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Research has identified xanthurenic acid as being a gametocyte-activating factor in malaria disease transmission. Gametogenesis can be induced in vitro with low concentrations of xanthurenic acid at pH concentrations of between 7.4 and 8.0 to 8.2. Further drugs research will be based on these findings in a bid to stop malaria transmission.

External ATP has been proposed to be an autocrine regulator of glucose-stimulated insulin secretion and responsible for the synchronization of the Ca2+ rhythmicity in the beta-cells required for a pulsatile release of insulin from the pancreas. The importance of external ATP for glucose-stimulated insulin release was evaluated in rats with the aid of 2-deoxy-N-methyladenosine-3,5-bisphosphate (MRS 2179), an inhibitor of the purinoceptors known to affect the Ca2+ signaling in beta-cells. The concentration of cytoplasmic Ca2+ was measured in single beta-cells and small aggregates with ratiometric fura-2 technique and the release of insulin recorded from isolated islets and the perfused pancreas. Addition of 1 micromol/l ATP induced premature cytoplasmic Ca2+ concentration ([Ca2+]i) oscillations similar to those found in beta-cells exposed to 20 mmol/l glucose. In most experiments, the presence of 10 micromol/l MRS 2179 did not remove the glucose-induced [Ca2+]i rhythmicity in single beta-cells or the synchronization seen in coupled cells. Nevertheless, the same concentration of MRS 2179 promptly interrupted the pulsatility (frequency 0.22 +/- 0.01/min) of insulin secretion, raising the total amounts released from the pancreas. Prolonged exposure of islets to 1 and 10 micromol/l MRS 2179 enhanced insulin secretion at 20 mmol/l glucose 33% (P < 0.05) and 63% (P < 0.01), respectively, without affecting the release at 3 mmol/l glucose. The results support the idea that neural ATP signals entrain the islets into a common rhythm resulting in pulsatile release of insulin and that glucose stimulation of the secretory activity is counteracted by accumulation of inhibitory ATP around the beta-cells. [ABSTRACT FROM AUTHOR]

The developed asymmetric monovalent bispecific IgG1 or Duet monoclonal antibody (Duet mAb) has two distinct fragment antigen-binding region (Fab) subunits that target two different epitope specificities sequentially or simultaneously. The design features include unique engineered disulfide bridges, knob-into-hole mutations, and kappa and lambda chains to produce Duet mAbs. These make it structurally and functionally complex, so one expects challenging developability linked to instability, degradation of products and pathways, and limited reports available. Here, we have treated the product with different sources of extreme stress over a lengthy period, including varying heat, pH, photo stress, chemical oxidative stress, accelerated stress in physiological conditions, and forced glycation conditions. The effects of different stress conditions on the product were assessed using various analytical characterization tools to measure product-related substances, post-translational modifications (PTMs), structural integrity, higher-order disulfide linkages, and biological activity. The results revealed degradation products and pathways of Duet mAb. A moderate increase in size, charge, and hydrophobic variants, PTMs, including deamidation, oxidation, isomerization, and glycation were observed, with most conditions exhibiting biological activity. In addition, the characterization of fractionated charge variants, including deamidated species, showed satisfactory biological activity. This study demonstrated the prominent stability of the Duet mAb format comparable to most marketed mAbs., Competing Interests: Declaration of competing interest All authors are or were employees of AstraZeneca and may hold stock ownership or interests in the company. Yasunori Watanabe and Lydia Dewis were employees of AstraZeneca during experiments and manuscript compilation., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Developing a knob-into-hole asymmetric bispecific IgG1 monoclonal antibody (mAb) poses manufacturing challenges due to the expression of chain pairing variants, also called mispaired species, in the desired product. The incorrect pairing of light and heavy chains could result in heterogeneous mispaired species of homodimers, heterodimers, light chain swapping, and low molecular weight species (LMWS). Standard chromatography, capillary electrophoretic, or spectroscopic methods poorly resolve these from the main variants. Here, we report a highly sensitive reverse-phase polyphenyl ultra-high-performance liquid chromatography (RP-UHPLC) method to accurately measure mispaired species of Duet mAb format, an asymmetric IgG1 bispecific mAb, for both process development and quality control analytical tests. Coupled with electrospray ionization mass spectrometry (ESI-MS), it enabled direct online characterization of mispaired species. This single direct assay detected diverse mispaired IgG-like species and LMWS. The method resolved eight disulfide bonds dissociated LMWS and three mispaired LMWS. It also resolved three different types of IgG-like mispaired species, including two homodimers and one heterodimer. The characterization and quantification simultaneously enabled the cell line selection that produces a lesser heterogeneity and lower levels of mispaired species with the desired correctly paired product. The biological activity assessment of samples with increased levels of these species quantified by the method exhibited a linear decline in potency with increasing levels of mispaired species in the desired product. We also demonstrated the utility of the technique for testing in-process intermediate materials to determine and assess downstream purification process capability in removing diverse mispaired IgG-like species and LMWS to a certain level during the downstream purification process. Our investigation demonstrates that adopting this method was vital in developing asymmetric bispecific mAb from the initial stage of cell line development to manufacturing process development. Therefore, this tool could be used in the control strategy to monitor and control mispaired species during manufacturing, thus improving the quality control of the final product., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘All authors are or were employees of AstraZeneca and may hold stock ownership or interests in the company. Yasunori Watanabe, Lydia Dewis, and Nikola Lonska were employees of AstraZeneca during experiments and manuscript compilation. reports financial support was provided by AstraZeneca UK Limited. All authors are or were employees of AstraZeneca and may hold stock ownership or interests in the company. Yasunori Watanabe, Lydia Dewis, and Nikola Lonska were employees of AstraZeneca during experiments and manuscript compilation. reports a relationship with AstraZeneca UK Limited that includes: employment. All authors are or were employees of AstraZeneca and may hold stock ownership or interests in the company. Yasunori Watanabe, Lydia Dewis, and Nikola Lonska were employees of AstraZeneca during experiments and manuscript compilation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.’., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The physical stability of peptide-based drugs is of great interest to the pharmaceutical industry. Glucagon-like peptide 1 (GLP-1) is a 31-amino acid peptide hormone, the analogs of which are frequently used in the treatment of type 2 diabetes. We investigated the physical stability of GLP-1 and its C-terminal amide derivative, GLP-1-Am, both of which aggregate into amyloid fibrils. While off-pathway oligomers have been proposed to explain the unusual aggregation kinetics observed previously for GLP-1 under specific conditions, these oligomers have not been studied in any detail. Such states are important as they may represent potential sources of cytotoxicity and immunogenicity. Here, we identified and isolated stable, low-molecular-weight oligomers of GLP-1 and GLP-1-Am, using size-exclusion chromatography. Under the conditions studied, isolated oligomers were shown to be resistant to fibrillation or dissociation. These oligomers contain between two and five polypeptide chains and they have a highly disordered structure as indicated by a variety of spectroscopic techniques. They are highly stable with respect to time, temperature, or agitation despite their noncovalent character, which was established using liquid chromatography-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results provide evidence of stable, low-molecular-weight oligomers that are formed by an off-pathway mechanism which competes with amyloid fibril formation., Competing Interests: Declaration of interests V.L. and A.G.S. are employees of AstraZeneca., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Fragmentation is a major degradation pathway ubiquitous to all therapeutic monoclonal antibody (mAb) and therefore, monitored throughout the manufacturing process. Here, we describe a three-step approach to 1) detect, 2) confirm and 3) characterize partially reduced fragment species in an immunoglobulin G1 (IgG1) mAb with prolonged hold time of harvested cell culture fluid (HCCF). Microchip capillary electrophoresis (MCE) and high-performance size exclusion chromatography (HPSEC) were used as fast and efficient screening methods to detect fragmentation. HPSEC was found to be underestimating fragmentation levels. To confirm and characterize the fragments, capillary electrophoresis-sodium dodecyl sulphate (CE-SDS) was employed. Interestingly, the absence of fragments in the reduced CE-SDS analysis suggested partial reduction of disulphide bonds contributing to fragmentation in this mAb lot. This was further confirmed using reverse phase high performance liquid chromatography (RP-HPLC) coupled with mass spectrometry, which established the presence of heavy-heavy-light (HHL), heavy-heavy (HH), light-light dimer (LL), light chain (LC) and half antibody (HL) fragments with good mass accuracy. In this study, we demonstrated a readily applicable systematic strategy to support process development and investigate anomalous events in manufacturing. An additional highlight of this work is the data-driven comprehensive comparison of modern and conventional analytical techniques for fragment analysis., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

High throughput screening methods have driven a paradigm shift in biopharmaceutical development by reducing the costs of good manufactured (COGM) and accelerate the launch to market of novel drug products. Scale-down cell culture systems such as shaken 24- and 96-deep-well plates (DWPs) are used for initial screening of hundreds of recombinant mammalian clonal cell lines to quickly and efficiently select the best producing strains expressing product quality attributes that fit to industry platform. A common modification monitored from early-stage product development is protein aggregation due to its impact on safety and efficacy. This study aims to integrate high-throughput analysis of aggregation-prone therapeutic proteins with 96-deep well plate screening to rank clones based on the aggregation levels of the expressed proteins. Here we present an automated, small-scale analytical platform workflow combining the purification and subsequent aggregation analysis of protein biopharmaceuticals expressed in 96-DWP cell cultures. Product purification was achieved by small-scale solid-phase extraction using dual flow chromatography (DFC) automated on a robotic liquid handler for the parallel processing of up to 96 samples at a time. At-line coupling of size-exclusion chromatography (SEC) using a 2.1 mm ID column enabled the detection of aggregates with sub-2 µg sensitivity and a 3.5 min run time. The entire workflow was designed as an application to aggregation-prone mAbs and "mAb-like" next generation biopharmaceuticals, such as bispecific antibodies (BsAbs). Application of the high-throughput analytical workflow to a shake plate overgrow (SPOG) screen, enabled the screening of 384 different clonal cell lines in 32 h, requiring < 2 μg of protein per sample. Aggregation levels expressed by the clones varied between 9 and 76%. This high-throughput analytical workflow allowed for the early elimination of clonal cell lines with high aggregation, demonstrating the advantage of integrating analytical testing for critical quality attributes (CQAs) earlier in product development to drive better decision making., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

There is an increasing emphasis on the critical evaluation of interbatch purity and physical stability of therapeutic peptides. This is due to concerns over the impact that product- and process-related impurities may have on safety and efficacy of this class of drug. Aspartic acid isomerization to isoaspartic acid is a common isobaric impurity that can be very difficult to identify without first synthesizing isoAsp peptide standards for comparison by chromatography. As such, analytical tools that can determine if an Asp residue has isomerized, as well as the site of isomerization within the peptide sequence, are highly sought after. Ion mobility-mass spectrometry is a conformation-selective method that has developed rapidly in recent years particularly with the commercialization of traveling wave ion mobility instruments. This study employed a cyclic ion mobility (cIMS) mass spectrometry system to investigate the conformational characteristics of a therapeutic peptide and three synthetic isomeric forms, each with a single Asp residue isomerized to isoAsp. cIMS was able to not only show distinct conformational differences between each peptide but crucially, in conjunction with a simple workflow for comparing ion mobility data, it correctly located which Asp residue in each peptide had isomerized to isoAsp. This work highlights the value of cIMS as a potential screening tool in the analysis of therapeutic peptides prone to the formation of isoAsp impurities.

Monoclonal antibodies (mAbs) are extremely complex due to the presence of structural modifications resulting from enzymatic and chemical reactions such as glycosylation, glycation, deamidation, isomerisation, oxidation, aggregation and fragmentation. Size and charge variants analysis are carried out from the early stages of drug development throughout product lifetime to investigate product degradation pathways and optimise process conditions. However, conventional analytical workstreams for size and charge variant characterization are both time and sample demanding, requiring the application of multiple analytical methods. This study presents the development of a novel 2D-LC/MS approach combining both aggregate and charge variant profiling of a mAb candidate in a single method. Aggregate quantification was performed in the first dimension ( 1 D) by size exclusion chromatography SEC, followed by online fraction transfer of the monomer peak to the second dimension ( 2 D) by a heart-cutting for charge variant analysis by cation exchange chromatography (CEX). Aiming to maximise the information obtained from minimal sample and time required for analysis, a salt-based separation with UV detection was developed for supporting the processing of a large number of samples to facilitate high-throughput process development (HTPD). In addition, a mass spectrometry (MS) compatible SEC-CEX separation was developed enabling online charge variant peak identification. This study presented the ability to multiplex mAb size and charge variants analysis by coupling SEC with CEX in a 2D-LC set-up. To date, this is the first 2D SEC-CEX-UV(-MS) application for intact mAb analysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Transgenic human monoclonal antibodies derived from humanized mice against different epitopes of the Middle East respiratory syndrome coronavirus (MERS-CoV), and chimeric llama-human bispecific heavy chain-only antibodies targeting the Rift Valley fever virus (RVFV), were produced using a CHO-based transient expression system. Two lead candidates were assessed for each model virus before selecting and progressing one lead molecule. MERS-7.7G6 was used as the model antibody to demonstrate batch-to-batch process consistency and, together with RVFV-107-104, were scaled up to 200 L. Consistent expression titers were obtained in different batches at a 5 L scale for MERS-7.7G6. Although lower expression levels were observed for MERS-7.7G6 and RVFV-107-104 during scale up to 200 L, product quality attributes were consistent at different scales and in different batches. In addition to this, peptide mapping data suggested no detectable sequence variants for any of these candidates. Functional assays demonstrated comparable neutralizing activity for MERS-7.7G6 and RVFV-107-104 generated at different production scales. Similarly, MERS-7.7G6 batches generated at different scales were shown to provide comparable protection in mouse models. Our study demonstrates that a CHO-based transient expression process is capable of generating consistent product quality at different production scales and thereby supports the potential of using transient gene expression to accelerate the manufacturing of early clinical material.

Background: Proteins are biomolecules that consist of sequences of amino acids (primary structure) which can further interact and cause the backbone to fold into more complex arrangements (secondary and tertiary structures). Any chemical alterations of the molecules after the translation of the messenger RNA code into a protein primary sequence are known as posttranslational modifications (PTMs). PTMs may affect the protein's functionality; thus it is necessary to identify them. PTMs of particular interest to the pharmaceutical industry include deamidation, oxidation, deglycosylation and isomerization, which may occur due to environmental stressors. However, they have proved challenging to identify quickly. Electronic and vibrational spectroscopies have proved valuable tools for studying higher-order structure and stability of proteins., Methods: In this work, circular dichroism (CD), infrared absorbance (IR) and Raman spectroscopies were applied to characterize antibody (mAb NIP 228) PTMs as a result of different stressors. Mass spectrometry was used to confirm the identity of modifications including the targeted ones., Results: Room temperature CD showed that the secondary structure was the same after all treatments, and temperature-controlled CD showed how protein stability was affected by modifications. Both Raman and IR analysis detected small differences between the reference and deglycosylated proteins, and clearly indicated the presence of other PTMs., Conclusion: This work required some novel computational approaches to pre-process Raman and IR spectra and a review of the band assignments for proteins existing in the literature., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The Zoonoses Anticipation and Preparedness Initiative (ZAPI) was set up to prepare for future outbreaks and to develop and implement new technologies to accelerate development and manufacturing of vaccines and monoclonal antibodies. To be able to achieve surge capacity, an easy deployment and production at multiple sites is needed. This requires a straightforward manufacturing system with a limited number of steps in upstream and downstream processes, a minimum number of in vitro Quality Control assays, and robust and consistent platforms. Three viruses were selected as prototypes: Middle East Respiratory Syndrome (MERS) coronavirus, Rift Valley fever virus, and Schmallenberg virus. Selected antibodies against the viral surface antigens were manufactured by transient gene expression in Chinese Hamster Ovary (CHO) cells, scaling up to 200 L. For vaccine production, viral antigens were fused to multimeric protein scaffold particles using the SpyCatcher/SpyTag system. In vivo models demonstrated the efficacy of both antibodies and vaccines. The final step in speeding up vaccine (and antibody) development is the regulatory appraisal of new platform technologies. Towards this end, within ZAPI, a Platform Master File (PfMF) was developed, as part of a licensing dossier, to facilitate and accelerate the scientific assessment by avoiding repeated discussion of already accepted platforms. The veterinary PfMF was accepted, whereas the human PfMF is currently under review by the European Medicines Agency, aiming for publication of the guideline by January 2022., (Copyright © 2021. Published by Elsevier Ltd.)

Host cell proteins (HCP) that co-purify with biologics produced in Chinese hamster ovary cells have been shown to impact product quality through proteolytic degradation of recombinant proteins, leading to potential product losses. Several problematic HCPs can remain in the final product even after extensive purification. Each recombinant cell line has a unique HCP profile that can be determined by numerous upstream and downstream factors, including clonal variation and the protein sequence of the expressed therapeutic molecule. Here, we worked with recombinant cell lines with high levels of copurifying HCPs, and showed that in those cell lines even modest downregulation (≤50%) of the difficult to remove HCP Cathepsin D, through stable short hairpin RNA interference or monoallelic deletion of the target gene using CRISPR-Cas9, is sufficient to greatly reduce levels of co-purifying HCP as measured by high throughput targeted LC-MS. This reduction led to improved product quality by reducing fragmentation of the drug product in forced degradation studies to negligible levels. We also show the potential of cell engineering to target other undesired HCPs and relieve the burden on downstream purification., (© 2021 Wiley Periodicals LLC.)

Bioconjugates are an important class of therapeutic molecules. To date, O- glycan-based metabolic glycoengineering has had limited use in this field, due to the complexities of the endogenous O- glycosylation pathway and the lack of an O- glycosylation consensus sequence. Here, we describe the development of a versatile on-demand O- glycosylation system that uses a novel, widely applicable 5 amino acid O- glycosylation tag, and a metabolically engineered UDP-galactose-4-eperimase (GALE) knock-out cell line. Optimization of the primary sequence of the tag enables the production of Fc-based proteins with either single or multiple O- glycans with complexity fully controlled by media supplementation. We demonstrate how the uniformly labeled proteins containing exclusively N-azido-acetylgalactosamine are used for CLICK chemistry-based bioconjugation to generate site-specifically fluorochrome-labeled antibodies, dual-payload molecules, and bioactive Fc-peptides for applications in basic research and drug discovery. To our knowledge, this is the first description of generating a site-specific O- glycosylation system by combining an O- glycosylation tag and a metabolically engineered cell line.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods., (© 2020 De Leoz et al.)

Glycation, the nonenzymatic reaction between the reducing sugar glucose and the primary amine residues on amino acid side chains, commonly occurs in the cell culture supernatant during production of therapeutic monoclonal antibodies (mAbs). While glycation has the potential to impact efficacy and pharmacokinetic properties for mAbs, the most common undesirable impact of glycation is on the distribution of charged species, often a release specification for commercial processes. Existing empirical approaches are usually insufficient to rationalize the effects of cell line and process changes on glycation. To address this gap, we developed a kinetic model for estimating mAb glycation levels during the cell culture process. The rate constant for glycation, including temperature and pH dependence, was estimated by fitting the kinetic model to time-course glycation data from bioreactors operated at different process settings that yielded a wide range of glycation values. The parameter values were further validated by independently estimating glycation rate constants using cell-free incubation studies at various temperatures. The model was applied to another mAb, by re-estimating the activation energy to account for effect of a glycation "hotspot". The model was further utilized to study the role of temperature shift as an approach to reduce glycation levels in the manufacturing process for mAb2. While a downshift in temperature resulted in lowering of glycation levels for mAb2, the model helped elucidate that this effect was caused due to contribution from changes in glucose consumption, mAb secretion and temperature, instead of a direct impact of temperature alone on the kinetic rate of glycation., (© 2019 American Institute of Chemical Engineers.)

Major histocompatibility complex (MHC) class I molecules present peptides, produced through cytosolic proteasomal degradation of cellular proteins, to cytotoxic T lymphocytes. In dendritic cells, the peptides can also be derived from internalized antigens through a process known as cross-presentation. The cellular compartments involved in cross-presentation remain poorly defined. We found a role for peptide trimming by insulin-regulated aminopeptidase (IRAP) in cross-presentation. In human dendritic cells, IRAP was localized to a Rab14+ endosomal storage compartment in which it interacted with MHC class I molecules. IRAP deficiency compromised cross-presentation in vitro and in vivo but did not affect endogenous presentation. We propose the existence of two pathways for proteasome-dependent cross-presentation in which final peptide trimming involves IRAP in endosomes and involves the related aminopeptidases in the endoplasmic reticulum.

Type 1 diabetes mellitus (T1DM) results from the destruction of beta cells by autoantigen-specific T cells. In the non-obese diabetic (NOD) mouse model, CD8+ T cells play an essential role in both the initial triggering of insulitis and its destructive phase, and proinsulin (PI) is one of the dominant target antigens (Ags). However, little is known about the beta cell epitopes presented by HLA class I molecules and recognized by human CD8+ T cells. We and other groups recently applied reverse immunology approaches to identify HLA class I-restricted PI epitopes. To establish an inventory of potential naturally processed epitopes, whole human PI or the transitional region between the B-chain and C-peptide were digested with purified proteasome complexes. By combining proteasome digestion data with epitope prediction algorithms, candidate epitopes restricted by HLA-A2.1 and other HLA class I molecules were identified. We validated immunogenicity and natural processing of the identified PI epitopes in HLA-A2.1-transgenic mice, while others demonstrated recognition of multiple PI epitopes by CD8+ T cells from T1DM and healthy subjects in the context of different HLA class I molecules. These results demonstrate the power of reverse immunology strategies for epitope discovery. DNA vaccination of HLA-transgenic mice may be another rapid and efficient reverse immunology approach to map additional epitopes derived from other T1DM Ags, such as IA-2 and glutamic acid decarboxylase 65 (GAD 65). Transfer of this information to Elispot- and MHC tetramer-based assay formats should allow to reliably detect and characterize autoreactive CD8+ T cell responses in T1DM, and may open new avenues for early T1DM diagnosis and immune intervention.

The generation of many HLA class I peptides entails a final trimming step in the endoplasmic reticulum that, in humans, is accomplished by two 'candidate' aminopeptidases. We show here that one of these, ERAP1, was unable to remove several N-terminal amino acids that were trimmed efficiently by the second enzyme, ERAP2. This trimming of a longer peptide required the concerted action of both ERAP1 and ERAP2, both for in vitro digestion and in vivo for cellular antigen presentation. ERAP1 and ERAP2 localized together in vivo and associated physically in complexes that were most likely heterodimeric. Thus, the human endoplasmic reticulum is equipped with a pair of trimming aminopeptidases that have complementary functions in HLA class I peptide presentation.

HIV proteins contain a multitude of naturally processed cytotoxic T lymphocyte (CTL) epitopes that concentrate in clusters. The molecular basis of epitope clustering is of interest for understanding HIV immunogenicity and for vaccine design. We show that the CTL epitope clusters of HIV proteins predominantly coincide with hydrophobic regions, whereas the noncluster regions are predominantly hydrophilic. Analysis of the proteasomal degradation products of full-length HIV-Nef revealed a differential sensitivity of cluster and noncluster regions to proteasomal processing. Compared with the epitope-scarce noncluster regions, cluster regions are digested by proteasomes more intensively and with greater preference for hydrophobic P1 residues, resulting in substantially greater numbers of fragments with the sizes and COOH termini typical of epitopes and their precursors. Indeed, many of these fragments correspond to endogenously processed Nef epitopes and/or their potential precursors. The results suggest that differential proteasomal processing contributes importantly to the clustering of CTL epitopes in hydrophobic regions.

The C-terminal fragment of merozoite surface protein-1 (MSP-1) of the mouse malaria parasite Plasmodium chabaudi chabaudi (AS) stimulates a weak CD4 T cell response when compared to the response to a more structurally simple region of the molecule. The tertiary structure of the C-terminal region of MSP-1 is maintained by five disulfide bonds. A peptide from this region could only be processed and loaded onto newly synthesized MHC class II molecules, whereas a peptide from the structurally simple region was available for loading onto recycling MHC class II. CD4(+) T cell hybridomas took longer to recognize an epitope derived from the disulfide-bonded region whether native parasite or recombinant MSP-1 antigen was used. Reduction of disulfide bonds in the C-terminal region subsequently allowed peptides to be loaded onto recycling MHC class II and greatly enhanced the rapidity of the T cell response. These data demonstrate that differential processing occurs intramolecularly in MSP-1, which may be responsible for the observed weak CD4 T cell responses against this region. The consequences of this in vivo may be that limited T cell help is available for protective antibody production which has important implications for designing vaccines based on MSP-1.

A modified form of antithrombin (AT) cleaved at the active site by thrombin (ATc) has been shown to be generated in vivo, corresponding to 1-4% of the circulating AT. An enzyme immunoassay has been developed for measuring ATc following plasma treatment with ammonium sulphate and heat denaturation of native AT. ATc plasma levels were found to be significantly higher (P = 0.003) in patients developing venous thromboembolism when compared to patients without such events or healthy controls (age and sex matched). In addition, ATc levels correlated with thrombin generation markers: thrombin-AT complex (r2 = 0.66, P = 0.005) and prothrombin fragment 1 + 2 (r2 = 0.58, P = 0.018), but, in contrast to both these markers, elevated ATc levels were detected for at least 2 weeks after the thromboembolic event. In conclusion, ATc appears to be a new marker for thrombin generation and overall activation of the coagulation system, having the advantage of being detected in the circulation for a longer period than other thrombin generation markers.