Epitope Competition Research Articles

Abstract 1535: A novel ELISA Kit:highly accuracy and sensitivity for GDF-15 detection

Abstract Growth/differentiation factor 15 (GDF-15) is a promising drug target and biomarker for cancer early screening or immunotherapy. Under normal conditions, GDF-15 is expressed in low concentrations in most organs. Increased protein levels are associated with disease states such as tissue hypoxia, inflammation, acute injury and oxidative stress. The development of GDF-15 detection kit can help pharmaceutical companies in the early stage of drug development. It can also facilitate the early screening of related diseases. Hence, we developed a novel GDF-15 detected ELISA kit based on double-antibody sandwich ELISA. Our kit has ultra-high sensitivity and specificity and the concentration of GDF-15 can be detected accurately in complex biological samples such as serum, plasma or urine. First of all, mice were immunized by GDF-15. After detecting the antibody titers in sera of the immunized mice, hybridoma technologies were used to produce GDF-15 monoclonal antibodies. 57 antibodies were analyzed by epitope competition, ELISA binding and KD binding detection. The specificity of pairwise candidates was screened to obtain optimal coating and detecting antibody pair. The detecting antibody was labeled with biotin which can be conjugated with streptavidin labeled HRP. Biotin-streptavidin labeling detection method can achieve signal amplification which offers an improved detection sensitivity. Our GDF-15 detection kit has better property in sensitivity and specificity. The detection range is from 10 pg/mL to 10000 pg/mL. LOD is 5 pg/mL and LOQ is 10 pg/mL. These parameters are better than many commercial test kits. Moreover, the concentration of GDF-15 can be detected accurately in complex clinical samples. We anticipate that this GDF-15 detection kit can be applied not only for companion diagnosis, but also for related drug development. Citation Format: Meng Liang, Feng He, Meng Zhang, Yinsong Ye, Shukun Chen, Jinying Ning, Feng Hao. A novel ELISA Kit:highly accuracy and sensitivity for GDF-15 detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1535.

Engineering a High-Affinity Anti-Methoxy Poly(ethylene glycol) (mPEG) Antibody for Sensitive Immunosensing of mPEGylated Therapeutics and mPEG Molecules.

Sensitive quantification of methoxy poly(ethylene glycol) (mPEG)-conjugated therapeutics for pharmacokinetic determination is critical for mPEGylated drug development. However, sensitive measurement of low-molecular-weight (lmw) mPEG compounds remains challenging due to epitope competition between backbone-specific anti-PEG antibodies. Here, we engineered a high-affinity methoxy-specific anti-mPEG antibody for sensitive quantification of free mPEG molecules and mPEGylated therapeutics. The affinity-enhanced h15-2Y antibody variant shows a 10.3-fold increase in mPEG-binding activity compared to parental h15-2b. h15-2Y-based sandwich ELISA can effectively quantify lmw mPEG5K and high-molecular-weight (hmw) mPEG20K at concentrations as low as 3.4 and 5.1 ng mL-1, respectively. Moreover, lmw mPEG compounds (560, 750, 1000, and 2000 Da) can be efficiently quantified via h15-2Y-based competitive ELISA with detection limits at nanomolar levels. This study provides a promising approach for application in the quantitative analysis of the various sizes of mPEG molecules to accelerate the timeline of mPEG-conjugated drug development.

A broadly neutralizing humanized ACE2-targeting antibody against SARS-CoV-2 variants

The successive emergences and accelerating spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages and evolved resistance to some ongoing clinical therapeutics increase the risks associated with the coronavirus disease 2019 (COVID-19) pandemic. An urgent intervention for broadly effective therapies to limit the morbidity and mortality of COVID-19 and future transmission events from SARS-related coronaviruses (SARSr-CoVs) is needed. Here, we isolate and humanize an angiotensin-converting enzyme-2 (ACE2)-blocking monoclonal antibody (MAb), named h11B11, which exhibits potent inhibitory activity against SARS-CoV and circulating global SARS-CoV-2 lineages. When administered therapeutically or prophylactically in the hACE2 mouse model, h11B11 alleviates and prevents SARS-CoV-2 replication and virus-induced pathological syndromes. No significant changes in blood pressure and hematology chemistry toxicology were observed after injections of multiple high dosages of h11B11 in cynomolgus monkeys. Analysis of the structures of the h11B11/ACE2 and receptor-binding domain (RBD)/ACE2 complexes shows hindrance and epitope competition of the MAb and RBD for the receptor. Together, these results suggest h11B11 as a potential therapeutic countermeasure against SARS-CoV, SARS-CoV-2, and escape variants.

Novel GLP-1/anti-apolipoprotein B bifunctional fusion protein alleviates diabetes and diabetic complications in combination with low-intensity ultrasound

AimsTo engineer and screen a novel GLP-1/anti-apolipoprotein B (apoB) bifunctional fusion protein with therapeutic potential on alleviating diabetes and diabetic complication in combination with low-intensity ultrasound. Main methodsAnti-apoB antibodies were screened by phage display technology and further fused to mutated GLP-1 (7–37) via light or heavy fusion to generate bifunctional fusion protein (termed aBG). The optimal design of aBG fusion protein was further confirmed by in vitro epitope competition assay and cAMP accumulation assay. Subsequently, chronic study in DIO mice were subjected to assess the long-term efficacy of screened fusion protein. Key findingsThe selected GLP-1/anti-apoB fusion protein, aBG-8, exerted either the highest binding affinities for GLP-1R and apoB, or the greatest LDL-C uptake capacity and GLP-1R activation activity. After 60-day treatment in DIO mice, aBG-8 was proved to exert the promising improvement on hyperglycemia, hyperlipidemia, and obesity in DIO mice. Furthermore, combined therapy of aBG-8 and low-intensity ultrasound could accelerate skin wound closure in diabetic mice. SignificanceA novel long-lasting bifunctional fusion molecule, aBG-8, was designed with the enormous potential on alleviating diabetes and diabetic complications in combination with low-intensity ultrasound.

Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay

Batch release testing for human and veterinary tetanus vaccines still relies heavily on methods that involve animals, particularly for potency testing. The quantity and quality of tetanus antigen present in these products is of utmost importance for product safety and clinical effect. Immunochemical methods that measure consistency of antigen content and quality, potentially as an indicator of potency, could be a better choice and negate the need for an in vivo potency test. These immunochemical methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against tetanus with a view to select antibodies that can be used for development of an in vitro potency immunoassay. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined using an in-house cell-based neutralisation assay to support prior in vivo potency data that was available for some, but not all, of the antibodies. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of “superiority” of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Characterisation of diphtheria monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay

Immunoassays are used for routine potency assessment of several vaccines, in some cases having been specifically developed as alternatives to in vivo potency tests. These methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against diphtheria with a view to select antibodies that can be used for development of an in vitro potency immunoassay for diphtheria vaccines. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined by a cell-based toxin neutralisation test and diphtheria toxin-domain recognition was determined by Western blotting. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of “superiority” of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Characterization and preclinical evaluation of a protease activated receptor 2 (PAR2) monoclonal antibody as a preventive therapy for migraine.

Migraine pain is thought to result from activation of meningeal nociceptors that might involve dural mast cell degranulation and release of proteases and pronociceptive mediators. Tryptase, the most abundant dural mast cell protease, has been demonstrated to stimulate dural mast cells, as well as trigeminal nociceptors by activating the protease activated receptor 2. Mast cell or neuronal protease activated receptors 2 may therefore represent a novel target for migraine treatment. In this study, we characterized and evaluated a novel protease activated receptor 2 monoclonal antibody as a preventive anti-migraine pain therapy in preclinical models. Flow cytometry, immunocytochemistry, calcium imaging, Homogeneous Time Resolved Technology (HTRF) epitope competition assay and serum pharmacokinetic (PK) assay in rats were performed to confirm the activity, specificity and in vivo stability of PAR650097, a novel anti- protease activated receptor 2 monoclonal antibody. In vivo assessment was performed in female C57BL/6J mice by evaluation of PAR650097 in preventing cutaneous allodynia elicited by (a) supradural injection of the protease activated receptor 2 agonist, Ser-Leu-Ile-Gly-Arg-Leu-amide trifluoroacetate (SLIGRL), or calcitonin gene-related (CGRP) peptide, and (b) induction of latent sensitization by priming with three daily episodes of restraint stress followed by challenge with a subthreshold inhalational exposure to umbellulone (UMB), a transient receptor potential ankyrin 1 (TRPA1) agonist. PAR650097 was administered as a pretreatment prior to the first restraint stress, umbellulone exposure, SLIGRL or calcitonin gene-related peptide injection. Additionally, fremanezumab, a calcitonin gene-related peptide antibody was administered as pre-treatment prior to supradural administration of calcitonin gene-related peptide or SLIGRL. In vitro, PAR650097 demonstrated rapid interaction with protease activated receptor 2, enabling it to fully inhibit protease-induced protease activated receptor 2 activation, in human and mouse cells, with high potency. Furthermore, PAR650097 was highly selective for protease activated receptor 2, demonstrating no affinity for protease activated receptor 1 protein and no functional effect on the activation of cellular protease activated receptor 1 with thrombin. In addition, PAR650097 had an acceptable PK profile, compatible with testing the effects of selective protease activated receptor 2 inhibition in vivo. In vivo, PAR650097 blocked cutaneous allodynia induced by either supradural SLIGRL or calcitonin gene-related peptide. Fremanezumab abolished cutaneous allodynia induced by supradural CGRP, and partially attenuated cutaneous allodynia induced by SLIGRL. Administration of PAR650097, before the first restraint stress episode, did not prevent the acute stress-induced cutaneous allodynia or restraint stress priming revealed by cutaneous allodynia induced by inhalational umbellulone. In contrast, PAR650097 prevented expression of cutaneous allodynia when given before the umbellulone challenge in restraint stress-primed animals. PAR650097 specifically inhibits endogenously expressed protease activated receptor 2 in human and mouse cells with high potency. This antibody has an acceptable PK profile in rodents and effectively blocked SLIGR-induced cutaneous allodynia. PAR650097 additionally prevented cutaneous allodynia induced by supradural calcitonin gene-related peptide, indicating that the protease activated receptor 2 receptor is a downstream consequence of calcitonin gene-related peptide actions. Fremanezumab effectively blocked calcitonin gene-related peptide-induced cutaneous allodynia and only partially reduced cutaneous allodynia induced by a protease activated receptor 2 activator, suggesting both calcitonin gene-related peptide-dependent and -independent mechanisms in promoting migraine pain. While PAR650097 did not prevent stress-induced cutaneous allodynia or priming, it effectively prevented cutaneous allodynia induced by a TRPA1 agonist in animals with latent sensitization. Activation of protease activated receptor 2, therefore, contributes to both calcitonin gene-related peptide-dependent and -independent mechanisms in promoting migraine-like pain. Therapeutic targeting of protease activated receptor 2 receptors may represent an anti-migraine pain strategy with a potentially broad efficacy profile.

Divergent memory responses driven by adenoviral vectors are impacted by epitope competition.

Adenoviral vectors induce robust epitope‐specific CD8+ T cell responses. Within the repertoire of responses generated both conventional memory evolution and the phenomenon of memory inflation are seen. The rules governing which epitopes inflate are not fully known, but may include a role for both antigen processing and competition. To investigate this, we looked at memory generated from vectors targeting the Gp33‐41 (KAVYNFATC/K9C) epitope from the gp of lymphocytic choriomeningitis virus (LCMV) in mice. This well‐described epitope has both the Gp33‐41 and Gp34‐41 epitopes embedded within it. Vaccination with a full‐length gp or a minigene Ad‐Gp33/K9C vector‐induced conventional memory responses against the immunodominant Gp33/K9C epitope but a strong inflationary response against the Gp34/A8C epitope. These responses showed sustained in vivo function, with complete protection against LCMV infectious challenge. Given the unexpected competition between epitopes seen in the minigene model, we further tested epitope competition using the full‐length Ad‐LacZ (β‐galactosidase) model. Generation of an Ad‐LacZ vector with a single amino acid disruption of the inflationary β‐gal96‐103/D8V epitope transformed the β‐gal497‐504/I8V epitope from conventional to inflationary memory. This work collectively demonstrates the importance of epitope competition within adenoviral vector inserts and is of relevance to future studies using adenoviral vectored immunogens.

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway.

The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease.

Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays

Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets.

Isolation of broadly neutralizing antibodies from single human memory B cell against human influenza A viruses (TECH2P.922)

Abstract During the past five years, broadly neutralizing antibodies (BnAbs) represent promising prophylactic and therapeutic treatments against influenza. Here, we have developed a novel method to isolate broadly neutralizing antibodies against influenza A virus from human memory B cells. Antigen-specific memory B cells were isolated from human PBMCs using tetramerized H3 (A/Brisbane/10/2007) hemagglutinin (HA) trimers. The H3-reactive single memory B cells were sorted into plates and stimulated in vitro. More than 40% sorted B cells produced on average 200 ng/ml IgG in the supernatant after 14 days. Supernatants from the expanded B cells were measured for their heterosubtypic binding specificity and neutralizing activity by MSD or a highly sensitive neutralization assay. The selective clones were recovered by single cell RT-PCR. Through screening 2688 memory B clones from 7 individuals, 11% clonable memory B cells were reactive with H3 hemagglutinin. Among them, H3/H7, H3/H7/H1 and H3/H7/H1/influenza B heterosubtypic binding population were 16%, 6.9% and 0.35%, respectively. A new broadly neutralizing Ab, 3I14 encoded by the IGHV3-30 germline gene, was further characterized and shown to possess cross-reactive binding and neutralization activity against both group 1 and group 2 influenza A viruses. Epitope mapping and competition assay revealed a highly conserved epitope located in the HA stalk. Passive transfer of this antibody also conferred in vivo protection.

Isolation of high-affinity, neutralizing anti-idiotype antibodies by phage and ribosome display for application in immunogenicity and pharmacokinetic analyses

Anti-idiotype antibodies against a therapeutic antibody are key reagents for the development of immunogenicity and pharmacokinetic (PK) assays during pre-clinical and clinical development. Here we have used a combination of phage and ribosome display to isolate a panel of monoclonal anti-idiotype antibodies with sub-nanomolar affinity and high specificity to a human anti-IgE monoclonal antibody. Anti-idiotype antibodies were enriched from scFv libraries using phage display, and a biochemical epitope competition assay was used to identify anti-idiotypes which neutralized IgE binding, which was essential for the intended use of the anti-idiotypes as positive controls in neutralizing anti-drug antibody (Nab) assays. The phage display-derived anti-idiotype antibodies were rapidly affinity-matured using a random point mutagenesis approach in ribosome display. Ten anti-idiotype antibodies with improved neutralizing activity relative to the parent antibodies displayed sub-nanomolar affinity for the anti-IgE antibody, representing up to 20-fold improvements in affinity from just two rounds of affinity-based selection. The optimized anti-idiotype antibodies retained the specificity of the parent antibodies, and importantly, were fit for purpose for use in PK and anti-drug antibody (ADA) assays. The approach we describe here for generation of anti-idiotype antibodies to an anti-IgE antibody is generically applicable for the rapid isolation and affinity maturation of anti-idiotype antibodies to any antibody-based drug candidate.

Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen

IgE antibodies specific for the major birch pollen allergen frequently cross-react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen (BP-SIT) induces IgG4 antibodies that inhibit IgE binding to Bet v 1. However, information on cross-reactivity of BP-SIT-induced Bet v 1-specific IgG4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross-reactivity, and IgE-blocking activity of Bet v 1-specific IgG4 antibodies emerging during conventional BP-SIT and whether IgG4-epitopes overlapped with IgE epitopes. IgE and IgG4 levels specific for Bet v 1, Mal d 1, and Cor a 1 were determined in 42 birch pollen-allergic patients before and during BP-SIT. Inhibition of IgE binding was studied by IgE-facilitated antigen-binding assays and basophil activation tests. Furthermore, inhibition of IgE-mediated activation of food allergen-reactive Bet v 1-specific T-cell lines was assessed. Competitive immunoscreening of phage-displayed peptides was applied to select mimotopes recognized by IgE and IgG4 antibodies, respectively. The resulting mimotopes were mapped on the surface of the 3D structure of the allergens using a computer-based algorithm. BP-SIT significantly increased Bet v 1- and food allergen-reactive IgG4 antibodies. In parallel, allergen-specific IgE levels decreased significantly. Sera containing food allergen-reactive IgG4 antibodies inhibited IgE binding, basophil activation, and IgE-mediated food allergen-induced T-cell proliferation. Predicted IgE and IgG4 epitopes on all allergens showed high overlap. Our results indicate that BP-SIT may induce Bet v 1-specific IgG4 antibodies that cross-react with related food allergens and inhibit IgE binding by epitope competition.

Epitope competition weakens specific T cell response to subdominant epitopes

Objective To elucidate the influences of epitope competition on the frequency and average intensity of specific T cell response.Methods C57BL/6 mice were immunized with either single epitope DNA vaccines (pSV-gag92 or pSV-env203) or fusion gene DNA vaccine (pSV-gag/env).Gag92and Env203 epitope-specific CD8 T cell responses were analyzed by intracellular cytokine staining assay.Results Gag92-specific IFN-γ+CD8 T cells that were induced by pSV-gag92 accounted for 0.415 00％ ±0.045 88％ of the total CD8 T cells,which was much more than that induced by pSV-gag/env of 0.058 67％ + 0.019 64％.Moreover,the mean fluorescence intensity of Gag92-specific TNF-α-IFN-γ+CD8 T cells (296.70+14.08) elicited by pSV-gag/env was significantly lower than that of Env203-specific TNF-α-IFN-γ+CD8 T cells (818.00+49.34).Conclusion Epitope competition could significantly decrease both the frequency and the average intensity of specific T cell response to subdominant epitopes. Key words: Single epitope DNA vaccine; Epitope competition; T cell response

Immunoreactivity of Two Novel Monoclonal Antibodies Against Human Inducible Co-stimulator Ligand

Human inducible co-stimulator ligand (GL50, CD275), also known as B7-H2 or ICOSL, is a positive co-stimulatory molecule of B7/CD28 superfamily, which plays a critical role in immune response. Here we generated two novel mouse anti-human GL50 monoclonal antibodies (MAbs) using classical hybridoma technology. The two MAbs (clones 2B4 and 4D11) were IgG1 (κ) and IgG2a (κ), respectively, and bound specifically to human GL50. Epitope competition assay showed that 2B4 and 4D11 bind to the same epitope of GL50, which is not recognized by the commercially available GL50 MAb (9F-8A4). Functionally, the two MAbs act as a blocker of T cell proliferation. Taken together, as useful tools, these two antibodies might be of great value for further exploration of the immune identification and function of GL50.

Three-dimensional Model of Human Platelet Integrin αIIbβ3 in Solution Obtained by Small Angle Neutron Scattering

Integrin alphaIIbbeta3 is the major membrane protein and adhesion receptor at the surface of blood platelets, which after activation plays a key role in platelet plug formation in hemostasis and thrombosis. Small angle neutron scattering (SANS) and shape reconstruction algorithms allowed formation of a low resolution three-dimensional model of whole alphaIIb beta3 in Ca(2+)/detergent solutions. Model projections after 90 degrees rotation along its long axis show an elongated and "arched" form (135 degrees) not observed before and a "handgun" form. This 20-nm-long structure is well defined, despite alphaIIb beta3 multidomain nature and expected segmental flexibility, with the largest region at the top, followed by two narrower and smaller regions at the bottom. Docking of this SANS envelope into the high resolution structure of alphaIIb beta3, reconstructed from crystallographic and NMR data, shows that the solution structure is less constrained, allows tentative assignment of the disposition of the alphaIIb and beta3 subunits and their domains within the model, and points out the structural analogies and differences of the SANS model with the crystallographic models of the recombinant ectodomains of alphaIIb beta3 and alphaV beta3 and with the cryo-electron microscopy model of whole alphaIIb beta3. The ectodomain is in the bent configuration at the top of the model, where alphaIIb and beta3 occupy the concave and convex sides, respectively, at the arched projection, with their bent knees at its apex. It follows the narrower transmembrane region and the cytoplasmic domains at the bottom end. AlphaIIb beta3 aggregated in Mn(2+)/detergent solutions, which impeded to get its SANS model.

Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein

Recent epidemics of West Nile virus (WNV) around the world have been associated with significant rates of mortality and morbidity in humans. To develop standard WNV diagnostic tools that can differentiate WNV from Japanese encephalitis virus (JEV), four monoclonal antibodies (MAbs) specific to WNV envelope (E) protein were produced and characterized by isotyping, reactivity with denatured and native antigens, affinity assay, immunofluorescence assay (IFA), and epitope competition, as well as cross-reactivity with JEV. Two of the MAbs (6A11 and 4B3) showed stronger reactivity with E protein than the others (2F5 and 6H7) in Western blot analysis. 4B3 could bind with denatured antigen, as well as native antigens in indirect ELISA, flow cytometry analysis, and IFA; whereas 2F5 showed highest affinity with native antigen. 4B3 and 2F5 were therefore used to establish an antigen capture-ELISA (AC-ELISA) detection system. The sensitivity of this AC-ELISA was 3.95 TCID 50/0.1 ml for WNV-infected cell culture supernatant. Notably, these MAbs showed no cross-reactivity with JEV, which suggests that they are useful for further development of highly sensitive, easy handling, and less time-consuming detection kits/tools in WNV surveillance in areas where JEV is epidemic.

Naive Precursor Frequencies and MHC Binding Rather Than the Degree of Epitope Diversity Shape CD8+ T Cell Immunodominance

The primary CD8(+) T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8(+) T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2K(b), resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F(1) hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8(+) T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33-41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8(+) T cell responses.

Relative dominance of epitope-specific CD8+ T cell responses in an F1 hybrid mouse model of respiratory syncytial virus infection

CD8+ cytotoxic T lymphocytes are key effectors of adaptive immunity for the control of virus infections. Epitope-specific responses are hierarchical and the rules for dominance are not well defined. Here we show that the H2-K d-restricted RSV M2 82–90 (K dM2 82–90) epitope dominates the H2-D b-restricted RSV M 187–195 (D bM 187–195) epitope and influences epitope-specific effector function in the acute and memory phases of the immune response to primary RSV infection in H-2 b/d hybrid mice. The hybrid mouse model provides a system to define rules of epitope hierarchy and better understand how antigen presentation and epitope competition affect the phenotype of effector and memory T cells.

The expression analysis of ICOS‐L on activated T cells and immature dendritic cells as well as malignant B cells and Grave’s‐disease‐derived thyroid tissues by two novel mAbs against human ICOS‐L

ICOS-L, a newly identified member of B7 superfamily, plays an important role in immune responses. In this article, we report on two novel mouse anti-human ICOS-L monoclonal antibodies (mAbs) named as 11C4 and 12B11, whose specificities were verified by methods of flow cytometry, western blotting, and epitope competition assay. The two mAbs bound to distinct ICOS-L epitopes on B cells. Interestingly, mAb 11C4 could well recognize ICOS-L molecule on activated T cells and Jurkat cell lines, which is different from commercial anti-ICOS-L mAb (clone number MIH12) and the other mAb 12B11. In addition, we found that the expression of ICOS-L molecule was only detected on the surface of immature monocyte-derived dendritic cells (Mo-DCs) and was sharply decreased after induction of mature Mo-DCs activated by tumor necrosis factor-alpha or CD40. Furthermore, we showed that 11C4 could effectively suppress the maturation of Mo-DCs in vitro as evidenced by the low expression of CD80, CD86, CD83, and human leukocyte antigen-DR, which suggested that ICOS-L may be involved in the maturation of Mo-DCs. Using immunohistochemistry staining with mAb 11C4, the expression of ICOS-L was found in B lymphoma tissues and thyroid tissues from the Grave's disease but not in thyroid adenoma and normal thyroid tissues.