Correlation of a commercial platform's results with post-vaccination SARS-CoV-2 neutralizing antibody response and clinical host factors.

INTRODUCTION: The objective of this study was to describe the correlation between the commercially available assay for anti-S1/RBD IgG and protective serum neutralizing antibodies (nAb) against SARS-CoV-2 in an adult population after SARS-CoV-2 vaccination, and determine if clinical variables impact this correlation. METHODS: We measured IgG anti-S1/RBD using the IgG-II CMIA assay and nAb IC50 values against SARS-CoV-2 WA-1 in sera serially collected post-mRNA vaccination in veterans and healthcare workers of the Veterans Affairs Connecticut Healthcare System (VACHS) between December 2020 and January 2022. The correlation between IgG and IC50 was measured using Pearson correlation. Clinical variables (age, sex, race, ethnicity, prior COVID infection defined by RT-PCR, history of malignancy, estimated glomerular filtration rate (GFR calculated using CKD-EPI equation) were collected by manual chart review. The impact of these clinical variables on the IgG-nAb correlation was analyzed first with univariable regression. Variables with a significance of p < 0.15 were analyzed with forward stepwise regression analysis. RESULTS: From 127 sera samples in 100 unique subjects (age 20-93 years; mean 63.83; SD 15.63; 29% female; 67% White), we found a robust correlation between IgG anti-S1/RBD and nAb IC50 (R2 = 0.83, R2adj = 0.70, p < 0.0001). Race, ethnicity, and a history of malignancy were not significant on univariable analysis. GFR (p < 0.05) and prior COVID infection (p < 0.001) had a significant impact on the correlation between IgG anti-S1/RBD and nAb IC50. Age (p = 0.06) and sex (p = 0.07) trended towards significance on univariable analysis, but were not significant on multivariable regression. CONCLUSIONS: There was a strong correlation between IgG anti-S1/RBD and nAb IC50 after SARS-CoV-2 vaccination. Clinical comorbidities, such as prior COVID infection and renal function, impacted this correlation. These results may assist the prediction of post-vaccination immune protection in clinical settings using cost-effective commercial platforms.

Search of a genomic sequence database for potential novel blood group antigens: Investigation into why some amino acid substitutions are not immunogenic.

BACKGROUND: Polypeptide blood group antigens are typically identified through investigation of the antibodies they induce. Human genome sequence databases are a new tool to identify AA substitutions that potentially create blood group antigens. STUDY DESIGN AND METHODS: The Erythrogene genomic sequence database was searched for missense mutations not known to be blood group antigens in the extracellular domains of selected RBC proteins in European populations. Any mutations found with prevalence of 1%-90% and not known to have induced antibodies in transfusion practice were analyzed using protein structural analysis and epitope prediction programs to determine why they apparently lack immunogenicity. RESULTS: Thirteen missense mutations not known to create blood group antigens were identified in the extracellular domains of Kell, BCAM, and RhD proteins, but not in RhCE, Urea Transporter 1 (Kidd), Atypical Chemokine Receptor 1 (Duffy), glycophorin A or glycophorin B. While 11 of the 13 mutations had low prevalence (<1%), a Kell Ser726Pro substitution and a BCAM Val196Ile substitution had predicted phenotype prevalences of 43.2% and 5.7%, respectively. Ser726Pro had multiple properties of a linear B-cell epitope, but possible suboptimal protein location for B-cell receptor binding and limited T-cell epitope possibilities. Val196Ile was not predicted to be in a linear B-cell epitope. CONCLUSION: Multiple potential new blood group antigens of low prevalence were identified. Whether they are antigenic remains to be determined. Two higher prevalence variants in Kell and BCAM are unlikely antigens, otherwise their antibodies presumably would already have been identified. Possible reasons for their poor immunogenicity were identified.

Relationship between B-cell epitope structural properties and the immunogenicity of blood group antigens: Outlier properties of the Kell K1 antigen.

BACKGROUND: The immunogenicities of polypeptide blood group antigens vary, despite most being created by single amino acid (AA) substitutions. To study the basis of these differences, we employed an immunoinformatics approach to determine whether AA substitution sites of blood group antigens have structural features typical of B-cell epitopes and whether the extent of B-cell epitope properties is positively related to immunogenicity. STUDY DESIGN AND METHODS: Fifteen structural property prediction programs were used to determine the likelihood of ß-turns, surface accessibility, flexibility, hydrophilicity, particular AA composition and AA pairs, and other B-cell epitope properties at AA substitution sites of polypeptide blood group antigens. RESULTS: AA substitution sites of Lua , Jka , E, c, M, Fya , C, and S were each located in regions with at least two structural features typical of B-cell epitopes. The substitution site of K, the most immunogenic non-ABO/D antigen, scored the lowest for most B-cell epitope properties and was the only one not predicted to be part of a linear B-cell epitope. The most immunogenic antigens studied (K, Jka , Lua , E) had B-cell epitope structural properties determined by the fewest programs; the least immunogenic antigens (e.g., Fya , S, C, c) had B-cell epitope properties according to the most programs. DISCUSSION: Counter to prediction, the immunogenicity of polypeptide blood group antigens was not positively related to B-cell epitope structural features present at their AA-substitution sites. Instead, it tended to be negatively related. The AA-substitution site of the most immunogenic non-ABO/D antigen, K, had the least B-cell epitope features.

Selective Delivery of Clinically Approved Tubulin Binding Agents through Covalent Conjugation to an Active Targeting Moiety.

The efficacy and tolerability of tubulin binding agents are hampered by their low specificity for cancer cells like most clinically used anticancer agents. To improve specificity, tubulin binding agents have been covalently conjugated to agents that target cancer cells to give actively targeted drug conjugates. These conjugates are designed to increase uptake of the drug by cancer cells while having limited uptake by normal cells, thereby improving efficacy and tolerability. Approaches used include an attachment to small molecules, polysaccharides, peptides, proteins, and antibodies that exploit the overexpression of receptors for these substances. Antibody targeted strategies have been the most successful to date, with six such examples having gained clinical approval. Many other conjugate types, especially those targeting the folate receptor, have shown promising efficacy and toxicity profiles in pre-clinical models and in early-stage clinical studies. Presented herein is a discussion of the success or otherwise of the recent strategies used to form these actively targeted conjugates.

Relationship of epitope glycosylation and other properties of blood group proteins to the immunogenicity of blood group antigens.

BACKGROUND: The intrinsic properties of polypeptide blood group antigens that determine their relative immunogenicities are unknown. Because size, composition, charge, dose, and epitope glycosylation affect the immunogenicity of other polypeptides, we examined whether similar properties were related to the immunogenicity of blood group antigens. STUDY DESIGN AND METHODS: Amino acid (AA) sequences of antithetical blood group antigens were searched for N- and O-glycosylation sites. Regression analysis was carried out to determine whether blood group protein properties, including total and ectodomain size, red blood cell (RBC) antigen site density, number of mismatched AAs between an antigen and its closest homolog, and differences in mass, charge, and hydrophobicity of the mismatched AAs, were related to immunogenicity. RESULTS: The immunogenicities of non-RhD polypeptide antigens were directly related to the total and ectodomain sizes of their carrier proteins. A negative power relationship existed between RBC antigen site density and immunogenicity, such that the most immunogenic antigens had the lowest site density. The strong immunogenicity of RhD was related to the number of AA mismatches between RhD and RhCE, to their cumulative hydrophobicity and electrostatic mismatch scores, and the cumulative AA mass difference. No N- or O-glycosylation differences were predicted for antithetical or homologous antigens, other than a previously known N-glycosylation difference between K and k. CONCLUSION: Epitope glycosylation appeared not to be a determinant of immunogenicity for blood group antigens, except possibly for K. The immunogenicity of blood group antigens was positively related to total and ectodomain sizes of blood group proteins and negatively related to antigen site density. Such findings should be considered hypothesis generating for future, more definitive studies.

Structural and functional impacts of amino acid substitutions that create blood group antigens: implications for immunogenicity.

BACKGROUND: The immunogenicities of polypeptide blood group antigens vary widely. One possible determinant of immunogenicity is antigenic foreignness. The goal was to employ alternative ways of assessing foreignness and determine whether foreignness was related to immunogenicity. STUDY DESIGN AND METHODS: Foreignness was assessed as the extent of protein functional disruption caused by the exofacial amino acid (AA) substitutions that create blood group antigens, using AA substitution prediction algorithms such as Meta-SNP and according to whether those substitutions were radical or conservative. RESULTS: AA substitutions that create the most immunogenic antigens had the highest Meta-SNP scores, predictive of greater protein structure and function changes. Four of the 11 exofacial AAs that distinguish the most immunogenic antigen, RhD, from RhCE, and substitutions creating four of the five next most immunogenic antigens had the highest Meta-SNP scores (0.293-0.649). Excluding the outlier Jka , the mean Meta-SNP score of the four most immunogenic non-RhD antigens (K, Lua , E, c) was 3.7-fold higher than the mean of the four least immunogenic (M, Fya , C, S), 0.459 versus 0.123 (p = 0.0026). Regression analysis revealed a relationship between immunogenicity and Meta-SNP score (R2 = 0.953). Actual protein functional disruption was predicted for the AA substitution creating the E antigen. An AA cluster at Positions 350, 353, and 354 of RhD was unique, containing radical substitutions according to two classification schemes and relatively high Meta-SNP scores (0.351-0.432). CONCLUSION: The immunogenicity of blood group antigens was related to the functional disruption caused by the AA substitutions that create the antigens, as measured by Meta-SNP score.

Platelet transfusion: a clinical practice guideline from the AABB.

BACKGROUND: The AABB (formerly, the American Association of Blood Banks) developed this guideline on appropriate use of platelet transfusion in adult patients. METHODS: These guidelines are based on a systematic review of randomized, clinical trials and observational studies (1900 to September 2014) that reported clinical outcomes on patients receiving prophylactic or therapeutic platelet transfusions. An expert panel reviewed the data and developed recommendations using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework. RECOMMENDATION 1: The AABB recommends that platelets should be transfused prophylactically to reduce the risk for spontaneous bleeding in hospitalized adult patients with therapy-induced hypoproliferative thrombocytopenia. The AABB recommends transfusing hospitalized adult patients with a platelet count of 10 × 109 cells/L or less to reduce the risk for spontaneous bleeding. The AABB recommends transfusing up to a single apheresis unit or equivalent. Greater doses are not more effective, and lower doses equal to one half of a standard apheresis unit are equally effective. (Grade: strong recommendation; moderate-quality evidence). RECOMMENDATION 2: The AABB suggests prophylactic platelet transfusion for patients having elective central venous catheter placement with a platelet count less than 20 × 109 cells/L. (Grade: weak recommendation; low-quality evidence). RECOMMENDATION 3: The AABB suggests prophylactic platelet transfusion for patients having elective diagnostic lumbar puncture with a platelet count less than 50 × 109 cells/L. (Grade: weak recommendation; very-low-quality evidence). RECOMMENDATION 4: The AABB suggests prophylactic platelet transfusion for patients having major elective nonneuraxial surgery with a platelet count less than 50 × 109 cells/L. (Grade: weak recommendation; very-low-quality evidence). RECOMMENDATION 5: The AABB recommends against routine prophylactic platelet transfusion for patients who are nonthrombocytopenic and have cardiac surgery with cardiopulmonary bypass. The AABB suggests platelet transfusion for patients having bypass who exhibit perioperative bleeding with thrombocytopenia and/or evidence of platelet dysfunction. (Grade: weak recommendation; very-low-quality evidence). RECOMMENDATION 6: The AABB cannot recommend for or against platelet transfusion for patients receiving antiplatelet therapy who have intracranial hemorrhage (traumatic or spontaneous). (Grade: uncertain recommendation; very-low-quality evidence).

Use of a cytokine-release assay to demonstrate loss of platelet secretory capacity during blood bank processing and storage.

CONTEXT: The extent to which changes in secretory function contribute to the storage lesion of platelets (PLTs) prepared for transfusion is not well described. OBJECTIVE: To develop a cytokine-release assay for the assessment of PLT secretory capacity during the preparation and storage of PLTs. DESIGN: Small volumes of PLT-rich plasma and PLT concentrate (PC) were prepared from whole blood (WB; N = 4 donors). Aliquots of WB, PLT-rich plasma, and PC were treated with 20 µM adenosine diphosphate or saline (control). Samples of WB-derived PCs obtained from a regional blood center were similarly stimulated at various storage times (N = 10 units). Plasma levels of RANTES (chemokine ligand 5; regulated on activation, normal T cell expressed and secreted) and PLT aggregation were measured following agonist addition. RESULTS: Adenosine diphosphate stimulated RANTES release from PLTs in fresh WB on average by 4.1-fold (P < .001), in PLT-rich plasma by 4.7-fold (P = .002), and in PC by 1.3-fold (P < .001). For blood center PCs, adenosine diphosphate failed to stimulate RANTES release at day 2 of storage or later (P ≥ .31). Baseline RANTES levels in the plasma/supernatant increased 660% during PC preparation (P = .02) and an additional 30% during subsequent storage (P < .001). Mean PLT aggregation decreased during processing from WB (95.6%) to PC (60.5%; P = .04). For blood center PCs, mean PLT aggregation decreased substantially from days 2 (41.0%) to 7 (2.3%; P < .001). CONCLUSIONS: A cytokine-release assay revealed a diminution in PLT secretory capacity during PC processing and storage, with complete elimination by day 2 of storage. Loss of PLT aggregability occurred more slowly. The cytokine-release assay may be a useful endpoint for optimizing PLT preparation and storage.

Record fragmentation due to transfusion at multiple health care facilities: a risk factor for delayed hemolytic transfusion reactions.

BACKGROUND: Patients transfused at more than one health care facility face safety risks, because their transfusion record is fragmented. Blood group antibodies documented at one facility may be unknown to others. Because many antibodies are evanescent, access to prior antibody records is important for preventing incompatible transfusions and delayed hemolytic reactions. The study goal was to quantify multisite transfusion activity and its impact on antibody record accuracy. STUDY DESIGN AND METHODS: Patients (n = 100) undergoing hospital transfusion testing were surveyed to determine the locations and dates of any prior transfusions. Also, transfusion records were examined to determine whether patients (n = 200) known to be alloimmunized at one hospital had antibody testing done at another nearby hospital and, if so, how often the results were discrepant. RESULTS: Twenty-three percent (23/100) of patients undergoing type-and-screen testing reported receiving transfusions at 24 other facilities. Locations of transfusions that occurred elsewhere were 54.2% (13/24) at eight other in-state hospitals, 12.5% in bordering states, 20.8% in more distant states, and 12.5% during military service. Twenty-one percent (42/200) of patients known to be alloimmunized at one hospital had antibody test results on record at another nearby hospital. Antibody discrepancies were noted in 64.3% (27/42) of cases. The most common discrepancy was the failure of one facility to detect an antibody. CONCLUSION: Multisite transfusions were common. For patients seen at both of two nearby hospitals, antibody records were frequently discrepant. The findings support the need for interfacility sharing of transfusion records, particularly at the regional level.

Optimising the delivery of tubulin targeting agents through antibody conjugation.

Despite their side effect profile, there currently remains a heavy reliance on traditional cytotoxics and particularly tubulin targeting agents in cancer chemotherapy. To address this concern, significant progress has been made in the selective delivery of drugs to the tumour site. This review will examine the published data in support of the hypothesis that forming antibody conjugates of tubulin targeting agents is an effective approach towards their more effective delivery to the tumour site. Particular emphasis will be placed on the diversity of concepts under investigation, the efficacy of resultant conjugates, evidence of decreased resistance and the side effect profiles of the conjugates.