral chicanery.