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Abstract: There is currently a significant interest in the identification and validation of HLA-restricted CTL epitopes, which are thought to have important implications for the development of preventive and/or therapeutic applications in bacterial or viral infections, autoimmune diseases, and cancer. To better facilitate epitope discovery and validation, we present a cell- and radioisotope-free HLA-A⁎0201 assay system which relies upon fluorescence polarization. The assay has the advantage of allowing real-time measurements in solution without separation steps. In this report, we directed our efforts towards enhancing the sensitivity and reproducibility of the assay by conducting an in-depth analysis of parameters critical for standardization. Initial experiments demonstrated that the attachment of a fluorescence moiety at positions 5 and 8 for 9-mers and positions 5 and 6 for 10-mers, respectively, does not interfere with ligand binding to soluble HLA-A⁎0201. In addition, it was found that their binding to HLA-A⁎0201 was very effective showing high affinity binding with K d''s between 10.7 to 21.8 nM and binding capacities of up to 37%. In order to deliver maximized responses, factors such as the regulation of thermal HLA activation parameters to initiate peptide exchange as well as the specific adjustment of assay components were identified. Overall, the results obtained clearly demonstrate high accuracy, sensitivity and reproducibility of the FP-based assay approach. With the need for both increased throughput and miniaturized volumes, this fully homogenous, fluorescent-type binding assay is expected to be useful for routine analysis of peptide binding to MHC class I as well as class II molecules. [Copyright &y& Elsevier]

Various approaches are currently proposed to successfully develop therapies for the prevention and treatment of infectious diseases and cancer. One of the most promising approaches is the development of vaccines that elicit cytotoxic T lymphocyte (CTL) responses. Consequently, identification and exact definition of molecular parameters involved in peptide-MHC class-I interactions of putative CTL epitopes are of prime importance for the development of immunomodulating compounds. To better facilitate epitope discovery, we developed and validated a novel state-of-the-art biochemical HLA-A*0201 assay, which is comprised of technologically advanced cutting edge reagents. The technique is based on competition and uses a FITC-labeled reference peptide and highly purified soluble HLA-A*0201 molecules to quantitatively measure the binding capacity of nonlabeled peptide candidates. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. During standardization, the problem of assay parameter variation is discussed, showing the dramatic influence of HLA and reference peptide concentrations as well as the choice of the reference peptide itself on IC50 determinations. For validation, a panel of 15 well-defined HLA-A*0201 ligands from various sources covering a broad range of binding affinities was tested. Binding data were used to compare against pre-existing quantitative assay systems. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with recombinant sHLA molecules is highly advantageous for the accurate assessment of peptide binding. Furthermore, the assay also features high-throughput screening capacity, providing uniquely efficient means of identifying and evaluating immune target molecules. [ABSTRACT FROM AUTHOR]

Measuring the interaction of class I human leukocyte antigens (HLA) and their peptide epitopes acts as a guide for the development of vaccines, diagnostics, and immune-based therapies. Here, we report the development of a sensitive biochemical assay that relies upon fluorescence polarization to indicate peptide interactions with recombinant soluble HLA proteins. It is a cell- and radioisotope-free assay that has the. advantage of allowing the direct, real-time measurement of the ratio between free and bound peptide ligand in solution without separation steps. Peptide/HLA assay parameters were established using several HLA A*0201-specific fluorescein isothiocyanate-labeled peptides. Optimal loading of synthetic peptides into fully assembled soluble HLA-A*0201 complexes was enabled by thermal destabilization at 53 °C for 15 min, demonstrating that efficient peptide exchange does not require the removal of endogenous peptides from the reaction environment. An optimal ratio of three β-2 microglobulin molecules per single HLA heavy chain was determined to maximize peptide binding. Kinetic binding studies indicate that soluble HLA-A*0201/peptide interactions are characterized by a range of moderate kon values (1 × 104 to 8.7 × 104 M-1 s-1) and slow koff values (1.9 × 10-4 to 4.3 × 10-4 s-1), consistent with parameters for native HLA molecules. Testing of the A*0201-specific peptides with 48 additional class I molecules demonstrates that the unique peptide binding behavior of individual HLA molecules is maintained in the assay. This assay therefore represents a versatile tool for characterizing the binding of peptide epitopes during the development of class I HLA-based vaccines and immune therapies. [ABSTRACT FROM AUTHOR]

Sperm protein 17 (Sp17) is a highly antigenic, testes-specific protein whose known function is to bind sperm to the zona pellucida. However, the Sp17 gene has been recently detected in normal non-testes tissues and malignant neoplasias. As the role of Sp17 in non-testes tissues is unknown, the characterization of the Sp17 gene in highly proliferating tissues may provide further insight into the regulation and alternative function of Sp17. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify the Sp17-1 transcript in multiple normal human tissues and cancer cell lines. Similarly, the Sp17-2 gene was examined by PCR. In addition, Northern and Western blot analyses were used to detect Sp17 mRNA and protein expression. The Sp17-1a and Sp17-1b transcripts were amplified from cancer cell lines. Similarly, an Sp17-2 transcript was also detected in cancer cell lines. Furthermore, Northern blot analysis revealed Sp17 mRNA expression in all cancer cell lines examined. However, Sp17 protein expression was not detected. The differential detection of the Sp17 transcripts in cancer cell lines as compared to normal non-testes tissues, suggests a potential pathogenic role for Sp17 in diseased cells. Moreover, the Sp17-2 transcript may be a marker for highly proliferating cells. Collectively, these data implicate Sp17 as a cancer testis antigen. [Copyright &y& Elsevier]

Sp17 was initially thought to be a sperm specific protein involved in the interaction of the spermatozoon with the oocyte''s surrounding extracellular glycoprotein matrix. Recent reports, however, indicate that Sp17 expression is neither testis-specific nor is it exclusively used for binding to the zona pellucida of the oocyte. In this study, we provide comprehensive characterization of the genomic structure of Sp17. We identified an intron-containing gene (Sp17-1) containing five exonic and four intronic sequences. Analysis of Sp17 transcripts using rapid amplification of DNA complementary to RNA (cDNA) ends (RACE) and polymerase chain reaction (PCR) techniques showed the presence of alternative polyadenylation resulting in the production of varying lengths of mRNAs as well as the usage of different transcriptional start sites. Moreover, an earlier description of the human Sp17 mRNA describing a splice variant could not be confirmed. Comparison to mouse Sp17 gene organization demonstrated a high degree of conservation, suggesting selective evolutionary pressure for this protein to retain a conserved gene architecture. Additionally, we identified a second gene (Sp17-2), whose most striking characteristic was the complete absence of introns. This Sp17-2 gene has likely arisen by reverse transcription (RT) of a spliced Sp17-1 mRNA with subsequent integration into the human genome. Its open reading frame (ORF) is interrupted by stop codons, giving rise to a pseudogene. Furthermore, Southern blot analysis of human genomic DNA indicated the possibility of additional Sp17 species within the human genome. [Copyright &y& Elsevier]

Acetylcholine mediates cell‐to‐cell communications in the skin. Human epidermal keratinocytes respond to acetylcholine via two classes of cell‐surface receptors, the nicotinic and the muscarinic cholinergic receptors. High affinity muscarinic acetylcholine receptors (mAChR) have been found on keratinocyte cell surfaces at high density. These receptors mediate effects of muscarinic drugs on keratinocyte viability, proliferation, adhesion, lateral migration, and differentiation. In this study, we investigated the molecular structure of keratinocyte mAChR and their location in human epidermis. Polymerase chain reaction amplification of cDNA sequences uniquely present within the third cytoplasmic loop of each subtype demonstrated the expression of the m1, m3, m4, and m5 mAChR subtypes. To visualize these mAChR, we raised rabbit anti‐sera to synthetic peptide analogs of the carboxyl terminal regions of each subtype. The antibodies selectively bound to keratinocyte mAChR subtypes in immunoblotting membranes and epidermis, both of which could be abolished by preincubating the anti‐serum with the peptide used for immunization. The immunofluorescent staining patterns produced by each antibody in the epidermis suggested that the profile of keratinocyte mAChR changes during epidermal turnover. The semiquantitative analysis of fluorescence revealed that basal cells predominantly expressed m3, prickle cells had equally high levels of m4 and m5, and granular cells mostly possessed m1. Thus, the results of this study demonstrate for the first time the presence of m1, m3, m4, and m5 mAChR in epidermal keratinocytes. Because keratinocytes express a unique combination of mAChR subtypes at each stage of their development in the epidermis, each receptor may regulate a specific cell function. Hence, a single cytotransmitter, acetylcholine, and muscarinic drugs may exert different biologic effects on keratinocytes at different stages of their maturation. [ABSTRACT FROM AUTHOR]

MHC class I molecules regulate brain development and plasticity in mice and HLA class I molecules are associated with brain disorders in humans. We investigated the relationship between plasma‐derived soluble human HLA class I molecules (sHLA class I), HLA class I serotypes and dementia. A cohort of HLA class I serotyped elderly subjects with no dementia/pre‐dementia (NpD, n = 28), or with dementia (D, n = 28) was studied. Multivariate analysis was used to examine the influence of dementia and HLA class I serotype on sHLA class I levels, and to compare sHLA class I within four groups according to the presence or absence of HLA‐A23/A24 and dementia. HLA‐A23/A24 and dementia, but not age, significantly influenced the level of sHLA class I. Importantly, the concurrent presence of HLA‐A23/A24 and dementia was associated with higher levels of sHLA class I (p < 0.001). This study has shown that the simultaneous presence of HLA‐A23/HLA‐A24 and dementia is associated with high levels of serum sHLA class I molecules. Thus, sHLA class I could be considered a biomarker of neurodegeneration in certain HLA class I carriers. [ABSTRACT FROM AUTHOR]

Aim The primary focus of HLA antibody testing for transplant patients is to assess a given patient’s potential risk for graft loss by determining the immunological status before and after transplantation. Since the physiological responses of the adaptive immune system against an HLA target is highly variable and antibody composition is distinct to each individual, accurate determination of specificities, titer and strengths of antibodies is of uttermost importance. Although MFI values provide a pragmatic approach in ranking responses, the method makes no attempt to explain titer and affinity influences in evaluating a response. To overcome such limitations, our goal was to demonstrate that titration analysis will allow better interpretation of individual antibody-HLA target interactions which will result in a better indicative risk assessment than MFI values. Methods By introduction of recombinant technology, soluble HLA ABC locus alleles were produced by a mammalian expression system and utilized to create a unique 120 single antigen assay. Utilizing this novel assay, a dose-response model was evaluated by performing multiple serological titration experiments probing the interaction of single HLA antigens with HLA antibodies. Results Results showed that the assay follows the principles of the dose-response concept not only demonstrating the binding of HLA antibodies as effectors to specific HLA targets with sigmoidal functions but also delivering potency values of their interaction. Potency analysis is a clinically relevant evaluation approach to determine titer in combination with strength of HLA antibodies independent of antibody efficacy levels. Reducing dose-response data to relative half maximum effect values showed the advantages of a fine-tuned data output by decreasing titration complexity, allowing semi-quantitative analysis and becoming independent of MFI values. Conclusions We believe that dose-response information will greatly support the identification of factors that can contribute to the individual characterization of sera antibodies and offer the potential of direct physical meaning that will resonate with clinical outcomes. This technique will likely lead to a better assessment of HLA titers and generate new insights for pre- and post-transplant monitoring. [ABSTRACT FROM AUTHOR]

Aim The identification of prognostic parameters for the clinical course of disease states is of considerable scientific and therapeutic relevance. Serum levels of soluble HLA (sHLA) class I molecules have been advocated at various occasions possessing such predictive potential. However, lack of quantitation of sHLA levels, unavailability of appropriate assay standards and small population sizes investigated have dominantly contributed to the inability to establish the physiological importance of the presence of sHLA in body fluids and to make accurate disease associations. In this work, we have redesigned the assay approach for sHLA class I detection and introduced optimized assay conditions including a sHLA standard of highest purity and structural integrity. Methods Applying a standardized detection methodology, serum samples were screened from one hundred normal volunteer blood donors with known HLA-phenotypes. In addition, plasmapheresis samples from various patients were utilized for large-scale sHLA purification using a W6/32-affinity column. Eluted probes were further analyzed applying multiple assay strategies. Results Our findings showed an average serum level of sHLA class I in normal individuals of 494 ng/ml [190–1402 ng/ml]. No significant differences in sHLA serum levels were observed between different blood types or between male and female donors. Closer investigation of the sHLA content of several plasmapheresis samples indicated that more than 75% of sHLA is complexed with other proteins and not available in free form. HLA specificity testing using mAb matched the determined allotype of the patients confirming the self-origin of the purified sHLA. Conclusions The design and development of assay methods is crucial to meet analytical performance requirements for intact sHLA quantitation. Extraction of sHLA from human blood is highly feasible and offers a valid approach for the elucidation of the relevance of sHLA-mediated immunoregulation. Analysis of HLA-complexed antibody species or just the simple presence of elevated sHLA amounts can provide a source of information to establish a foundation for unique diagnostic approaches to gain insights into immunopathological processes for transplantation and for diseases such as cancer, autoimmunity, allergy, and viral diseases. [ABSTRACT FROM AUTHOR]

Aim Some earlier studies indicated that monoclonal Abs can be neutralized and inhibition measured with a bioassay design structured to reduce 95–100% of the maximal signal at 100 μ g/ml sHLA if operated in the mid linear range of the response signal. The aim of this investigation was to determine whether the theoretical concepts describing the basic nature of neutralization of HLA antibodies could be applied to more complex serological specimen resulting in clinically useful information. Methods Sera specimens were tested by individually adding different combinations of sHLA molecules as neutralizing agents prior to response measurement. After subtraction of the resulting inhibitory profile from the non-treated reference profile, a neutralization profile was generated, visualizing Ab responses directly related to the applied neutralizer allele. Results As shown, sHLA-A ∗ 02:01 was able to 100% neutralize A2-related responses, leaving other HLA/antibody interactions untouched. In this example, when also subtracting B ∗ 08:01 and B ∗ 49:01, a near complete inhibition of the reference profile was achieved. Minor assay-to-assay background fluctuations of 10% can generally be observed. Significantly, HLA antigens considered directly responsible for the immunizing event generally showed a complete inhibition of the reference profile, whereas partial inhibitions were seen as indicator of HLA antibody species present with different antigen/epitope correlation. Conclusion Creating a neutralizer profile subtracting the inhibitory profile from the reference profile is most helpful for better analytical assessment of signal patterns, delivering a positive view of the blocking event, ultimately allowing better interpretation of antibody populations. As demonstrated, complex reactivity patterns could be reduced to more simplifying outputs. We believe that reductive profiling techniques will greatly assist in obtaining more accurate and meaningful data in the assessment of risk for immunologic rejection. [ABSTRACT FROM AUTHOR]

Aim The measurement of rejection-causing HLA Abs in serological samples of HLA sensitized individuals has clinical relevance in the evaluation of pre- and post-transplant patients. However, due to the increasing demand for more accurate and meaningful data, the determination of MFI values alone is not sufficient anymore to evaluate a patient’s risk potential. In this study, we aimed to demonstrate the enormous complexity of Ab patterns in serological specimen. Methods To visualize variations and different levels of HLA Ab patterns, several investigative tools were applied including HLA Ab-detection assays, neutralization experiments, sera titration analysis and comparative information from human monoclonal Ab profiles derived from single B cells. Results In tier 1, neutralization experiments clearly demonstrated that original response patterns could be deconvoluted into individual sub-patterns and single alleles identified as primary cause of an immunizing event. Depending on exposure, one or several antigens were generally involved. Tier 2 patterns were originated from a single epitope harbored by the immunizing antigen of origin that naturally cross-reacts with related epitope-sharing antigens. Currently, over 100 human monoclonal Ab patterns were collected defining such epitopes and being used to further resolve Tier 1 patterns by a pattern recognition algorithm. Tier 3 involves either individual epitopes shared among related antigens or Abs that recognize a specific HLA/Ab interaction but not necessarily at the same affinity. Tier 4 includes diverse epitope patterns that appear using different isotype-recognizing detection systems. Tier 5 refers to experiments conducted under a time interval, visualizing the appearance/disappearance of different Ab species in time. Conclusion We believe that the knowledge gained about the apparent complexity of specific immune patterns will help us to reach the next chapter of patient care and contribute to a better risk management in Ab-related graft rejection. [ABSTRACT FROM AUTHOR]

Organs transplanted across donor‐specific HLA antibodies (DSA) are associated with a variety of clinical outcomes, including a high risk of acute kidney graft rejection. Unfortunately, the currently available assays to determine DSA characteristics are insufficient to clearly discriminate between potentially harmless and harmful DSA. To further explore the hazard potential of DSA, their concentration and binding strength to their natural target, using soluble HLA, may be informative. There are currently a number of biophysical technologies available that allow the assessment of antibody binding strength. However, these methods require prior knowledge of antibody concentrations. Our objective within this study was to develop a novel approach that combines the determination of DSA‐affinity as well as DSA‐concentration for patient sample evaluation within one assay. We initially tested the reproducibility of previously reported affinities of human HLA‐specific monoclonal antibodies and assessed the technology‐specific precision of the obtained results on multiple platforms, including surface plasmon resonance (SPR), bio‐layer interferometry (BLI), Luminex (single antigen beads; SAB), and flow‐induced dispersion analysis (FIDA). While the first three (solid‐phase) technologies revealed comparable high binding‐strengths, suggesting measurement of avidity, the latter (in‐solution) approach revealed slightly lower binding‐strengths, presumably indicating measurement of affinity. We believe that our newly developed in‐solution FIDA‐assay is particularly suitable to provide useful clinical information by not just measuring DSA‐affinities in patient serum samples but simultaneously delivering a particular DSA‐concentration. Here, we investigated DSA from 20 pre‐transplant patients, all of whom showed negative CDC‐crossmatch results with donor cells and SAB signals ranging between 571 and 14899 mean fluorescence intensity (MFI). DSA‐concentrations were found in the range between 11.2 and 1223 nM (median 81.1 nM), and their measured affinities fall between 0.055 and 24.7 nM (median 5.34 nM; 449‐fold difference). In 13 of 20 sera (65%), DSA accounted for more than 0.1% of total serum antibodies, and 4/20 sera (20%) revealed a proportion of DSA even higher than 1%. To conclude, this study strengthens the presumption that pre‐transplant patient DSA consists of various concentrations and different net affinities. Validation of these results in a larger patient cohort with clinical outcomes will be essential in a further step to assess the clinical relevance of DSA‐concentration and DSA‐affinity. [ABSTRACT FROM AUTHOR]

Aim Dendritic Cells (DC) mediate anti-tumor immunity, and DC-based immunotherapies represent a promising way to fight cancer. However, questions about the optimal manufacturing process and delivery strategies still remain. So far, DC-based immunotherapies have only been tested for maturational state and expression of select CD markers. However, these characteristics do not establish successful peptide loading or actual presentation of peptides by HLA. To date, class I HLA presentation of peptides on loaded DC has only been confirmed using indirect immune monitoring techniques such as ELISPOT and tetramer staining. Here, we present the first quantitative assay designed for ICT-107, a DC immunotherapy being tested in glioblastoma, that directly assesses peptide presentation on pulsed DC. This assay may prove valuable for monitoring quality and potency of DC-based immunotherapies via a reliable, reproducible and straightforward method. Methods We generated a T cell receptor mimic monoclonal antibody (TCRm), RL13A, specific for HLA-A∗01:01 in complex with MAGE-1 peptide (EADPTGHSY; “EAD9”). Next, mature DC from three different HLA-A1+ patients were pulsed with 20 μg/ml of EAD9 peptide. Loaded DC were then stained with PE-labeled RL13A. QuantiBRITE PE-Beads were used to establish a standard curve so that the number of HLA-A1/EAD9 complexes could subsequently be determined. Results Flow cytometric staining of peptide-pulsed DC with RL13A showed a clear shift in MFI compared to patient-matched, unpulsed DC. Using the QuantiBRITE PE-bead standard curve, the number of HLA-A1/EAD9 on the surface of ICT-107 was determined. All 3 lots of ICT-107 presented a comparable number of HLA-A1/EAD complexes (1200–2500 copies) on the surface. Conclusions The data from ICT-107 provide proof of concept that an HLA-specific, quantitative assay can be used to directly confirm presentation of the peptides on DC-based immunotherapies. TCRm staining enables detection as well as quantification of HLA/peptide complexes presented by peptide-pulsed DC, and the assay can now be used to characterize the DC immunotherapy, ICT-107. Future applications for this assay include both the optimization of the manufacturing process and serving as a release assay for HLA-A1+ DC-based therapeutics containing the MAGE-1 EAD9 peptide. R. Buchli: Employee; Company/Organization; Pure MHC LLC. W.H. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure MHC LLC. [ABSTRACT FROM AUTHOR]

Aim Rhesus macaques are a commonly used primate model for the study of viral infection. The class I MHC of these animals has been well studied at the genetic level. However functional ligandome characteristics such as peptide binding motif and length distribution have been elucidated in only a handful of macaque MHC (Mamu). Historically, Mamu ligandome characteristics have mimicked HLA-B despite the MAMU-A designation. Here we selected a high-frequency, uncharacterized, allele Mamu-A1∗004:01 to define the ligandome characteristics of this allele for comparison to HLA class I. Method Soluble MAMU-A1∗004:01 and HLA-B57:01 constructs were created and transfected these into the class I deficient cell line 721.221. sMHC harvested from transfected cells was affinity purified and ligand pools were isolated with an acid boil followed by 3 kDa cut-off ultrafiltration. Ligand pools were separated with two-dimentional LCMS. Data was acquired with a triple TOF mass spectrometer in information-dependant acquisition mode. Peptide sequences from the resulting MS2 fragment spectra were identified using the PEAKS and Mascot algorithms at a False Discovery Rate (FDR) of 1%. Results In this study, we identified 1447 ligands for Mamu-A1∗004. The peptide binding motif for MAMU-A1∗004 shows the P2 anchor as S, T, A and a PΩ anchor of W or F. This motif is strikingly similar to the HLA-B∗57:01 peptide binding motif. Indeed a comparison of the Mamu-A1∗004 and HLA-B∗57:01 eluted ligands from the same cell line shows that 32% of the Mamu-A1∗004 ligands are shared/identical with HLA-B∗57:01 ligands. Further, we established a Mamu class I peptide binding assay and demonstrated that known HLA-B57 HIV ligands bind to MAMU-A1∗004 with the same affinities. Although these allomorphs are similar in their motif, the length preferences are different: Compared to HLA-B57, Mamu-A1∗004 prefers nonamers and shows less tolerance for long (>13 amino acids) ligands. Conclusion Here we found that a common Rhesus macaque allele Mamu-A1∗004 is functionally homologous to HLA-B∗57:01. In humans, HLA-B∗57:01 is considered to be an important protective allele in HIV infections. These data enable the study of an HLA-B∗57:01-like molecule in non-human primate models of SIV infection. C. McMurtrey: Consultant; Company/Organization; Pure Protein LLC . R. Buchli: Employee; Company/Organization; Pure Protein LLC . W. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure Protein LLC. [ABSTRACT FROM AUTHOR]

Aim Successful pairing of a transplant recipient with a donor organ depends on accurate prediction of risk factors for immunologic rejection. As such, there is much interest in accurate clinical measurement of HLA Ab specificities in recipient sera. However, it has become apparent that current methodologies are still surrounded by shortcomings. Here we offer a new solution that is based on recent advancements made in the application of microarray technology in clinical diagnostics, to increase the precision and sensitivity of HLA Ab serology. The aim of this study was to demonstrate the coupling of recombinant soluble HLA proteins to a novel microarray technology as a new, simplified, inexpensive and accurate approach to transplant serology. Methods In order to validate and optimize sHLA microarray performance, multiple 12 × 12 microarrays were fabricated by microfluidic spotting of highly purified soluble HLA class I and II molecules under a range of conditions chosen to optimize protein stabilization on the microarray surface. Of particular interest were the evaluation of maximal signal and background intensities, signal uniformity between different HLA alleles and the structural integrity of the HLA proteins subsequent to microfluidic attachment to the microarray surface. Results HLA protein printed in this array system yielded >65,000 MFI with essentially no background signal. Signal uniformity among alleles tested was consistently below a 10% spread. As assessed by structure-specific antibodies HC-10 and W6/32, it was determined that the native dimer configuration predominated subsequent to microarray printing. Accuracy and reproducibility required for a low cost HLA antibody-screening array was validated with over 200 samples, resulting in a high degree of concordance with known monoclonal Ab patterns and established clinical reference sera. Conclusion We have successfully demonstrated that the combination of microarray and soluble HLA technology has resulted in a simple, accurate, and low cost method for high-performance multiplexed HLA Ab detection, which with additional development in progress will be ready to accommodate very high resolution HLA Ab screening, at time of transplantation and as a routine follow-up test to monitor the earliest stages of graft rejection. [ABSTRACT FROM AUTHOR]

Aim: Due to the increasing demand for more accurate and meaningful data in antibody detection assays, which includes quantitative measures of antibody titers, we evaluated a series of serological specimens to demonstrate the benefit of antibody titration analysis. We show that by identifying the linear range of the assay, semi-quantitative observations are possible where titer comparisons can be easily made and saturation issues such as “prozon effects” are eliminated. Methods: To determine linear range and half maximal effective concentrations (EC50), serological specimens from highly sensitized patients were diluted to eight different concentrations and their MFI signal determined using an in-house, 120-allele Luminex-based single specificity solid phase assay platform. Results: Our results show that individual, single-allele titration curves can be generated to establish optimal performance range. We found that titration analysis has two major benefits: (1) eliminating serological interferences at high concentrations, and (2) semi-quantitative observations within the linear range. According to our test series, most sera will cause interferences and high background if tested undiluted. Accurate determinations can only be made by applying serial dilutions to reach highest signal-to-background ratios. Conclusions: Since the physiological responses of the adaptive immune system against an HLA target are highly variable and antibody composition is distinct to each individual, accurate determination of specificities, titer and strengths of antibodies has been extremely difficult. Titration analysis offers the possibility to evaluate several of such functional traits of antibodies to any given HLA molecule allowing better interpretation of individual antibody populations in a quantitative matter. This project will likely lead to a better assessment of HLA titers and generate new insights for post-transplant monitoring and early recognition of immunologic rejection. [Copyright &y& Elsevier]

Aim: Antibody concentration, isotype, epitope specificity, cross-reactivity, and the ability to fix complement have all been implicated as factors that contribute to the pathogenicity of anti-HLA antibodies. Since humoral responses are typically polyclonal in nature, multiple epitopes with overlapping cross-reactivities are generally observed. In this study, we demonstrate that applying different combinations of soluble HLA (sHLA) as neutralizing agents, specific individual inhibitory patterns can be obtained resulting in the deconvolution of complex reaction patterns. Methods: A two-tier approach was applied to profile serological specimen from sensitized patients. (1) A sera specimen was titrated to establish the linear range of the sera antibody reactivities. (2) A dilution corresponding to a half-maximal signal output was applied and pure sHLA added to neutralize a given allele pattern. For signal evaluation, an in-house, 120-allele Luminex-based single specificity solid phase assay platform was utilized. Results: A scheme of different HLA combinations was applied for each sera to create individual neutralization patterns. Our results show that complex reactivity patterns can be reduced to more simplified outputs allowing better interpretation of antibody populations. Single neutralization patterns correspond well with individual eplets. Furthermore, this technique was also of use in evaluating false positive/negative assay signals obtained by other assay systems. Conclusions: The power of deconvolution will greatly assist in obtaining more accurate and meaningful data to assess transplant patients and also add new insights into anti-HLA antibody profiling and epitope structure analysis. Further experiments using the titration model determining half maximal effective concentrations (EC50) for sera titers and half maximal inhibitory concentrations (IC50) for HLA inhibition may result in a mathematical engraftment factor applied in the assessment of risk for immunologic rejection. [Copyright &y& Elsevier]

Aim: The apparent clinical and research utility of soluble HLA (sHLA) underscores the need to further investigate immunological effects triggered by injections into mice. Developing reliable methods for immune response monitoring is crucial for individualizing therapies aimed at extending organ survival and improving patient health. In this study, we examined the humoral immune response elicited by immunization of sHLA into mice. Methods: The method involved five steps: (1) production/purification of injectable grade sHLA; (2) immunization of 9 female BALB/c mice with 25, 50, and 150 μg of sHLA-A*02:01; (4) injections at day 0, 21, 42, 63; (5) blood samples taken at day 0, 21, 42, 63, 72; (3) monitoring immunized mice for HLA-specific antibody production by an in-house, 120-allele Luminex-based single specificity solid phase assay. Results: Our results indicated that all mice immunized with sHLA-A*02:01 triggered an immune response. Titration analysis allowed appropriate measurements of semi-quantitative relationships between samples at different stages. It was noted that mice with 150 μg injection doses were capable to elicit high antibody titers as early as 21 days. Mice at lower injection doses had a slower time course. The average immune response distribution between loci was found to be highest in A alleles followed by C and weakest in B alleles. Overall, data indicate the generation of antibodies of broad specificity rather than against allogeneic determinants. Conclusions: Low variability between Balb/c mice is explained by the fact that all mice have an identical genetic makeup. These findings underscore the potential importance of polymorphisms in the MHC locus on immune responses. Furthermore, this data may allow increasing accuracy and efficiency in the determination of immunological responses that could help monitor occurrences observed in humans. In addition, injectable sHLA may serve as quality reagents in the creation of therapeutic/diagnostic anti-HLA antibodies or to induce tolerance. [Copyright &y& Elsevier]

Aim: Classification of allele overlapping peptide-binding specificities has become an important issue in vaccine design with direct implications concerning population coverage. In this study, we used a combinatoric approach not only to discover epitope/HLA combinations suitable to be utilized in peptide binding assays, but also to evaluate the degree of overlapping peptide binding capacities among the alleles tested. Methods: A set of 20 different FITC-labeled reference peptides was applied in combination with over 50 sHLA alleles to determine their binding capabilities. During the process, each peptide candidate was incubated with activated sHLA, and the peptide/HLA interaction was monitored over time using Fluorescence Polarization (FP). This assay combination allows the direct measurement of the ratio between free and bound labeled ligand in solution without any separation steps. The technique of FP is based on the principal that if a fluorescent-labeled peptide binds to the sHLA molecule of higher molecular weight, polarization values will increase due to the slower molecular rotation of the bound probe. Results: Data analysis showed that the majority of peptide candidates were capable of binding with various degrees of overlap, reflecting the ability of HLA class I alleles to share the binding of identical peptides. The approach resulted in the identification of over 40 new peptide/HLA combinations applicable for epitope discovery and characterization. However, comparison to published binding motifs and supertype classifications showed several inconsistencies indicating the need of more refinement in their definition. Conclusions: Our approach suggests that broadly cross-reactive peptide epitopes can be identified and greatly enhance the effectiveness of future vaccine designs by providing a more extensive population coverage. They will also have a profound benefit in the understanding of antigenic peptide selection, degeneration and discrimination during T-cell mediated immune responses. [Copyright &y& Elsevier]

Abstract: Immunizing events including pregnancy, transfusions, and transplantation promote strong alloantibody responses to HLA. Such alloantibodies to HLA preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays detect alloreactive antibodies, yet key attributes including antibody concentration and isotype remain largely unexplored. The goal here was to provide a detailed profile of allogeneic antibodies to class II HLA. Methodologically, alloantibodies were purified from sensitized patient sera using an HLA-DR11 immunoaffinity column and subsequently categorized. Antibodies to DR11 were found to fix complement, exist at a median serum concentration of 2.3μg/mL, consist of all isotypes, and isotypes IgG2, IgM, and IgE were elevated. Because multimeric isotypes can confound diagnostic determinations of antibody concentration, IgM and IgA isotypes were removed and DR11-IgG tested alone. Despite removal of multimeric isotypes, patient-to-patient antibody concentrations did not correlate with MFI values. In conclusion, allogeneic antibody responses to DR11 are comprised of all antibody isotypes at differing proportions, these combined isotypes fix complement at nominal serum concentrations, and enhancements other than the removal of IgM and IgA multimeric isotypes may be required if MFI is to be used as a means of determining anti-HLA serum antibody concentrations in diagnostic clinical assays. [Copyright &y& Elsevier]

Abstract: Major histocompatibility complex (MHC) class I molecules bind and display peptide antigens on the cell surface. CD8+ T lymphocytes recognize peptides in association with class I proteins to initiate a cytotoxic immune response. To understand the specificity of such immune responses and to facilitate the development of therapies for disease, it is important to identify MHC-presented peptides. In this study, platelets, easily obtainable and often associated with immune-mediated disease, were selected to identify MHC class I–associated peptides. MHC-associated peptides presented on platelets of normal individuals and individuals with idiopathic thrombocytopenic purpura (ITP) were characterized. ITP is characterized by the premature immune destruction of platelets. It is associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane GPIIb/IIIa or GPIb/IX. In addition to characterizing five fully and several partially sequenced peptides from platelets, the peptide GPRGA(L/I)S(L/I)(L/I) was identified from four of the five ITP patients. The anchor motif of this peptide correlates with the presence of the HLA-B7 allele. A BLAST search identified this peptide as GPIb (4–12). In conclusion, platelets from normal and ITP individuals can present peptides from general cellular proteins and platelet specific proteins, such as GPIb, to the immune system via MHC class I. [Copyright &y& Elsevier]

Neoantigens are novel peptide sequences resulting from somatic mutations in tumors that upon loading onto major histocompatibility complex (MHC) molecules allow recognition by T cells. Accurate neoantigen identification is thus critical for designing cancer vaccines and predicting response to immunotherapies. Neoantigen prioritization relies on correctly inferring whether the presenting peptide sequence can successfully induce an immune response. An important, yet underappreciated, variable in neoantigen-prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient's specific HLA alleles. While a subset of peptide positions are presented to the T-cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T-cell responses. However, a systematic method for determining anchor locations and application of these to evaluate neoantigens has not been reported. Here, we calculated high probability anchor positions for 328 HLA alleles using a reference dataset generated from clinical and TCGA patient samples. Results showed clusters of different anchor trends among the HLA alleles analyzed. A subset of anchor results were orthogonally validated using protein crystallography structures. Analysis of 923 tumor samples showed that 7-41% of neoantigen candidates were potentially misclassified in the neoantigen selection process and can be rescued using allele-specific knowledge of anchor positions. Representative anchor trends have been experimentally validated using peptide-MHC stability assays and competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, we hope to formalize and streamline the identification process for relevant clinical studies. [ABSTRACT FROM AUTHOR]

HLA specific antibodies vary in their pathogenicity and this is likely to be the net effect of constant chain usage, quantity, specificity, and affinity. Here we have measured the affinity of human monoclonal antibodies for a range of HLA proteins. Purified antibodies and ligands allowed dynamic interactions to be measured directly by surface plasmon resonance. Physiochemical differences between pairs of ligands were quantified using electrostatic mismatch and hydrophobic mismatch scores. All antibodies were characterized by fast on-rates and slow off rates but with a wide range of association rates (k on, 3.63–24.25 × 10 5 per mol per second) and dissociation rates (k off , 0.99–10.93 × 10 −3 per second). Dissociation constants (K D ) ranged from 5.9 × 10 −10  M to 3.0 × 10 −8  M. SN320G6 has approximately a twenty-fold greater affinity for HLA A2 compared with SN607D8, but has a similar affinity for HLA-A2 and B57. In contrast, SN607D8 has greater than a twofold greater affinity for HLA-A2 compared with A68. Similarly, WK1D12 has about a threefold greater affinity for HLA-B27 compared with B7. The higher affinity interactions correlate with the specificity of stimulating antigen. This is the first study to directly measure the binding kinetics and affinity constants for human alloantibodies against HLA. [ABSTRACT FROM AUTHOR]

Aim Mapping anti-HLA antibody epitopes has commonly been done by comparing antibody reactivity patterns to a panel of naturally occurring HLA allelic variants such as the solid phase HLA antibody screening panels. Information including the donor’s immunizing alleles and the recipient-derived HLA further helps to refine HLA antibody epitopes. While these methods are inexpensive and widely available, they represent an indirect means of mapping those amino acids on HLA that are critical to antibody recognition and binding. Here we test a complementary direct approach to measure the ‘foot print’ of an antibody at it interacts with HLA. A recently developed method called protein painting was used to map the interaction of mouse monoclonal antibody BB7.2 with HLA-A*02:01. Methods Briefly, purified soluble HLA-A*02:01 was incubated with and without BB7.2. Protein mixtures were incubated with the trypsin inhibitor Remazol Brilliant Blue (RBB), trypsin digested, and tryptic peptide fragments of HLA-A*02:01 were analyzed using LCMS. In this manner BBB protects solvent accessible Arg and Lys residues from trypsin digestion. When HLA-A*02:01 is first incubated with BB7.2, Arg and Lys residues covered by the antibody are not painted by BBB and therefore they remain trypsin susceptible. Results A mass spectrometric analysis of the resulting trypic peptides revealed that BB7.2 protects R44, R97, R108, R111, R131, and K144 from painting. These residues are primarily located on lateral edge of the alpha-2 alpha helix as shown on the three dimensional structure of HLA-A*02:01. In a solid phase assay, BB7.2 reacts exclusively with A2 and A28 allomorphs, data that identifies the well defined 144TKH epitope. The protein painting data shown here are consistent with the 144TKH epitope as K144 was protected from RBB binding by BB7.2. Conclusion We demonstrate a new method for direct mapping of anti-HLA antibody epitopes that is complementary to solid phase assays. Furthermore, protein painting reveals not only the critical residues for antibody binding, but the surrounding amino acids covered by the antibody. R. Buchli : Employee; Company/Organization; Pure Protein LLC. W. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure Protein LLC. [ABSTRACT FROM AUTHOR]

For the quantification of HLA-specific memory B cells from peripheral blood of sensitized individuals, a limited number of methods are available. However, none of these are capable of detecting memory B cells directed at HLA class II molecules. Since the majority of antibodies that occur after transplantation appear to be specific for HLA class II, our aim was to develop an assay to detect and quantify HLA class II-specific memory B cells from peripheral blood. By using biotinylated soluble HLA class II molecules as detection agent, we were able to develop an HLA class II-specific memory B cell ELISPOT assay. The assay was validated using B cell-derived hybridomas that produce human monoclonal antibodies directed at specific HLA class II molecules. In pregnancy-immunized females, we found memory B cell frequencies ranging from 25 to 756 spots per 10 6 B cells specific for the immunizing paternal HLA class II molecules, whereas in non-immunized males no significant spot formation was detected. Here, we present a novel ELISPOT assay for quantifying HLA class II-specific memory B cells from peripheral blood. This technique provides a unique tool for monitoring the HLA class II-specific memory B cell pool in sensitized transplant recipients. [ABSTRACT FROM AUTHOR]

Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)-mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+ T cells into strategies designed to enhance T cell immunity. [ABSTRACT FROM AUTHOR]

Aim: Not all anti-HLA antibodies are detrimental to graft survival, yet it is difficult to pinpoint detrimental anti-HLA antibodies. Here, we provide a tool for isolating anti-HLA antibodies from complex patient sera to enable the functional and phenotypic characterization of these immunoglobulins. Anti-HLA antibodies were isolated from several sensitized patients and characterized for functional activity and antibody classification. Methods: A soluble version of the class II molecule HLA-DR11 was produced in mammalian cells, purified, covalently coupled to a solid support, and used to recover anti-HLA-DR11 antibodies from the sera of sensitized patients. Sera from patients sensitized by a renal graft were compared to sera from patients not sensitized by an organ. Anti-HLA-DR11 antibodies from these patients were quantified, assayed for HLA specificity and cross reactivity, quantified, and isotyped. Results: HLA-DR11immunoaffinity purification yielded antibodies specific for HLA-DR11 while antibodies for HLA-DQ specificities flowed through the column. Serum concentrations of total anti-HLA-DR11 antibodies did not differ between the two groups and ranged from 5.9μg/ml – 467ng/ml. All patients had anti-HLA-DR11 antibodies from every isotype with enrichments in IgM, IgG2, IgG3, IgE and underrepresentation of IgG1 as compared to bulk serum antibodies. IgG2 was more prevalent in patients whose sensitizing event was a renal graft compared to patients not sensitized by an organ. Conclusions: HLA immunoaffinity chromatography facilitated the purification anti-HLA antibodies from patient sera so they could be assayed apart from sera. The resulting data show that there are qualitative but not quantitative differences in anti-HLA antibodies between patients who were sensitized with a renal graft as compared to patients not sensitized with an organ. These data establish HLA immunoaffinity columns as a tool for isolating anti-HLA antibodies so that the nature of these antibodies can be determined. McMurtrey: Pure Protein: Consultant. Buchli: Pure Protein: Employee. Cate: Pure Protein: Employee. Osborn: Pure Protein: Employee. VanGundy: Pure Protein: Employee. Mojsilovic: Pure Protein: Employee. Hildebrand: Pure Protein: Science Med Advisor. [Copyright &y& Elsevier]

Aim: Strong humoral responses to allogeneic HLA preclude organ transplantation, promote hyperacute rejection, and contribute to chronic transplant rejection. Therapies exist to prevent B cells from secreting antibodies, and bulk antibody removal is also an option, but there is no means to specifically deplete antibodies that recognize an allogeneic HLA molecule while leaving humoral immunity largely intact. Here, we tested a sHLA Antibody Removal Column (SHARC) for removing anti-HLA antibodies from liter volumes of patient samples. Methods: Approximately 70 milligrams of the soluble class II molecule HLA-DRB1∗11:01/DRA1∗01:01 (DR11) produced in mammalian cells was covalently coupled to a solid support matrix. The HLA-DR11 matrix was packed into a column for the purpose of removing anti-DR11antibodies from patient plasma. 2.5L of patient plasma containing anti-DR11 antibodies was run over the column at a clinically relevant flow rate of 40ml/min. Fractions of the flow through were taken and anti-HLA-DR11 antibodies were measured using a single antigen bead assay. Results: When patient plasma recognizing multiple HLA was passed over the DR11 matrix, antibodies specific for DR11were removed while antibodies for other class II (DQ) passed through the SHARC intact. Additionally there was no change in bulk antibody concentration after the plasma was passed over the column. DR11 specific antibodies that bound to the column were recovered by elution and retained their reactivity for DR11 as well as other specificities with expected shared cross reactivity. Conclusions: Native class II molecules were coupled to an affinity matrix, antibodies specific for class II HLA were removed from patient plasma, and the depleted antibodies were recovered intact from the column. These data represent a proof of concept for selective antibody desensitization as a potential therapeutic. McMurtrey: Pure Protein: Consultant. Buchli: Pure Protein: Employee. VanGundy: Pure Protein: Employee. Hildebrand: Pure Protein: Science Med Advisor. [Copyright &y& Elsevier]

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity. [ABSTRACT FROM AUTHOR]

Elevated levels of human leukocyte antigen (HLA) proteins have been reported in several pathologic conditions that are associated with increased concentrations of white blood cells (e.g., infection, inflammation, and lymphoproliferative disorders). The mechanisms by which HLA proteins are solubilized from cell membranes are insufficiently understood. We hypothesized that HLA proteins may be cleaved from cell membranes by insufficiently inhibited leukocytic elastase, as expected in alpha-1 antitrypsin deficiency (A1ATD), resulting in elevated plasma levels of soluble HLA (sHLA) proteins. Using an enzyme-linked immunosorbent assay, we measured sHLA II levels in the peripheral blood of patients with A1ATD with or without co-existing chronic obstructive pulmonary disease (COPD), with COPD only, and in a control group. Mean (±SD) sHLA II plasma levels were 110 ± 200 pg/mL in patients with A1ATD and COPD (Group 1), 10 ± 30 pg/mL in patients with COPD without A1ATD (Group 2), 70 ± 90 pg/mL in patients with A1ATD without COPD (Group 3), and 10 ± 30 pg/mL in healthy donors (Group 4). Soluble HLA II plasma levels were significantly higher in Group 1 ( P = .001) and Group 3 ( P = .002) versus Group 4. Our preliminary results suggest that leukocytic elastase and probably other proteinases solubilize HLA proteins from cell membranes. This mechanism would operate in inflammation with elevated leukocytic elastase levels but more so with inflammation and A1ATD, where elastase would be insufficiently inhibited. If this mechanism is verified, plasma sHLA levels could potentially be used to measure cell damage due to proteinases and, therefore, for monitoring the therapeutic efficacy of alpha-1 antitrypsin (A1AT) augmentation therapy. [ABSTRACT FROM AUTHOR]

To escape immune recognition, viruses acquire amino acid substitutions in class I human leukocyte antigen (HLA)-presented cytotoxic T-lymphocyte (CTL) epitopes. Such viral escape mutations may (i) prevent peptide processing, (ii) diminish class I HLA binding, or (iii) alter T-cell recognition. Because residues 418 to 426 of the hypervariable influenza A virus nucleoprotein (NP418-426) epitope are consistently bound by class I HLA and presented to CTL, we assessed the impact that intraepitope sequence variability has upon T-cell recognition. CTL elicited by intranasal influenza virus infection were tested for their cross-recognition of 20 natural NP418-426 epitope variants. Six of the variant epitopes, of both H1N1 and H3N2 origin, were cross-recognized by CTL while the remaining NP418-426 epitope variants escaped targeting. A pattern emerged whereby variability at position 5 (P5) within the epitope reduced T-cell recognition, changes at P4 or P6 enabled CTL escape, and a mutation at P8 enhanced T-cell recognition. These data demonstrate that substitutions at P4 and/or P6 facilitate influenza virus escape from T-cell recognition and provide a model for the number, nature, and location of viral mutations that influence T-cell cross-recognition. [ABSTRACT FROM AUTHOR]

Cytotoxic T lymphocytes (CTL) limit influenza virus replication and prevent morbidity and mortality upon recognition of HLA class I presented epitopes on the surface of virus infected cells, yet the number and origin of the viral epitopes that decorate the infected cell are unknown. To understand the presentation of influenza virus ligands by human MHC class I molecules, HLA-B*O7O2-presented viral peptides were directly identified following influenza infection. After transfection with soluble class I molecules, peptide ligands unique to infected cells were eluted from isolated MHC molecules and identified by comparative mass spectrometry (MS). Then CTL were gathered following infection with influenza and viral peptides were tested for immune recognition. We found that the class I molecule B*0702 presents 3-6 viral ligands following infection with different strains of influenza. Peptide ligands derived from the internal viral nucleoprotein (NP[sub418-426] and NP[sub473-481]) and from the internal viral polymerase subunit PB1 (PB1[sub329-337]) were presented by B*0702 following infection with each of 3 different influenza strains; ligands NP[sub418-426], NP[sub473--481], and PB1[sub329-337] derived from internal viral proteins were consistently revealed by class I HLA. In contrast, ligands derived from hemagglutinin (HA) and matrix protein (Ml) were presented intermittently on a strain-by-strain basis. When tested for immune recognition, HLA-B*O7O2 transgenic mice responded to NP[sub418-426] and PB1[sub329-337] consistently and NP[sub473-481] intermittently while ligands from HA and Ml were not recognized. These data demonstrate an emerging pattern whereby class I HLA reveal a handful of internal viral ligands and whereby CTL recognize consistently presented influenza ligands. [ABSTRACT FROM AUTHOR]

Summary: Dendritic cells (DCs) are highly specialized antigen-presenting cells of the immune system that are efficient at presenting peptide-antigen for the activation of T cells and are often the cell type of choice for vaccine targeting by virtue of high expression levels of MHC and costimulatory molecules. Since the level of peptide–MHC complex significantly influences stimulation of T cells, a proof-of-concept potency assay was developed to directly examine the presentation and density of MHC class I peptides derived from the processing of a model tumor antigen, (hCGβ), on the surface of DCs. In this study we first generated antibodies (TCR mimics or TCRm) to two peptide-HLA-A*0201 epitopes derived from hCGβ designated as TMT (40–48) and GVL (47–55). Characterization of each TCRm by ELISA and flow cytometric analysis, demonstrated specific binding to soluble recombinant HLA-A2 protein and HLA-A2.1+ T2 cells loaded with relevant peptide. TCRm reactive against the TMT and GVL epitopes blocked granzyme-B production by peptide-specific cytotoxic T lymphocytes (CTL) lines further supporting their recognition specificity. For the assessment of antigen presentation function, human immature monocyte-derived DCs (iDCs) were treated with the mannose receptor targeting vaccine, B11-hCGβ and matured with Poly I:C. The TMT and GVL epitope reactive CTL lines responded to vaccine-treated but not vehicle-treated mature DCs (mDCs) with TMT and GVL TCRm specifically blocking IFN-γ production. The TCRm were then used to directly confirm specific peptide–MHC complexes on mDCs. TCRm staining of vaccine-treated mDCs showed detection of the TMT and GVL peptide-HLA-A2 complexes. These findings demonstrate that TCRms may be important tools for determining the potency of DC-based vaccines. [Copyright &y& Elsevier]

Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) loaded with viral peptides are a potential form of immunotherapy of human immunodeficiency virus type 1 (HIV-1) infection. We show that DCs derived from blood monocytes of subjects with chronic HIV-1 infection on combination antiretroviral drug therapy have increases in expression of HLA, T-cell coreceptor, and T-cell activation molecules in response to the DC maturation factor CD40L comparable to those from uninfected persons. Mature DCs (mDCs) loaded with HLA A*0201-restricted viral peptides of the optimal length (9-mer) were more efficient at activating antiviral CD8+ T cells than were immature DCs or peptide alone. Optimal presentation of these exogenous peptides required uptake and vesicular trafficking and was comparable in DCs derived from HIV-1-infected and uninfected persons. Furthermore, DCs from HIV1-infected and uninfected persons had similar capacities to process viral peptides with C-terminal and N-terminal extensions through their proteasomal and cytosolic pathways, respectively. We conclude that DCs derived from HIV-1-infected persons have similar abilities to process exogenous peptides for presentation to CD8+ T cells as those from uninfected persons. This conclusion supports the use of DCs loaded with synthetic peptides in immunotherapy of HIV-1 infection. [ABSTRACT FROM AUTHOR]

Aim: Class I human leukocyte antigen molecules (A, B and C) reveal intracellular viruses by presenting viral peptide epitopes at the cell surface. At this point the number of viral ligands that decorate the HLA of an infected cell and biases in the targeting of these viral/HLA complexes by cytotoxic T lymphocytes is not understood. Our goal is to determine the number of viral ligands presented by the HLA of infected cells and to assess T cell decisions in responding to available targets. Methods: A human epithelial cell line was transfected with three different soluble class I HLA-A and three HLA-B constructs. HLA-producing clones were expanded and grown in bioreactors and infected with West Nile virus (WNV) or were left uninfected. HLA/peptide complexes were purified from WNV-infected and uninfected cells. To identify WNV derived HLA ligands, peptides were isolated, comparatively mapped by mass spectroscopy and sequenced by MSMS. Results: Of the 19 ligands identified in this study, 15 were eluted from HLA-A complexes and 4 were eluted from HLA-B. HLA-A∗02:01 and A∗11:01 each presented 6 viral peptides. Viral epitopes sampled by class I HLA were dispersed fairly evenly throughout different viral proteins (C, M, E, NS2b, NS3, NS4b and NS5). T cells from infected individuals tended to respond strongly to 1-2 particular viral ligands with minimal T cell activity to other viral epitopes. Conclusions: These data indicate that HLA-A molecules tend to present the majority of WNV-derived ligands to CTL, suggesting that HLA-A and HLA-B may play different roles in the development of viral immunity. Class I HLA was unbiased, sampling peptides from various locations throughout the virus. While T cell activity was detected to most viral ligands, a clear hierarchy demonstrates that particular viral epitopes are key determinants of immune protection. These data will guide the development of vaccines, diagnostics, and therapeutics. Buchli: Pure Protein L.L.C.: Employee. [Copyright &y& Elsevier]

Aim: Therapies exist for the bulk removal of antibodies in sensitized transplant patients, but there are no means to specifically deplete antibodies that recognize a specific HLA molecule. Current bulk antibody removal procedures may leave a patient compromised immunologically and more susceptible to opportunistic infections. The goal of this project was to create specific HLA-A∗02:01 removal matrices and demonstrate their ability to capture defined anti-HLA antibodies. Methods: Soluble class I HLA-A∗02:01 molecules were produced in a native conformation in mammalian cells, purified by affinity chromatography, coupled to a Sepharose matrix, and loaded into a column enclosure. Various columns were created and tested with an automated chromatography system by initial injection of various HLA antibodies under different loading conditions. After an extensive column wash, the bound antibodies were released by a pH shift and quantitated. Loading and elution profiles were recorded. Results: Initial tests show that HLA columns maintain their structural integrity and function. Multiple passes of the antibody W6/32, recognizing only intact HLA molecules, resulted in consistent and repeatable binding patterns. During the entire performance process, several parameters were identified, determining column saturation, capacity and efficiency values. Specificity was shown by removing a typical A∗02:01 recognition pattern from an A2 sera. In addition, treatment conditions were established to reuse the device. Conclusions: Anti-HLA antibody removal devices are highly efficient in selectively depleting HLA antibodies. Capable of removing up to 1 mg per ml of matrix depending on the avidity of the antibody makes this device very efficient for research studies of HLA antibodies and provides promise for therapeutic applications. Furthermore, this device might also find an application in HLA diagnostics, allowing for the deconvolution of serologic samples and providing a new way to evaluate high PRA patients. [Copyright &y& Elsevier]

Aim: Many sensitized transplant patients have HLA-specific antibodies due to previous graft failures, blood transfusions, and pregnancies. These humoral responses are typically polyclonal in nature, antibody composition is distinct to each individual, and it is difficult to precisely determine antibody specificity. Distinct antibodies cannot be readily isolated for individual characterization. In this study we isolate distinct anti-HLA antibodies to evaluate their phenotypic and functional traits. Methods: After characterizing sera from highly sensitized patients with a single antigen assay, sera were passed over HLA-specific separation devices made by coupling purified soluble HLA to a sepharose matrix. A scheme of multiple separation rounds involving loading and elution cycles with different HLA allele combinations was applied. This process resulted in mono-specific patterns of HLA reactivity measured by a Luminex-based bead assay. Results: Our results show that complex patterns of sera reactivity can be reduced to more simplified outputs allowing better interpretation of individual antibody populations. Separation of IgG, IgA, and IgM further assisted in the interpretation of these responses. Furthermore, this technique was also of use in evaluating false positive/negative assay signals, strengthening otherwise weak antibody reactivity, and eliminating background interference. Conclusions: Antibody concentration, isotype, epitope specificity, cross-reactivity, and the ability to fix complement have all been implicated as factors that contribute to the pathogenicity of anti-HLA antibodies following organ transplantation. The deconvolution of antibody reactivity patterns can assist in the assessment of risk factors for immunologic rejection as well as provide new insights into anti-HLA antibody profiling and epitope structure analysis. [Copyright &y& Elsevier]

Aim: Degraded or denatured HLA proteins can contribute to false positive, false negative, and inconsistent MFI signals. To provide recommendations for improvements in the manufacturing process, HLA protein degradation was systematically studied from the perspective of physical instability; changes in higher order structure following acid and base hydrolysis, thermal degradation, reagent changes, and freeze-thaw cycles were analyzed. The goal was to identify optimal physical conditions for maintaining HLA integrity. Methods: To detect significant changes in the quality and function of soluble HLA, specific bead sets loaded with different allele specificities were created and tested under stress conditions using the Luminex platform. Various monoclonal antibodies as well as sera specimen were used to probe the effects of pH variations and temperature ranges, specific chemical agents, or working conditions. Results: These HLA stress studies showed that the exposure of soluble HLA loaded beads tolerated temperatures up to 50°C with no detectable loss of activity. Freezing did cause a reduction in activity that could be overcome by using stabilizers such as glycerol. Favorable results were also found while investigating the pH range, with optimal performance up to pH 10.5; a basic environment is preferred. Acidic environments down to pH 5.5 were accepted, but bead activity declined below pH 5.5. Conclusions: Stress testing can identify optimal conditions and prevent deleterious conditions. Here, a number of analytical techniques characterized alterations in HLA protein primary structure, physical stability, and activity. Our results indicate optimal conditions, propose stability-indicating methodologies, and provide a means for testing HLA protein activity and potency. The question of how much degradation is acceptable is still pending – we found that a level above 90% intact HLA is preferred. Overall, this study provides valuable information to improve future formulations and manufacturing processes. [Copyright &y& Elsevier]

Aim: Solid phase antibody detection assays are used in most clinical histocompatibility laboratories. Recent studies have shown that current methodologies can be inconsistent within and between laboratories. Understanding the source of assay inconsistency will promote standardization. In this study, we investigated secondary antibody systems and HLA integrity inconsistencies to facilitate a standardization strategy for HLA antibody detection assays. Methods: A soluble HLA-based assay was tested in a three step protocol; (1) binding of soluble HLA to a solid support, (2) incubation of the coupled HLA with sera/antibody, and (3) visualization of anti-HLA antibodies using a detection system. Nearly 200 soluble HLA molecules provided the platform content and more than 40 positive and negative controls were included to address Ig isotype specificity, HLA structural integrity on the platform, capacity of the support, and additional contributing factors. Results: Consistency challenges in anti-HLA antibody testing are multi-layered. In the secondary antibody detection system, anti-IgG PE antibodies crossreact with IgM controls resulting in non-proportional signal amplification because of IgM’s pentameric nature. Furthermore, variation in the avidity of tested secondary antibodies influences a consensus MFI. Commercial bead systems compared to soluble HLA revealed variation in bead HLA content. Structural disintegration of particular HLA impaired bead capacity by two logs, altered MFI saturation, and a linear detection range could only be achieved by testing various dilutions of sera samples. Conclusions: First, identifying assay inconsistencies and then standardizing methods, reagents, and controls to eliminate these inconsistencies will achieve concordance within and between laboratories. Here, we characterize variability in secondary antibodies, bead specificities, and HLA integrity on bead preparations. Having characterized these assay variables, options for assay standardization are discussed. [Copyright &y& Elsevier]

Aim: Solid phase antibody detection assays using single antigens represent a cutting edge technology for screening the sera of transplant patients. Considering the large number of HLA targets, it has been difficult to QC all single allelic HLA entities with well characterized serological reagents. Our aim is to identify, generate, and characterize agreeable sera/antibodies for the quality control of single HLA antigens produced by various manufacturers. Methods: Monospecific antibodies are considered the most appropriate means for the quality control of single antigens. A number of human and mouse monoclonal antibodies represent excellent reagents for quality assessment. However, monoclonal antibodies are not available for many HLA. Here, deconvolution of serological specimens, isolating mono-specific anti-HLA antibodies by an absorption-elution technology further assisted in increasing the reagent pool. We then utilize these monospecific antibodies for single HLA antigen QC purposes. Results: We characterized HLA monospecific antibodies from dozens of candidate reagents. The purified antibodies were highly specific for specific HLA targets. Initial titration curves indicate an optimal performance range with a specific HLA. Thereafter, each monospecific reagent was screened for its HLA cross-reactive pattern. Crossreactivity coincides with known epitopes and suggests that clinically relevant and irrelevant epitopes are indicated. Conclusions: Well-characterized sera/antibodies provide not only a consensus for antibody specificity, helping to standardize and QC existing HLA platforms, but also allow a consensus for the strength (MFI) of the antibody-HLA interaction. The provision of multiple HLA monospecific antibodies that can be harvested from patient sera by individual laboratories will enable multiple HLA single antigen QC. Furthermore, harvest of HLA monospecific sera will provide insights into HLA epitope structures, supporting the efforts to understand crossreactive HLA in the clinical lab. [Copyright &y& Elsevier]

Aim: Single antigen beads (SAB) assays are routinely used to identify anti-HLA antibodies and the level of detection is commonly communicated as mean fluorescence intensity (MFI). Many factors may contribute to an MFI value, including antibody concentration and interference of non-IgG isotypes. Here we purified polyclonal anti-HLA antibodies from sensitized patients and tested the influence of antibody concentration and non-IgG isotypes on MFI. Methods: Using an HLA-DR11 affinity column, we purified anti-HLA-DR11 antibodies from 13 sensitized human sera. The purified anti-HLA antibodies were then tested on a SAB assay using a secondary that only recognizes IgG isotypes. IgG isotypes were separated from IgM and IgA isotypes via size exclusion HPLC. Reduction of multimeric antibodies (IgM, IgA) to monomers was preformed with 100mM DTT at 4°C overnight with a total antibody concentration at 1mg/ml. Results: Serial dilution experiments with affinity purified anti-HLA antibodies demonstrated a strong correlation between antibody concentration and MFI within a single patient. However, between patients, the MFI value did not correlate to an antibody concentration – MFI was not comparable between patients. When IgM and IgA multimers were separated from IgG, a significant increase in MFI was observed compared to the original mix of isotypes. Furthermore, reduction of IgM and IgA into monomers with IgG did not impact MFI values as the monomeric mix provided an MFI matching the IgG monomer only. Conclusions: Multiple factors influence SAB MFI. Here we tested the influence of both antibody concentration and the presence of IgM, and IgA on MFI. We show that MFI correlates with antibody concentration within a particular patient but not between patients. We also show that mixing IgM and IgA multimers (but not monomers) with IgG antibodies interferes with the assay by reducing MFI. Together these data exemplify the complexity of anti-HLA antibodies and factors that influence the SAB that is used to detect them. Cate: Pure Protein: Employee. McMurtrey: Pure Protein: Consultant. Buchli: Pure Protein: Employee. VanGundy: Pure Protein: Employee. Osborn: Pure Protein: Employee. Mojsilovic: Pure Protein: Employee. Hildebrand: Pure Protein: Science Med Advisor. [Copyright &y& Elsevier]

Aim: While it is generally accepted that HLA antibodies represent significant risk factors for transplant rejection and graft failure, it has become apparent that current methodologies are not delivering all the answers to accurately assess transplant patients. Over the years, many controversies and shortcomings have been discussed concerning solid phase antibody detection assays but only a few improvements have been implemented. To better accommodate the increasing demand for more accurate and meaningful data, we set out to develop a new generation of solid phase single antigen assays using soluble HLA. Methods: A single specificity solid phase assay platform was created utilizing 120 soluble HLA alleles and a large panel of control proteins, all of which were validated by qualified monoclonal antibodies. The assays were optimized in regard to sera incubations, wash steps and PE-detection. In some cases, serological specimens were treated using supplemental techniques to reduce sera complexity or remove interference and background problems. Results: In several case studies, more distinctive reaction patterns compared to other assay manufacturers could be demonstrated. The insight into isotype distribution provided helpful facts to draw further conclusions regarding the patient’s immunological state. The usage of soluble HLA demonstrated a higher capacity in sera titration experiments while lack of substantial structural disintegration reduced the false positive rate allowing better definition of single cross-reactive patterns. Conclusions: The complex nature of human sera has set new challenges in the evaluation of the phenotypic and functional traits of antibodies. A large diverse panel of highly purified functionally intact HLA antigens along with a definitive set of control proteins is essential to satisfy the increasing demand for meaningful immunological assessment of transplant patients. [Copyright &y& Elsevier]