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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 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: 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: 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]

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: 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]