Low-affinity Antibodies Research Articles

COMMON MECHANISMS OF SPECIFIC HUMORAL IMMUNE RESPONSE’ SHAPING AND SUSTAINING BY THE EXAMPLE OF IMMUNE RESPONSE TO MEASLES AND RUBELLA VIRUSES

Abstract. T follicular helper cells (Tfh) are a CD4+ Th cell subset promoted the cognate control of antigen-specific B cell immunity. Upon first contact with antigen-primed B cells, Tfh can support either extrafollicularly differentiation into short-lived plasma cells (PC) or enter follicles to form germinal centers (GC). Signaling lymphocytic activation molecule (SLAM) interaction between Tfh and activated B-cells is essential for GC development. Within GC, Tfh regulates the fate of antigen-specific GC B cells expressing high-affinity B cell receptors to develop memory B cell (Bm) or long-lived PC. Short-lived PC produce low-affinity IgM and IgG3 early antibodies. Both Bm and long-lived PC have high-affinity class-switched IgA and IgG, predominantly IgG1 antibodies. Measles virus uses human SLAM-molecule as a cellular receptor. SLAM is expressed on dendritic cells and activated B and T-cells. This is an important regulator of the isotype switching and antibody affinity maturation, especially IgG3-IgG1 switching. Development of long-term humoral immunity, charac terized by the formation of high-affinity predominantly IgG1 antibodies, is a critical component of protective immunity to pathogens and the major goal of vaccination. However, the mechanisms involved in the shaping and sustaining of long-term humoral immunity remain poorly understood.

Affinity enhancement of antibodies: how low-affinity antibodies produced early in immune responses are followed by high-affinity antibodies later and in memory B-cell responses.

The antibodies produced initially in response to most antigens are high molecular weight (MW) immunoglobulins (IgM) with low affinity for the antigen, while the antibodies produced later are lower MW classes (e.g., IgG and IgA) with, on average, orders of magnitude higher affinity for that antigen. These changes, often termed affinity maturation, take place largely in small B-cell clusters (germinal center; GC) in lymphoid tissues in which proliferating antigen-stimulated B cells express the highly mutagenic cytidine deaminase that mediates immunoglobulin class-switching and sequence diversification of the immunoglobulin variable domains of antigen-binding receptors on B cells (BCR). Of the large library of BCR-mutated B cells thus rapidly generated, a small minority with affinity-enhancing mutations are selected to survive and differentiate into long-lived antibody-secreting plasma cells and memory B cells. BCRs are also endocytic receptors; they internalize and cleave BCR-bound antigen, yielding peptide-MHC complexes that are recognized by follicular helper T cells. Imperfect correlation between BCR affinity for antigen and cognate T-cell engagement may account for the increasing affinity heterogeneity that accompanies the increasing average affinity of antibodies. Conservation of mechanisms underlying mutation and selection of high-affinity antibodies over the ≈200 million years of evolution separating bird and mammal lineages points to the crucial role of antibody affinity enhancement in adaptive immunity.

51. Myasthenia gravis – Identifying risk factors leading to a more severe course of the disease in a cohort of 45 patients

Introduction Myasthenia gravis is an autoimmune disease that causes neuromuscular transmission disorder manifested by muscle weakness and fatigue, in particular predilection muscle groups. Most cases are mediated by T lymphocyte-dependent polyclonal antibody directed against acetylcholine receptors. Approximately 10–15% of cases are etiological associated with thymoma. AChR antibodies in serum are detectable in 80–90% of patients. Of the remaining seronegative MG forms are most commonly detected antibodies to MuSK (30–70%) and especially in thymoma antistriatal, antititin and antiryanodin antibodies. Some cases are associated with low affinity antibodies against AChR. Diagnostic and therapeutic procedures in medicine has recently developed in a fast rate. The goal of our observations was to assess how the reflected developments in the area of the approach to patients with this diagnosis affected our department and to try identifying risk factors leading to a more severe course of the disease. Patients and methods The study included 45 patients (27 men, 18 women) followed in our department from 2009 to the present. The diagnosis was established on the basis of history, clinical examination and EMG findings. In this retrospective study was used material available in our archives. Results We present a cohort of patients with myasthenia gravis diagnosed and treated in our department. In our patients the EMG findings correspond with the severity of the disorder. We found that the higher titers of the antibodies against AChR led to a more severe course of the disease. Conclusion Despite advances in diagnosis and treatment of myasthenia, this still represents a challenge for physicians due to the relatively variable course and clinical findings. The aim of our study was to refer to this variability and point out not full understanding the pathogenesis of myasthenia. We determined risk factors leading to a more serious course of the disease in our patients.

Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants

Antibodies to transferrin receptor (TfR) have potential use for therapeutic entry into the brain. We have shown that bispecific antibodies against TfR and β-secretase (BACE1 [β-amyloid cleaving enzyme-1]) traverse the blood-brain barrier (BBB) and effectively reduce brain amyloid β levels. We found that optimizing anti-TfR affinity improves brain exposure and BACE1 inhibition. Here we probe the cellular basis of this improvement and explore whether TfR antibody affinity alters the intracellular trafficking of TfR. Comparing high- and low-affinity TfR bispecific antibodies in vivo, we found that high-affinity binding to TfR caused a dose-dependent reduction of brain TfR levels. In vitro live imaging and colocalization experiments revealed that high-affinity TfR bispecific antibodies facilitated the trafficking of TfR to lysosomes and thus induced the degradation of TfR, an observation which was further confirmed in vivo. Importantly, high-affinity anti-TfR dosing induced reductions in brain TfR levels, which significantly decreased brain exposure to a second dose of low-affinity anti-TfR bispecific. Thus, high-affinity anti-TfR alters TfR trafficking, which dramatically impacts the capacity for TfR to mediate BBB transcytosis.

Dynamic Quantification of Antigen Molecules on Cells with Flow Cytometry

Flow cytometry is a powerful tool for the quantification of target molecules on cells. Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium. A calibration procedure, with carefully prepared reagents is needed to convert the intensity of the fluorescence signal to the number of target antigens. The new method presented here may be more robust and has application to lower affinity antibodies and non-equilibrium labeling conditions.We have developed a novel kinetic approach to the antigen quantification problem. Instead of using a static calibration system, we analyzed fluorescence profile dynamics measured by flow cytometry in the general case of reversible antibody-antigen binding. The model considers the full fluorescence profile on a distribution of cells. Experimental data obtained with the LSRII cytometer were fitted by the diffusion-reaction mathematical model using the Levenberg-Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the reaction rate constants and the number of target antigens per cell. Results were compared with the QuantiBRITE calibration system, which uses calibration beads with known amounts of covalently bound PE and unimolar (1:1) anti-Ag-PE reagents bound to capture beads or cells under saturating staining conditions.Our approach is independent of specially prepared calibration beads and antibody reagents and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions.

Chimeric DNA Vaccines: An Effective Way to Overcome Immune Tolerance.

The fact that cancer immunotherapy is considered to be a safe and successful weapon for use in combination with surgery, radiation, and chemotherapy treatments means that it has recently been chosen as Breakthrough of the Year 2013 by Science editors. Anticancer vaccines have been extensively tested, in this field, both in preclinical cancer models and in the clinic. However, tumor-associated antigens (TAAs) are often self-tolerated molecules and cancer patients suffer from strong immunosuppressive effects, meaning that the triggering of an effective anti-tumor immune response is difficult. One possible means to overcome immunological tolerance to self-TAAs is of course the use of vaccines that code for xenogeneic proteins. However, a low-affinity antibody response against the self-homologous protein expressed by cancer cells is generally induced by xenovaccination. This issue becomes extremely limiting when working with tumors in which the contribution of the humoral rather than the cellular immune response is required if tumor growth is to be hampered. A possible way to avoid this problem is to use hybrid vaccines which code for chimeric proteins that include both homologous and xenogeneic moieties. In fact, a superior protective anti-tumor immune response against ErbB2+ transplantable and autochthonous mammary tumors was observed over plasmids that coded for the fully rat or fully human proteins when hybrid plasmids that coded for chimeric rat/human ErbB2 protein were tested in ErbB2 transgenic mice. In principle, these findings may become the basis for a new rational means of designing effective vaccines against TAAs.

Low-affinity IgM antibodies lacking somatic hypermutations are produced in the secondary response of C57BL/6 mice to (4-hydroxy-3-nitrophenyl)acetyl hapten

Class-switched memory B cells, which are generated through the processes of somatic hypermutation (SHM) and affinity-based selection in germinal centers, contribute to the production of affinity-matured IgG antibodies in the secondary immune response. However, changes in the affinity of IgM antibodies during the immune response have not yet been studied, although IgM(+) memory B cells have been shown to be generated. In order to understand the relationship between IgM affinity and the recall immune response, we prepared hybridomas producing anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) IgM antibodies from C57BL/6 mice and from activation-induced cytidine deaminase (AID)-deficient mice. Binding analysis by ELISA showed that mAbs obtained from the secondary immune response contained IgM mAbs with affinity lower than the affinity of mAbs obtained from the primary response. By analyzing sequences of the IgM genes of hybridomas and plasma cells, we found many unmutated VH genes. VH genes that had neither tyrosine nor glycine at position 95 were frequent. The repertoire change may correlate with the lower affinity of IgM antibodies in the secondary response. The sequence and affinity changes in IgM antibodies were shown to be independent of SHM by analyzing hybridomas from AID-deficient mice. A functional assay revealed a reciprocal relationship between affinity and complement-dependent hemolytic activity toward NP-conjugated sheep RBCs; IgM antibodies with lower affinities had higher hemolytic activity. These findings indicate that lower affinity IgM antibodies with enhanced complement activation function are produced in the secondary immune response.

Allergy's Achilles' heel?

Allergic reactions to otherwise innocuous substances involve a complex interplay of molecular interactions—some strong, some weak. This study reveals a key role for low-affinity antibodies and thus a possible point of weakness that may be exploited for therapeutic intervention.

IgM autoantibodies: Roquin and Bob1ng to a different tune

Pathogenic autoantibodies directed against ubiquitous self-antigens such as immunoglobulin-G (IgG) anti-double-stranded DNA (anti-dsDNA) antibodies in systemic lupus erythematosus are produced by plasma cells that have undergone immunoglobulin isotype switching and affinity maturation in the germinal centre (GC).1 These autoantibodies form circulating immune complexes that are often deposited in the skin, kidneys and joints where they activate complement to cause tissue damage. In contrast, IgM (and IgA) natural autoantibodies are produced in the steady-state from birth by plasma cells derived from B1 cells, and are ‘hardwired’ to be polyreactive, low-affinity antibodies with relatively low pathogenic potential.2, 3 Nevertheless, IgM autoantibodies bound to antigen are still capable of activating the classical complement pathway and causing diseases such as autoimmune haemolytic anaemias due to cold agglutinins. In this issue, Corcoran and coworkers4 remind us of this by showing that sanroque mice harbouring mutations in Rc3h1/Roquin1 develop a GC-independent IgM-mediated autoimmune disease when crossed with mice deficient for the transcription factor Obf1/Ocab/Bob1 (Figure 1). These IgM anti-dsDNA antibodies are deposited in the kidneys where they cause glomerulonephritis. This remarkable observation adds to the growing list of insights into the regulation of autoantibody responses in the sanroque ENU-mutant mouse model, which is characterized by excessive follicular T helper (Tfh) cell activity and spontaneous GC development.5 Previous studies using this model have focussed on the role of specific genes and cellular interactions involved in the production of GC-dependent autoantibodies, such as the Bcl6, Sh2d1a, Cd28 and Icos.6, 7 However, the current study neatly turns this on its head by showing that sanroque mice develop an even more severe fatal autoimmunity when Obf1 is also deficient and they are unable to form GCs and secrete IgG autoantibodies. This unexpected finding is all the more intriguing given that Obf1 deficiency by itself does not cause autoimmunity, and can also prevent the GC-dependent development of lupus-like autoimmune diseases in aged Ailos−/− 8 and MRL-lpr mice.9

Hepatitis B Virus Exposure During Childhood in Cameroon, Central African Republic and Senegal After the Integration of HBV Vaccine in the Expanded Program on Immunization

More than 2 billion people worldwide have been exposed to hepatitis B virus (HBV). To prevent these infections, Senegal and Cameroon integrated the HBV vaccine into their Expanded Program on Immunization (EPI) in 2005, as did the Central African Republic (CAR) in 2008. We evaluated the prevalence of HBV exposure and infection after the integration of the HBV vaccine in the EPI. An observational cross-sectional study was conducted among the hospitalized children 3 months to 6 years of age in Cameroon, CAR and Senegal. Plasma was collected for the detection of anti-HBc, anti-HBs and hepatitis B surface antigen in children with anti-HBc and anti-HBs. Between April 2009 and May 2010, 1783 children were enrolled, 19.4% of whom were anti-HBc positive. The percentage of children with anti-HBc was 44.4% among the children younger than 6 months, decreasing after 6 months to reach 18.8% at 12 months. This decline was followed by a rapid increase in anti-HBc positivity rate in CAR observed as early as 12 months of age compared with Cameroon and Senegal, where the anti-HBc increased between 18 and 36 months of age, respectively. The prevalence of hepatitis B surface antigen-positive children was significantly higher in CAR than that in Cameroon and Senegal (5.1% versus 0.7% and 0.2%; P < 0.001). Socioeconomic level, age and country were factors associated with the presence of anti-HBc. Passive transfer of anti-HBc maternal antibodies versus HBV exposure could be differentiated as early as 12 months of age. The low prevalence of anti-HBc and hepatitis B surface antigen among children born after the integration of HBV vaccine in the EPI in Cameroon and Senegal suggests a positive impact of HBV vaccination.

ChemInform Abstract: Designing a New Antifungal Glycoconjugate Vaccine

AbstractReview: 93 refs.

Designing a new antifungal glycoconjugate vaccine

Bacterial capsular polysaccharides have been used as effective vaccines for adults but infants and seniors respond poorly to these immunogens because pure polysaccharides are unable to activate T-cells resulting in antibodies of low affinity and poor immunological memory. These deficiencies are addressed by conjugate vaccines composed of bacterial polysaccharide covalently attached to protein carriers such as tetanus or diphtheria toxoids. These vaccines activate T-cells and have been hugely effective in reducing the incidence of dangerous infectious diseases such as bacterial meningitis in infants and adults. The methods of conjugation often produce conjugate vaccines that contain polysaccharides with several cross links to one or more protein carrier molecules. Synthesis of large oligosaccharides derivatized to achieve single site attachment to protein has established the minimal size of an oligosaccharide that is required to produce protective antibody. Several examples are described where such oligosaccharides range in size from 8 to 16 monosaccharides. A more limited set of examples show that protective antibodies may be elicited by conjugate vaccines composed of tri and tetrasaccharide epitopes. One example is the β-mannan present in the phosphomannan glycoprotein of Candida albicans. Reverse engineering a protective antibody that recognizes this oligosaccharide revealed that a disaccharide was the minimal epitope. Further epitope mapping by functional group modification established that the internal mannose residue of a disaccharide was involved in the most important antibody-disaccharide interactions. NMR binding studies in combination with homology modeling of the bound β-mannan antigen suggested an optimum oligosaccharide for inclusion in a conjugate vaccine. Several conjugate vaccines were developed to test these conclusions. Immunization of mice or rabbits with conjugates containing disaccharide or trisaccharide conjugated to immunogenic proteins followed by live challenge experiments showed that the vaccines reduced fungal burden. The results of these and other studies suggest new approaches to novel synthetic conjugate vaccines.

Development of Antibody Medicines by Bio-venture: Lesson from License Negotiations with Mega Pharmacies

The current method of antibody production is mainly the hybridoma method, in which mice are immunized with an excess amount of antigen for a short period to promote activation and proliferation of B-lymphocytes producing the antibodies of interest. Because of the excess antigen, those producing low-affinity antibodies are activated. In contrast, human blood B-lymphocytes are activated through natural immune reactions, such as the reaction to infection. B-lymphocytes are stimulated repeatedly with a small amount of antigen, and thus only those producing high-affinity antibodies are activated. Consequently, the lymphocytes producing the high-affinity antibodies are accumulated in human blood. Therefore, human lymphocytes are an excellent source of high-affinity antibodies. Evec, Inc. has established a unique method to produce high-affinity antibodies from human lymphocytes using Epstein-Barr virus (EBV), which induces the proliferation of B-lymphocytes. The method first induces the proliferation of B-lymphocytes from human blood using EBV, and then isolates those producing the antibodies of interest. The key features of the Evec technique are: 1) development of a lymphocyte library consisting of 150 donors' lymphocytes from which donors suited to develop the antibodies of interest can be selected in 4 days; and 2) development of a sorting method and cell microarray method for selecting lymphocyte clones producing the target antibodies. Licensing agreements have been concluded with European and Japanese pharmaceutical companies for two types of antibody. This paper describes Evec's antibody technology and experience in license negotiations with Mega Pharmacies.

Adjuvant dependence of APS pathology-related low-affinity antibodies during secondary immune response to tetanus toxoid in BALB/c mice

One of the established animal models for autoimmune disease antiphospholipid syndrome (APS) is TTd hyperimmunization of mice. Tetanus toxoid (TTd) and plasma protein β2GPI share structural homology so that immunization with TTd induces appearance of cross-reactive antibodies. In this paper, we have investigated the presence and dynamic of fluctuation of specific (anti-TTd) and auto (anti-β2GPI) antibodies induced in BALB/c mice during secondary immune response after TTd immunization with alhydrogel or glycerol as adjuvants. In addition, we followed the induced reproductive pathology as a sign of autoimmune outcome. We show undoubtedly adjuvant dependance of (1) level of induced anti-TTd IgG antibodies, (2) changes in levels of low-affinity anti-β2GPI IgG antibodies, and (3) change in fecundity and fertility during secondary immune response. These findings once more indicate the importance of chosen adjuvants used for successful immunization and eventual autoantibody outcome, this time associated with the processes involving low affinity, natural antibodies.

Stratification of Antibody-Positive Subjects by Antibody Level Reveals an Impact of Immunogenicity on Pharmacokinetics

The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic. The antibody responses displayed a wide range of relative concentrations (30 ng/mL to >13 μg/mL) and peaked at various times during the study. To evaluate the impact of immunogenicity on PK, AMG 317 concentration data were analyzed following stratification by dose group, time point, antibody status (positive or negative), and antibody level (relative concentration). With dose group as a stratifying variable, a moderate reduction in AMG 317 levels (<50%) was observed in antibody-positive subjects when compared to antibody-negative subjects, but the difference was not statistically significant in all dose groups. The most significant reduction in AMG 317 levels was revealed when antibody data was stratified by both time point and antibody level. In general, high ADA concentrations (>500 ng/mL) and later time points (week 12) were associated with significantly (up to 97%) lower trough AMG 317 concentrations. The use of quasi-quantitative antibody data and appropriate statistical methods was critical for the most comprehensive evaluation of the impact of immunogenicity on PK.

Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory

One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell-dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell-dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen.

The origin of IgE memory and plasma cells

The origin of IgE memory and plasma cells

Myasthenia gravis masquerading as acute sixth nerve paresis following head trauma – or vice versa? A case report

Aim: To report a case of sudden-onset diplopia, blurred vision and an inability of the left eye to fully abduct following trauma. Subsequent examinations showed significant variability leading to a possible diagnosis of myasthenia gravis. Methods: This is a case report of a 7-year-old boy with a 2-day history of blurred vision, horizontal diplopia and headaches following a bump to the left side of his head that presented as a left sixth nerve paresis. The history, orthoptic findings, investigations and possible aetiologies are discussed. Results: Orthoptic assessments initially revealed a marked left esotropia with restriction on left abduction. Computed tomography (CT) scans and magnetic resonance imaging (MRI) were both normal, as was monitoring of the patient’s intracranial pressures. Further orthoptic assessments demonstrated normal ocular movements on some visits whilst on other visits a restriction of left abduction was still shown. The patient then developed a vertical element with ptosis with significant variability at each visit, leading to a possible diagnosis of ocular myasthenia gravis. He was started on pyridostigmine and improvement was observed. However, low-affinity acetylcholine receptor antibodies, MuSK antibodies, Tensilon test and nerve fibre stimulation were negative and pyridostigmine was stopped. Conclusion: A question mark still hangs over the aetiology of this case. After a year of monitoring it was decided that the most suitable management for this patient was botulinum toxin. Following one injection the patient is currently straight and demonstrating stereopsis.

Old questions and novel clues: Complexity of IgE repertoires

Old questions and novel clues: Complexity of IgE repertoires

Structural Basis for Differential Neutralization of Ebolaviruses

There are five antigenically distinct ebolaviruses that cause hemorrhagic fever in humans or non-human primates (Ebola virus, Sudan virus, Reston virus, Taï Forest virus, and Bundibugyo virus). The small handful of antibodies known to neutralize the ebolaviruses bind to the surface glycoprotein termed GP1,2. Curiously, some antibodies against them are known to neutralize in vitro but not protect in vivo, whereas other antibodies are known to protect animal models in vivo, but not neutralize in vitro. A detailed understanding of what constitutes a neutralizing and/or protective antibody response is critical for development of novel therapeutic strategies. Here, we show that paradoxically, a lower affinity antibody with restricted access to its epitope confers better neutralization than a higher affinity antibody against a similar epitope, suggesting that either subtle differences in epitope, or different characteristics of the GP1,2 molecules themselves, confer differential neutralization susceptibility. Here, we also report the crystal structure of trimeric, prefusion GP1,2 from the original 1976 Boniface variant of Sudan virus complexed with 16F6, the first antibody known to neutralize Sudan virus, and compare the structure to that of Sudan virus, variant Gulu. We discuss new structural details of the GP1-GP2 clamp, thermal motion of various regions in GP1,2 across the two viruses visualized, details of differential interaction of the crystallized neutralizing antibodies, and their relevance for virus neutralization.