Tuft-Cell-Derived Leukotrienes Drive Rapid Anti-helminth Immunity in the Small Intestine but Are Dispensable for Anti-protist Immunity.

Helminths, allergens, and certain protists induce type 2 immune responses, but the underlying mechanisms of immune activation remain poorly understood. In the small intestine, chemosensing by epithelial tuft cells results in the activation of group 2 innate lymphoid cells (ILC2s), which subsequently drive increased tuft cell frequency. This feedforward circuit is essential for intestinal remodeling and helminth clearance. ILC2 activation requires tuft-cell-derived interleukin-25 (IL-25), but whether additional signals regulate the circuit is unclear. Here, we show that tuft cells secrete cysteinyl leukotrienes (cysLTs) to rapidly activate type 2 immunity following chemosensing of helminth infection. CysLTs cooperate with IL-25 to activate ILC2s, and tuft-cell-specific ablation of leukotriene synthesis attenuates type 2 immunity and delays helminth clearance. Conversely, cysLTs are dispensable for the tuft cell response induced by intestinal protists. Our findings identify an additional tuft cell effector function and suggest context-specific regulation of tuft-ILC2 circuits within the small intestine.

A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing.

Identification of specific epitopes targeted by neutralizing antibodies is essential to advance epitope-based vaccine design strategies. We report a facile methodology for rapid epitope mapping of neutralizing antibodies (NAbs) against HIV-1 Envelope (Env) at single-residue resolution, using Cys labeling, viral neutralization assays, and deep sequencing. This was achieved by the generation of a library of Cys mutations in Env glycoprotein on the viral surface, covalent labeling of the Cys residues using a Cys-reactive label that masks epitope residues, followed by infection of the labeled mutant virions in mammalian cells in the presence of NAbs. Env gene sequencing from NAb-resistant viruses was used to accurately delineate epitopes for the NAbs VRC01, PGT128, and PGT151. These agreed well with corresponding experimentally determined structural epitopes previously inferred from NAb:Env structures. HIV-1 infection is associated with complex and polyclonal antibody responses, typically composed of multiple antibody specificities. Deconvoluting the epitope specificities in a polyclonal response is a challenging task. We therefore extended our methodology to map multiple specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as in an HIV-1-infected elite neutralizer capable of neutralizing tier 3 pseudoviruses with high titers. The method can be readily extended to other viruses for which convenient reverse genetics or lentiviral surface display systems are available.

Leukotriene D4 paradoxically limits LTC4-driven platelet activation and lung immunopathology.

BACKGROUND: The 3 cysteinyl leukotrienes (cysLTs), leukotriene (LT) C4 (LTC4), LTD4, and LTE4, have different biologic half-lives, cellular targets, and receptor specificities. CysLT2R binds LTC4 and LTD4in vitro with similar affinities, but it displays a marked selectivity for LTC4in vivo. LTC4, but not LTD4, strongly potentiates allergen-induced pulmonary eosinophilia in mice through a CysLT2R-mediated, platelet- and IL-33-dependent pathway. OBJECTIVE: We sought to determine whether LTD4 functionally antagonizes LTC4 signaling at CysLT2R. METHODS: We used 2 different in vivo models of CysLT2R-dependent immunopathology, as well as ex vivo activation of mouse and human platelets. RESULTS: LTC4-induced CD62P expression; HMGB1 release; and secretions of thromboxane A2, CXCL7, and IL-33 by mouse platelets were all were blocked by a selective CysLT2R antagonist and inhibited by LTD4. These effects did not depend on CysLT1R. Inhaled LTD4 blocked LTC4-mediated potentiation of ovalbumin-induced eosinophilic inflammation; recruitment of platelet-adherent eosinophils; and increases in IL-33, IL-4, IL-5, and IL-13 levels in lung tissue. In contrast, the effect of administration of LTE4, the preferred ligand for CysLT3R, was additive with LTC4. The administration of LTD4 to Ptges-/- mice, which display enhanced LTC4 synthesis similar to that in aspirin-exacerbated respiratory disease, completely blocked the physiologic response to subsequent lysine-aspirin inhalation challenges, as well as increases in levels of IL-33, type 2 cytokines, and biochemical markers of mast cell and platelet activation. CONCLUSION: The conversion of LTC4 to LTD4 may limit the duration and extent of potentially deleterious signaling through CysLT2R, and it may contribute to the therapeutic properties of desensitization to aspirin in aspirin-exacerbated respiratory disease.

Cysteinyl maresins regulate the prophlogistic lung actions of cysteinyl leukotrienes.

BACKGROUND: Cysteinyl leukotrienes (CysLTs) are potent prophlogistic mediators in asthmatic patients; however, inhibition of CysLT receptor 1 is not a consistently effective treatment, suggesting additional regulatory mechanisms. Other cysteinyl-containing lipid mediators (LMs) derived from docosahexaenoic acid, namely maresin conjugates in tissue regeneration (MCTRs), were recently discovered. Therefore their production and actions in the lung are of considerable interest. OBJECTIVE: We sought to determine MCTR production, bioactions, and mechanisms in the human lung and in patients with experimental allergic airway inflammation. METHODS: LM metabololipidomic profiling of the lung was performed by using liquid chromatography with tandem mass spectrometry. Donor-derived human precision-cut lung slices were exposed to leukotriene (LT) D4, MCTRs, or both before determination of airway contraction. The actions of exogenous MCTRs on murine allergic host responses were determined in the setting of ovalbumin- and house dust mite-induced lung inflammation. RESULTS: Lipidomic profiling showed that the most abundant cysteinyl LMs in healthy human lungs were MCTRs, whereas CysLTs were most prevalent in patients with disease. MCTRs blocked LTD4-initiated airway contraction in human precision-cut lung slices. In mouse allergic lung inflammation MCTRs were present with temporally regulated production. With ovalbumin-induced inflammation, MCTR1 was most potent for promoting resolution of eosinophils, and MCTR3 potently decreased airway hyperreactivity to methacholine, bronchoalveolar lavage fluid albumin, and serum IgE levels. MCTR1 and MCTR3 inhibited lung eosinophilia after house dust mite-induced inflammation. CONCLUSION: These results identified lung MCTRs that blocked human LTD4-induced airway contraction and promoted resolution of murine allergic airway responses when added exogenously. Together, these findings uncover proresolving mechanisms for lung responses that can be disrupted in patients with disease.

Role of the Cysteine 81 Residue of Macrophage Migration Inhibitory Factor as a Molecular Redox Switch.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory and tumor-promoting cytokine that occurs in two redox-dependent immunologically distinct conformational isoforms. The disease-related structural isoform of MIF (oxMIF) can be specifically and predominantly detected in the circulation of patients with inflammatory diseases and in tumor tissue, whereas the ubiquitously expressed isoform of MIF (redMIF) is abundantly expressed in healthy and diseased subjects. In this article, we report that cysteine 81 within MIF serves as a "switch cysteine" for the conversion of redMIF to oxMIF. Modulating cysteine 81 by thiol reactive agents leads to significant structural rearrangements of the protein, resulting in a decreased ß-sheet content and an increased random coil content, but maintaining the trimeric quaternary structure. This conformational change in the MIF molecule enables binding of oxMIF-specific antibodies BaxB01 and BaxM159, which showed beneficial activity in animal models of inflammation and cancer. Crystal structure analysis of the MIF-derived EPCALCS peptide, bound in its oxMIF-like conformation by the Fab fragment of BaxB01, revealed that this peptide adopts a curved conformation, making the central thiol protein oxidoreductase motif competent to undergo disulfide shuffling. We conclude that redMIF might reflect a latent zymogenic form of MIF, and formation of oxMIF leads to a physiologically relevant, i.e., enzymatically active, state.

The inhibition of 5-Lipoxygenase (5-LO) products leukotriene B4 (LTB4) and cysteinyl leukotrienes (cysLTs) modulates the inflammatory response and improves cutaneous wound healing.

To analyze the participation of the enzyme 5-lipoxygenase (5-LO) in skin repair, WT wounds were compared to those in 5-LO deficient mice (5-LO-/-), which presented faster closure and reduced inflammatory infiltrate in the skin, together with increased CD4 regulatory T cells markers in the draining lymph nodes. The 5-LO-/- wounds also had diminished TNF-α, CCL11, CCL7, CCL2, CXCL9, CCR1 and CXCR2 mRNA expression in the lesions, besides differential extracellular matrix remodeling. Furthermore, when cysteinyl leukotriene (cysLT) and leukotriene (LTB4) receptors were antagonized in WT mice, there was a remarkable reduction in TNF-α expression and faster skin healing, similarly to the findings in 5-LO-/- animals. Finally, our results suggested that 5-LO products, in special cysLT and LTB4, underline skin inflammation that follows skin injury and their neutralization may be an important strategy to improve cutaneous healing.

Allergenicity of acrolein-treated shrimp tropomyosin evaluated using RBL-2H3 cell and mouse model.

BACKGROUND: Food processing effects can modify protein functional properties. However, protein was oxidized inevitably by lipid peroxidation during food processing. Acrolein, a primary by-product of lipid peroxidation, can modify the structural and functional properties of protein. The aim of the research was to analyze the effect of acrolein on allergenicity of TM, a major allergen in shrimp. RESULTS: The overall allergenic effects of acrolein-treated TM were evaluated using female BALB/c mice and a mediator-releasing RBL-2H3 cell line. Acrolein-treated TM significantly decreased TM-specific immunoglobulin E/G1 levels, and histamine and mMCP-1 release in mouse serum. Release of inflammatory mediators such as ß-hexosaminidase, histamine, cysteinyl leukotriene and prostaglandin D2 was clearly suppressed after acrolein treatment. CONCLUSION: These results indicate that acrolein-induced tropomyosin modification can decrease the allergenicity of TM. This reduction contributes to allergenic potential changes in shrimp during processing and preservation. © 2018 Society of Chemical Industry.

High-affinity pan-specific monoclonal antibodies that target cysteinyl leukotrienes and show efficacy in an acute model of colitis.

Cysteinyl leukotrienes (CysLTs) are a small family of biological signaling lipids produced by active leukocytes that contribute to diverse inflammatory disease states as a consequence of their engagement with dedicated G protein-coupled receptors. Immunization of mice with a CysLT-modified hapten carrier protein yielded novel monoclonal antibodies that display variable binding affinity to CysLTs. Solution binding assays indicated differing specificities among the antibodies tested, with antibody 10G4 displaying a preference for leukotriene C4 (LTC4). X-ray crystallography of a humanized 10G4 Fab fragment in complex with LTC4 revealed that binding induces a hook-like conformation within the hydrocarbon tail of the lipid arachidonic acid moiety. Specific hydrogen bonding to the LTC4 carboxylate groups further stabilized the complex, while a water molecule mediated a hydrogen bond network that connected the N-terminal arm of l-glutathione to both the arachidonyl carboxylate of LTC4 and the antibody heavy chain. Prophylactic administration of two anti-CysLT antibodies in mice followed by challenge with LTC4 demonstrated their in vivo efficacy against acute inflammation in a vascular permeability model. 10G4 ameliorated the effects of acute dextran sulfate sodium-induced colitis, suggesting that anti-CysLT antibodies could provide a therapeutic benefit in the treatment of inflammatory diseases.

Platelet-driven leukotriene C4-mediated airway inflammation in mice is aspirin-sensitive and depends on T prostanoid receptors.

Cysteinyl leukotrienes (cysLTs) are bronchoconstricting lipid mediators that amplify eosinophilic airway inflammation by incompletely understood mechanisms. We recently found that LTC4, the parent cysLT, potently activates platelets in vitro and induces airway eosinophilia in allergen-sensitized and -challenged mice by a platelet- and type 2 cysLT receptor-dependent pathway. We now demonstrate that this pathway requires production of thromboxane A2 and signaling through both hematopoietic and lung tissue-associated T prostanoid (TP) receptors. Intranasal administration of LTC4 to OVA-sensitized C57BL/6 mice markedly increased the numbers of eosinophils in the bronchoalveolar lavage fluid, while simultaneously decreasing the percentages of eosinophils in the blood by a TP receptor-dependent mechanism. LTC4 upregulated the expressions of ICAM-1 and VCAM-1 in an aspirin-sensitive and TP receptor-dependent manner. Both hematopoietic and nonhematopoietic TP receptors were essential for LTC4 to induce eosinophil recruitment. Thus, the autocrine and paracrine functions of thromboxane A2 act downstream of LTC4/type 2 cysLT receptor signaling on platelets to markedly amplify eosinophil recruitment through pulmonary vascular adhesion pathways. The findings suggest applications for TP receptor antagonists in cases of asthma with high levels of cysLT production.

A camel anti-lysozyme CDR3 only domain antibody selected from phage display VHH library acts as potent lysozyme inhibitor.

Mimetics of antibody-binding sites represent particularly interesting targets, however they are difficult to identify. In most cases, naturally derived CDR3 peptides show a much lower activity and affinity. In this study, we identified a CDR3 domain antibody with framework 3 (FR3) and FR4 in the flank by screening a lysozyme-immunized phage display VHH library. This antibody has a potent enzyme inhibiting activity and high thermal stability. With sequence alignment and site-directed mutagenic analysis, we found that the cysteine residue at amino acid position 88 in FR3 might play a key role in maintaining the stability of the CDR3 antibody. The small-sized CDR3 domain antibody might act as a new scaffold for affinity transfer, hence making a useful contribution to the understanding of antigen-antibody interactions.

Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils.

BACKGROUND: Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. OBJECTIVES: To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. METHODS: Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. RESULTS: Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1ß increased the expression of PLA2G10 by eosinophils. CONCLUSIONS: These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma.

Aspirin activation of eosinophils and mast cells: implications in the pathogenesis of aspirin-exacerbated respiratory disease.

Reactions to aspirin and nonsteroidal anti-inflammatory drugs in patients with aspirin-exacerbated respiratory disease (AERD) are triggered when constraints upon activated eosinophils, normally supplied by PGE2, are removed secondary to cyclooxygenase-1 inhibition. However, the mechanism driving the concomitant cellular activation is unknown. We investigated the capacity of aspirin itself to provide this activation signal. Eosinophils were enriched from peripheral blood samples and activated with lysine ASA (LysASA). Parallel samples were stimulated with related nonsteroidal anti-inflammatory drugs. Activation was evaluated as Ca2+ flux, secretion of cysteinyl leukotrienes (CysLT), and eosinophil-derived neurotoxin (EDN) release. CD34+ progenitor-derived mast cells were also used to test the influence of aspirin on human mast cells with measurements of Ca2+ flux and PGD2 release. LysASA induced Ca2+ fluxes and EDN release, but not CysLT secretion from circulating eosinophils. There was no difference in the sensitivity or extent of activation between AERD and control subjects, and sodium salicylate was without effect. Like eosinophils, aspirin was able to activate human mast cells directly through Ca2+ flux and PGD2 release. AERD is associated with eosinophils maturing locally in a high IFN-γ milieu. As such, in additional studies, eosinophil progenitors were differentiated in the presence of IFN-γ prior to activation with aspirin. Eosinophils matured in the presence of IFN-γ displayed robust secretion of both EDN and CysLTs. These studies identify aspirin as the trigger of eosinophil and mast cell activation in AERD, acting in synergy with its ability to release cells from the anti-inflammatory constraints of PGE2.

Dectin-2 regulates the effector phase of house dust mite-elicited pulmonary inflammation independently from its role in sensitization.

The myeloid C-type lectin receptor Dectin-2 directs the generation of Th2 and Th17 immune responses to the house dust mite Dermatophagoides farinae through the generation of cysteinyl leukotrienes and proinflammatory cytokines, respectively, but a role for Dectin-2 in effector phase responses has not been described. In this study, we demonstrate that administration of the Dectin-2 mAb solely at the time of D. farinae challenge abrogated eosinophilic and neutrophilic inflammation in the bronchoalveolar lavage fluid and Th1, Th2, and Th17 inflammation in the lung of previously sensitized mice. Furthermore, Dectin-2 null mice (Clec4n(-/-)) sensitized with the adoptive transfer of D. farinae-pulsed wild-type (WT) bone marrow-derived dendritic cells (DCs) also had less D. farinae-elicited pulmonary inflammation, supporting an effector function for Dectin-2. The protection from pulmonary inflammation seen with the Dectin-2 mAb or in Clec4n(-/-) mice was associated with little or no reduction in lung-draining lymph node cells or their cytokine production and with no reduction in serum IgE. WT and Clec4n(-/-) mice recipients, sensitized with D. farinae-pulsed WT bone marrow-derived DCs, had comparable levels of D. farinae-elicited IL-6, IL-23, TNF-α, and cysteinyl leukotrienes in the lung. By contrast, D. farinae-elicited CCL4 and CCL8 production from pulmonary CD11c(+)CD11b(+)Ly6C(+) and CD11c(+)CD11b(+)Ly6C(-)CD64(+) monocyte-derived DCs was reduced in Clec4n(-/-) recipients. Addition of CCL8 at the time of D. farinae challenge abrogated the protection from eosinophilic, neutrophilic, and Th2 pulmonary inflammation seen in Clec4n(-/-) recipients. Taken together, these results reveal that Dectin-2 regulates monocyte-derived DC function in the pulmonary microenvironment at D. farinae challenge to promote the local inflammatory response.

Identification of the immunodominant epitope region in phospholipase A2 receptor-mediating autoantibody binding in idiopathic membranous nephropathy.

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Recent clinical studies established that >70% of patients with idiopathic (also called primary) MN (IMN) possess circulating autoantibodies targeting the M-type phospholipase A2 receptor-1 (PLA2R) on the surface of glomerular visceral epithelial cells (podocytes). In situ, these autoantibodies trigger the formation of immune complexes, which are hypothesized to cause enhanced glomerular permeability to plasma proteins. Indeed, the level of autoantibody in circulation correlates with the severity of proteinuria in patients. The autoantibody only recognizes the nonreduced form of PLA2R, suggesting that disulfide bonds determine the antigenic epitope conformation. Here, we identified the immunodominant epitope region in PLA2R by probing isolated truncated PLA2R extracellular domains with sera from patients with IMN that contain anti-PLA2R autoantibodies. Patient sera specifically recognized a protein complex consisting of the cysteine-rich (CysR), fibronectin-like type II (FnII), and C-type lectin-like domain 1 (CTLD1) domains of PLA2R only under nonreducing conditions. Moreover, absence of either the CysR or CTLD1 domain prevented autoantibody recognition of the remaining domains. Additional analysis suggested that this three-domain complex contains at least one disulfide bond required for conformational configuration and autoantibody binding. Notably, the three-domain complex completely blocked the reactivity of autoantibodies from patient sera with the full-length PLA2R, and the reactivity of patient sera with the three-domain complex on immunoblots equaled the reactivity with full-length PLA2R. These results indicate that the immunodominant epitope in PLA2R is exclusively located in the CysR-FnII-CTLD1 region.

Cysteine oxidation impairs systemic glucocorticoid responsiveness in children with difficult-to-treat asthma.

BACKGROUND: The mechanisms underlying glucocorticoid responsiveness are largely unknown. Although redox regulation of the glucocorticoid receptor (GR) has been reported, it has not been studied in asthmatic patients. OBJECTIVE: We characterized systemic cysteine oxidation and its association with inflammatory and clinical features in healthy children and children with difficult-to-treat asthma. We hypothesized that cysteine oxidation would be associated with increased markers of oxidative stress and inflammation, increased features of asthma severity, decreased clinically defined glucocorticoid responsiveness, and impaired GR function. METHODS: PBMCs were collected from healthy children (n = 16) and children with asthma (n = 118) aged 6 to 17 years. Children with difficult-to-treat asthma underwent glucocorticoid responsiveness testing with intramuscular triamcinolone. Cysteine, cystine, and inflammatory chemokines and reactive oxygen species generation were quantified, and expression and activity of the GR were assessed. RESULTS: Cysteine oxidation was present in children with difficult-to-treat asthma and accompanied by increased reactive oxygen species generation and increased CCL3 and CXCL1 mRNA expression. Children with the greatest extent of cysteine oxidation had more features of asthma severity, including poorer symptom control, greater medication use, and less glucocorticoid responsiveness despite inhaled glucocorticoid therapy. Cysteine oxidation also modified the GR protein by decreasing available sulfhydryl groups and decreasing nuclear GR expression and activity. CONCLUSIONS: A highly oxidized cysteine redox state promotes a posttranslational modification of the GR that might inhibit its function. Given that cysteine oxidation is prevalent in children with difficult-to-treat asthma, the cysteine redox state might represent a potential therapeutic target for restoration of glucocorticoid responsiveness in this population.

Human-derived cathelicidin LL-37 directly activates mast cells to proinflammatory mediator synthesis and migratory response.

Cathelicidins, a family of antimicrobial peptides, are well known for their role in host defense, particularly against bacteria. Apart from direct killing of microbes through the membrane disruption, cathelicidins can also exert immunomodulatory effects on cells involved in inflammatory processes. Considering the important role of mast cells in inflammation, the aim of this study was to determine whether LL-37, human-derived cathelicidin, can induce mast cell activation. We have observed that LL-37 directly stimulates mast cell to degranulation and production of some proinflammatory cytokines, but fails to induce cysteinyl leukotriene generation and release. We have also documented that LL-37 acts as a strong mast cell chemoattractant. In intracellular signaling in mast cells activated by LL-37 participates PLC/A2 and, in part, MAPKs, and PI3K. In conclusion, our results indicate that cathelicidins may enhance antibacterial inflammatory response via attracting mast cell to pathogen entry site and via induction of mast cell-derived mediator release.

Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.

Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

Cysteine racemization on IgG heavy and light chains.

Under basic pH conditions, the heavy chain 220-light chain 214 (H220-L214) disulfide bond, found in the flexible hinge region of an IgG1, can convert to a thioether. Similar conditions also result in racemization of the H220 cysteine. Here, we report that racemization occurs on both H220 and L214 on an IgG1 with a λ light chain (IgG1λ) but almost entirely on H220 of an IgGl with a κ light chain (IgG1κ) under similar conditions. Likewise, racemization was detected at significant levels on H220 and L214 on endogenous human IgG1λ but only at the H220 position on IgG1κ. Low but measurable levels of D-cysteines were found on IgG2 cysteines in the hinge region, both with monoclonal antibodies incubated under basic pH conditions and on antibodies isolated from human serum. A simplified reaction mechanism involving reversible ß-elimination on the cysteine is presented that accounts for both base-catalyzed racemization and thioether formation at the hinge disulfide.

Modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevent cross-linked multimer formation and potency loss.

BACKGROUND: Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form. RESULTS: The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25 °C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach. CONCLUSION: rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.