Sagacious Epitope Selection for Vaccines, and Both Antibody-Based Therapeutics and Diagnostics: Tips From Virology and Oncology

The target of an antibody plays a significant role in the success of antibody-based therapeutics and diagnostics, and to an extent, that of vaccine development. This importance is focussed on the target binding site – epitope, where epitope selection as a part of design thinking beyond traditional antigen selection using whole cell or whole protein immunisation can positively impact success. With purified recombinant protein production and peptide synthesis to display limited/selected epitopes, intrinsic factors that can affect the functioning of resulting antibodies can be more easily selected for. Many of these factors stem from the location of the epitope that can affect accessibility of the antibody to the epitope at a cellular or molecular level, direct inhibition of target antigen activity, conservation of function despite escape mutations, and even non-competitive inhibition sites. Through the incorporation of novel computational methods for predicting antigen changes to model-informed drug discovery and development, superior vaccines and antibody-based therapeutics or diagnostics can now be more easily designed to mitigate failures. With detailed examples, this review highlights the new opportunities, factors and methods of predicting antigenic changes for consideration in sagacious epitope selection.

Sagacious Epitope Selection for Vaccines, and Both Antibody-Based Therapeutics and Diagnostics: Tips From Virology and Oncology

The target of an antibody plays a significant role in the success of antibody-based therapeutics and diagnostics, and to an extent, that of vaccine development. This importance is focussed on the target binding site – epitope, that sagacious epitope selection in a form of design thinking beyond traditional antigen selection using whole cell or whole protein immunisation can positively improve success. Intrinsic factors that can affect the functioning of resulting antibodies can be more easily selected for with purified recombinant protein production and peptide synthesis to display limited/selected epitopes. Many of these factors stem from the location of the epitope that can affect the accessibility of the antibody to the epitope at a cellular or molecular level, direct inhibition of target antigen activity, conservation of function despite escape mutations, and even non-competitive inhibition sites. Through the incorporation of novel computational methods for predicting antigen changes to model-informed drug design development, superior vaccines and antibody-based therapeutics or diagnostics can now be more easily designed, mitigating failures. With detailed examples, this review highlights the new opportunities, factors and methods of predicting antigenic changes for consideration in sagacious epitope selection.

Alterations of Plasma Complement C3b, C5b and VWF, ADAMTS13 in Patients with Graft-Versus-Host Disease and Thrombotic Microangiopathy after Hematopoietic Stem-Cell Transplantation

Background: Transplant-associated microangiopathy (TMA) is an uncommon but devastating complication in patients undergoing hematopoietic stem-cell transplantation (HSCT). It may be confused with severe graft-versus-host disease (GVHD), infection, and other transplant related thrombotic diseases. Limited studies have shown the changes in plasma VWF/ADAMTS13 or complement activation markers in patients after HSCT. However, the role of VWF/ADAMTS13 and complement activation in patients with TMA and GVHD is not fully understood. Current study is to investigate the alterations of plasma levels of C3b, C5b and VWF/ADAMTS13 in patients with TMA and to explore their roles in the pathogenesis and early diagnosis of transplant-associated TMA. Methods: From 2011 to 2014, 14 patients with TMA were enrolled into the study in a single medical center. 71 other patients following HSCT were recruited as control subjects including 11 cases of hepatic vein occlusion disease (VOD), 20 cases of severe infections, 20 cases of severe II-IV° GVHD and 20 cases without complications. Blood sample was collected before transplantation and at the onset of transplantation related complication. Fluorescence resonance energy transfer substrate (FRETS)-VWF73 assay detected plasma ADAMTS13 activity. Collagen-binding assay and latex immunoassay determined VWF activity and VWF antigen, respectively. Plasma VWF multimer was determined by agarose gel electrophoresis and Western blot. Plasma levels of complement C3b and C5b were measured with ELISA. Results: Compared with the levels before transplantation, plasma ADAMTS13 activity and VWF antigen or activity in the patients with TMA did not differ from those who developed TMA, with infection or GVHD or without any complication (p> 0.05). However, plasma ADAMTS13 activity decreased and the ratio of VWF antigen/activity increased significantly in patients with VOD (p< 0.05). Plasma VWF multimer distribution was similar in patients with infection, GVHD or without complication, but ultralarge multimers of VWF was present in patients with TMA and VOD. Plasma levels of complement C3b was increased in patients after HSCT (198.46 ng/ml ±14.78 ng/ml) compared with healthy subjects (85.02 ng/ml±8.50ng/ml) (p< 0.05), but exhibited no difference in the other groups. Plasma C3b increased significantly in patients with TMA and GVHD (p<0.05). The plasma levels of C3b were higher in the TMA group (480.70 ng/ml±66.76ng/ml) than the GVHD group (298.50 ng/ml ±32.06 ng/) (p< 0.05). Also, plasma levels of C5b in patients with TMA were significantly increased (1059.49 ng/ml ±85.57 ng/ml) as compared with those before transplantation (653.19ng/ml ±44.91ng/ml) and other groups (p< 0.05). Conclusions: We conclude that plasma ADAMTS 13 activity and the ratio of VWF antigen/activity remained stable in the patients with transplant-associated TMA, but the levels of complement C3b and C5b, particularly the C5b, increased significantly, suggesting the critical role of complement pathway in the pathogenesis of TMA. C5b may be a specific biomarker for early diagnosis of TMA but C3b is a marker for both TMA and GVHD. Disclosures No relevant conflicts of interest to declare.

Tissue Factor Expression in Obese Type 2 Diabetic Subjects and Its Regulation by Antidiabetic Agents

Objective.Increased coagulation activation may contribute to the high incidence of cardiovascular complications observed in obese and type 2 diabetes (T2D) subjects. Although tissue factor (TF), the primary initiator of coagulation is increased in obesity, its expression in adipose tissues and its association with metabolic parameters are unclear. We sought to compare TF expression in plasma and adipose tissues of obese subjects with and without T2D, its correlation with metabolic parameters, and regulation in response to antidiabetic drugs.MethodsSubjects were recruited from diabetes clinics and adipose tissue was obtained by needle biopsy of lower subcutaneous abdominal depot. For the intervention study, subjects were randomized into treatment groups with rosiglitazone or metformin for 4 months.Results.Plasma TF antigen, activity, and adipose TF mRNA were greater in obese T2D subjects compared with obese nondiabetics. Plasma TF activity correlated with fasting insulin, glucose, and free fatty acids, (FFAs), and adipose TF mRNA correlated with plasma FFA. Plasma TF activity was reduced by metformin and increased with rosiglitazone treatment.Conclusions.Specific diabetes-related metabolic parameters, but not obesity per se, are correlated with TF expression. Regulation of TF activity by different classes of antidiabetic drugs may relate to protective or adverse cardiovascular outcomes.