Differences In Epitope Specificity Research Articles

Humoral immunity to SARS-CoV-2 elicited by combination COVID-19 vaccination regimens.

The SARS-CoV-2 pandemic prompted a global vaccination effort and the development of numerous COVID-19 vaccines at an unprecedented scale and pace. As a result, current COVID-19 vaccination regimens comprise diverse vaccine modalities, immunogen combinations, and dosing intervals. Here, we compare vaccine-specific antibody and memory B cell responses following two-dose mRNA, single-dose Ad26.COV.2S, and two-dose ChAdOx1, or combination ChAdOx1/mRNA vaccination. Plasma-neutralizing activity, as well as the magnitude, clonal composition, and antibody maturation of the RBD-specific memory B cell compartments, showed substantial differences between the vaccination regimens. While individual monoclonal antibodies derived from memory B cells exhibited similar binding affinities and neutralizing potency against Wuhan-Hu-1 SARS-CoV-2, there were significant differences in epitope specificity and neutralizing breadth against viral variants of concern. Although the ChAdOx1 vaccine was inferior to mRNA and Ad26.COV.2S in several respects, biochemical and structural analyses revealed enrichment in a subgroup of memory B cell neutralizing antibodies with distinct RBD-binding properties resulting in remarkable potency and breadth.

Fine-Specificity Epitope Analysis Identifies Contact Points on Ricin Toxin Recognized by Protective Monoclonal Antibodies.

Ricin is a fast-acting protein toxin classified by the Centers for Disease Control and Prevention as a biothreat agent. In this report, we describe five new mouse mAbs directed against an immunodominant region, so-called epitope cluster II, on the surface of ricin's ribosome-inactivating enzymatic subunit A (RTA). The five mAbs were tested alongside four previously described cluster II-specific mAbs for their capacity to passively protect mice against 10× LD50 ricin challenge by injection. Only three of the mAbs (LE4, PH12, and TB12) afforded protection over the 7-d study period. Neither binding affinity nor in vitro toxin-neutralizing activity could fully account for LE4, PH12, and TB12's potent in vivo activity relative to the other six mAbs. However, epitope mapping studies by hydrogen exchange-mass spectrometry revealed that LE4, PH12, and TB12 shared common contact points on RTA corresponding to RTA α-helices D and E and β-strands d and e located on the back side of RTA relative to the active site. The other six mAbs recognized overlapping epitopes on RTA, but none shared the same hydrogen exchange-mass spectrometry profile as LE4, PH12, and TB12. A high-density competition ELISA with a panel of ricin-specific, single-domain camelid Abs indicated that even though LE4, PH12, and TB12 make contact with similar secondary motifs, they likely approach RTA from different angles. These results underscore how subtle differences in epitope specificity can significantly impact Ab functionality in vivo. ImmunoHorizons, 2018, 2: 262-273.

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies.

In this report we investigated, within a group of closely related single domain camelid antibodies (VH Hs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast-acting toxin and biothreat agent. The V1C7-like VH Hs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin-neutralizing activities. Using the X-ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta-based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1R29G mutant was largely devoid of toxin-neutralizing activity (TNA). However, the TNA of the V1C7G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen-deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.

Generation and characterization of new monoclonal antibodies targeting the PHF1 and AT8 epitopes on human tau

Tauopathies are a group of neurodegenerative disorders, including Alzheimer’s disease, defined by the presence of brain pathological inclusions comprised of abnormally aggregated and highly phosphorylated tau protein. The abundance of brain tau aggregates correlates with disease severity and select phospho-tau epitopes increase at early stages of disease. We generated and characterized a series of novel monoclonal antibodies directed to tau phosphorylated at several of these phospho-epitopes, including Ser396/Ser404, Ser404 and Thr205. We also generated phosphorylation independent antibodies against amino acid residues 193–211. We show that most of these antibodies are highly specific for tau and strongly recognize pathological inclusions in human brains and in a transgenic mouse model of tauopathy. They also reveal epitope-specific differences in the biochemical properties of Alzheimer’s disease sarkosyl-insoluble tau. These new reagents will be useful for investigating the progression of tau pathology and further as tools to target the cellular transmission of tau pathology.

IgG anti-tTG responses in different autoimmune conditions differ in their epitope targets and subclass usage.

Antibodies of the IgA class directed against the enzyme tissue transglutaminase (tTG) are highly specific for coeliac disease (CD). IgG antibodies to tTG also occur in CD, and have also been reported in autoimmune diseases such as type 1 diabetes mellitus and Crohn's disease. In comparison to the IgA anti-tTG response, little is known of the IgG anti-tTG response in terms of epitope specificity and IgG subclass usage. The aim of this study was to investigate and compare epitopes recognised by CD and non-CD IgG anti-tTG antibodies, and determine the relative proportions of the IgG subclasses comprising this response. IgG anti-tTG positive individuals who did not have CD were identified by screening groups of patients with type I diabetes mellitus, Crohn's disease and granulomatosis with polyangiitis. Results from ELISA blocking experiments and mutant tTG antigens demonstrate that non-CD IgG anti-tTG bind different epitopic determinants to CD IgG anti-tTG. The IgG subclass usage of coeliac disease and type 1 diabetes was dominated by IgG1, whereas this IgG subclass was infrequently a component of the IgG anti-tTG response in diseases such as granulomatosis with polyangiitis and Crohn's disease. The differences in epitope specificity and subclass usage of IgG anti-tTG observed between CD and non-CD individuals may be due to the differing mechanisms underlying tTG autoimmunity.

Promotion of a subdominant CD8 T cell response during murine gammaherpesvirus 68 infection in the absence of CD4 T cell help.

CD8 and CD4 T cells are each critically important for immune control of murine gammaherpesvirus 68 (γHV68) infection. In immunocompetent mice, acute γHV68 infection results in lifelong latency, but in the absence of CD4 T cell help, mice succumb to viral recrudescence and disease. However, the requirements for CD4 T cell help in the generation and maintenance of antiviral CD8 T cell responses are incompletely understood, and it is unclear whether there are epitope-specific differences in the requirement of CD8 T cells for CD4 help. In this report, we characterized the CD8 T cell response to γHV68 in major histocompatibility complex (MHC) class II(-/-) mice, which lack CD4 T cells, or after antibody-mediated depletion of CD4 T cells. All antiviral CD8 T cells exhibited marked upregulation of surface expression of the inhibitory receptor programmed death-1 (PD-1), but surprisingly, while the immunodominant memory response appeared to be functionally impaired, helpless CD8 T cells of a subdominant specificity had increased numbers and enhanced functionality. Thus, we demonstrate differential requirements for CD4 help in the antiviral CD8 T cell response to a latent gammaherpesvirus. Importance: γHV68 is a mouse pathogen closely related to the oncogenic human γHVs, which infect a majority of the world's population. Reactivation of these viruses from latency can lead to complications, disease, and even death. CD4 T cells are required for complete immune control of long-term infection, in part by providing key signals to dendritic cells that in turn instruct optimal antiviral CD8 T cell responses. We have investigated multiple virus-specific CD8 T cell responses during infection and identified a subdominant CD8 T cell response that is numerically and functionally enhanced in the absence of CD4 T cell help. This occurs in spite of high surface expression of an inhibitory receptor and in contrast to the immunodominant response, which is impaired. Our data suggest that signals from CD4 T cells are important in maintaining the CD8 T cell hierarchy during γHV infections.

AB0025 Proteomic analysis of anti-ccp immunoglobulins for the identification of rheumatoid arthritis patients requiring early aggressive treatment with anti-tnf

BackgroundAntibodies against citrullinated peptides (ACPA) are a hallmark of rheumatoid arthritis (RA) patients and are associated with poor outcome. ACPA are usually detected using the anti-CCP test, identifying antibodies against...

Molecular determinants of humoral immune specificity for the occupational allergen, methylene diphenyl diisocyanate

Methylene diphenyl diisocyanate (MDI), a low molecular weight chemical important for producing polyurethane foam, coatings, and elastomers is a major cause of occupational asthma, however, mechanisms of disease pathogenesis remain poorly understood. This study characterizes the rearranged germline and hypervariable region cDNA of new anti-MDI secreting hybridomas derived from mice immunized with MDI-conjugated to autologous serum proteins. Six IgG1 secreting clones were identified in initial screening ELISAs, based on differential binding to MDI conjugated human albumin vs. mock exposed albumin. The mAbs secreted by the hybridomas also recognized MDI conjugated to other model proteins (e.g. ovalbumin, transferrin), but did not bind unconjugated proteins, or protein conjugates prepared with other isocyanates (e.g. TDI, HDI). The mAbs displayed MDI-dose dependent binding in ELISA and Western blot, and exhibited varying degrees of cross-competition, suggesting differences in epitope specificity. The cDNA encoding the monoclonal antibodies reveal clonal differences in the CDR3 regions, germline gene usage, and patterns of somatic hypermutation related to epitope specificity. Together, the data provide new insight into the molecular determinants of humoral MDI specificity, and characterize anti-MDI IgG1 mAbs that may be developed into useful diagnostic reagents.

Defective CD8 T cell memory generated during latent gammaherpesvirus infection (105.11)

Abstract Antiviral CD8 T cells are essential for protection during murine gammaheresvirus-68 (γHV68) infection. We recently identified a large panel of epitope-specific CD8 T cell responses that were differentially regulated by antigen presentation during the establishment of viral latency, but how these T cell responses are maintained throughout latent infection is unclear. Here we show that recent thymic emigrants are primed during latency, accumulate over time, and can proliferate in response to experimentally-induced viral reactivation. We found that in contrast to ORF6487- and ORF61524-specific responses, CD8 T cells specific for the ORF75c940 epitope primed during latency were deficient in their ability to respond during reactivation. This defect was not a result of impaired generation of ORF75c940-specific cells during latency or a lack of antigen presentation during reactivation, but rather appears to be a consequence of reduced proliferation following stimulation. The finding of epitope-specific differences in proliferative capacity suggests that the functionality of latency-primed responses may be regulated by differential antigen presentation or viral gene expression during latency. These findings have important implications for the development of therapeutic vaccine strategies against latent gammaherpesvirus infections.

Antigenic changes in human albumin caused by reactivity with the occupational allergen diphenylmethane diisocyanate

Diphenylmethane diisocyanate (MDI), the chemical commonly used as a cross-linking agent in commercial polyurethane production, is a well-recognized cause of asthma. Reaction products between MDI and “self” proteins are hypothesized to act as antigens capable of inducing airway inflammation and asthma; however, such MDI antigens remain incompletely understood. We used a variety of analytical methods to characterize the range of MDI–albumin reaction products that form under physiological conditions. Sites of MDI conjugation on antigenic MDI–albumin products, as defined by serum immunoglobulin G (IgG) from MDI-exposed workers, were determined by high-performance liquid chromatography (HPLC) followed by tandem mass spectrometry (MS/MS). The data identified 14 MDI conjugation sites (12 lysines and 2 asparagines) on human albumin and highlight reaction specificity for the second lysine in dilysine (KK) motifs, and this may be a common characteristic of “immune-sensitizing” chemicals. Several of the MDI conjugation sites are not conserved in albumin from other species, and this may suggest species differences in epitope specificity for self protein (albumin)–isocyanate conjugates. The study also describes new applications of contemporary proteomic methodology for characterizing and standardizing MDI–albumin conjugates destined for use in clinical research.

Problems with GH assays and strategies toward standardization

Disorders affecting GH secretion--either GH deficiency or GH excess (acromegaly)--are biochemically defined through peak or nadir concentrations of human GH in response to dynamic tests. Immunoassays employing polyclonal or monoclonal antibodies are routinely used for the analysis of GH concentrations, and many different assays are available on the market today. Unfortunately, the actual value reported for the GH concentration in a specific patient's sample to a large extent depends on the assay method used by the respective laboratory. Variability between assay results exceeds 200%, limiting the applicability of consensus guidelines in clinical practice. Reasons for the heterogeneity in GH assay results include the heterogeneity of the analyte itself, the availability of different preparations for calibration, and the interference from matrix components such as GH-binding protein. Furthermore, the reporting of results in mass units or international units together with the application of variable conversion factors led to confusion. International collaborations proposed measures to improve the comparability of assay results, recommending the use of a single, recombinant calibrator for all assays and reporting only in mass units as first steps. However, because of the differences in epitope specificity of antibodies used in different assays, method-specific cut-off levels for dynamic tests might remain necessary to correctly interpret and compare results from different laboratories.

The Alloantigenic Sites of α3α4α5(IV) Collagen: PATHOGENIC X-LINKED ALPORT ALLOANTIBODIES TARGET TWO ACCESSIBLE CONFORMATIONAL EPITOPES IN THE α5NC1 DOMAIN

The Alloantigenic Sites of α3α4α5(IV) Collagen: PATHOGENIC X-LINKED ALPORT ALLOANTIBODIES TARGET TWO ACCESSIBLE CONFORMATIONAL EPITOPES IN THE α5NC1 DOMAIN

Coordinated Function of Murine Cytomegalovirus Genes Completely Inhibits CTL Lysis

Murine CMV (MCMV) encodes three viral genes that interfere with Ag presentation (VIPRs) to CD8 T cells, m04, m06, and m152. Because the functional impact of these genes during normal infection of C57BL/6 mice is surprisingly modest, we wanted to determine whether the VIPRs are equally effective against the entire spectrum of H-2(b)-restricted CD8 T cell epitopes. We also wanted to understand how the VIPRs interact at a functional level. To address these questions, we used a panel of MCMV mutants lacking each VIPR in all possible combinations, and CTL specific for 15 H-2(b)-restricted MCMV epitopes. Only expression of all three MCMV VIPRs completely inhibited killing by CTL specific for all 15 epitopes, but removal of any one VIPR enabled lysis by at least some CTL. The dominant interaction between the VIPRs was cooperation: m06 increased the inhibition of lysis achieved by either m152 or m04. However, for 1 of 15 epitopes m04 functionally antagonized m152. There was little differential impact of any of the VIPRs on K(b) vs D(b), but a surprising degree of differential impact of the three VIPRs for different epitopes. These epitope-specific differences did not correlate with functional avidity, or with timing of VIPR expression in relation to Ag expression in the virus replication cycle. Although questions remain about the molecular mechanism and in vivo role of these genes, we conclude that the coordinated function of MCMV's three VIPRs results in a powerful inhibition of lysis of infected cells by CD8 T cells.

Epitope-specific T-cell responses and allergic phenotypes: implications for T-cell peptide therapy

In the 1990s, elucidation of the primary amino acid sequence of several major allergens using molecular cloning techniques opened the door to T-cell epitope mapping studies. Such analyses underscored the complexity of the allergen-specific T-cell repertoire and the challenges to using allergen-derived peptides to identify epitope-specific differences associated with allergic and nonallergic responses. This review highlights important factors that may influence the nature of epitope-specific T-cell responses observed in vitro. These include the properties of the allergen, genetics of the host and selection of patients with defined allergic phenotypes based on serum antibody profiles and skin test reactivity. By taking these factors into account, T-cell epitope-specific differences associated with distinct allergic phenotypes can be identified. Observations at the T-cell epitope level undermine the Th1/Th2 paradigm as a model for the development of allergic versus nonallergic responses. Instead, they support the mounting data that point to a network of interactions between T helper cells and regulatory T cells, which controls the allergic response. The ability of peptides that localize to polypeptide chain 2 of the major cat allergen, Fel d 1, to preferentially induce interleukin-10 and interferon-γ is discussed. Mechanisms whereby specific allergen-derived peptides may modify the T-cell repertoire and influence the immune outcome are also outlined. Further investigation of allergen-derived T-cell epitopes is warranted in order to optimize the design of peptide vaccines for the treatment of allergic disease.

CD4+T-Cell Responses to Epstein-Barr Virus (EBV) Latent-Cycle Antigens and the Recognition of EBV-Transformed Lymphoblastoid Cell Lines

There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4+ T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4+ T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4+ epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4+ T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4+ clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4+ T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.

Quantitative determination of immunoglobulin G specific for group B streptococcal beta C protein in human maternal serum.

The beta C protein of group B streptococci (GBS) elicits antibody that is protective against GBS challenge in animals and is considered to be a potential component of a GBS conjugate vaccine. We developed a quantitative enzyme-linked immunosorbent assay to measure beta-specific serum immunoglobulin G (IgG) levels and used it to compare beta-specific IgG in a group of mothers of neonates with invasive type Ib/beta GBS disease and a group of mothers colonized with Ib/beta strains whose neonates remained well. beta-Specific IgG concentrations from these 2 groups were similar. To investigate differences in beta-specific antibody in animals and humans, protein fragments were generated that corresponded to major regions within the beta C protein. A single major region was predominantly recognized in human and rabbit serum samples. Thus, in contrast to immunized animals, no relationship was seen between levels of naturally acquired human beta-specific IgG and protection from neonatal disease. This difference was not explained by a major difference in epitope specificity.

CD8(+) lymphocytes respond to different HIV epitopes in seronegative and infected subjects.

HIV-1-specific cytotoxic T-lymphocyte (CTL) responses have been detected at a low frequency in many HIV-1-exposed, persistently seronegative (HEPS) subjects. However, it is unclear how CTLs could protect against HIV acquisition in HEPS subjects, when high levels of circulating CTL fail to prevent disease progression in most seropositive subjects. To address this issue we studied CD8(+) lymphocyte responses to a panel of HIV-1 CTL epitopes in 91 HEPS and 87 HIV-1-infected Nairobi sex workers. HIV-specific responses in seropositive women focused strongly on epitopes rarely or never recognized in HEPS subjects, who targeted epitopes that were subdominant or unrecognized in infected women. These differences in epitope specificity were restricted by only those HLA class I alleles that are associated with a reduced risk of HIV-1 infection in this cohort. Late seroconversion in HEPS donors was associated with a switch in epitope specificity and/or immunodominance to those epitopes preferentially recognized by HIV-1-infected women. The likelihood of detecting HIV-1-specific responses in HEPS women increased with the duration of viral exposure, suggesting that HIV-1-specific CD8(+) responses are acquired over time. The association between differential recognition of distinct CTL epitopes and protection from HIV-1 infection may have significant implications for vaccine design.

Antineutrophil cytoplasmic antibodies to proteinase 3 in Wegener's granulomatosis: Epitope analysis using synthetic peptides

Antineutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are strongly associated with Wegener's granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity nor to functional effects of these antibodies in vitro, suggesting differences in epitope specificity. To define relevant epitopes for PR3-ANCA, sera of WG patients were analyzed on their reactivity to linear peptides of PR3. Fifty linear peptides of 15 amino acids in length with an overlap of 10 aa spanning the entire PR3 sequence were synthesized. Sera of 27 WG patients with active disease and 27 age- and sex-matched healthy controls, eight anti-PR3 monoclonal antibodies (mAbs), and a rabbit anti-PR3 serum were tested by enzyme-linked immunosorbent assay for reactivity to PR3 peptides. Rabbit anti-PR3 serum recognized three distinct peptide areas, whereas none of the anti-PR3 mAbs bound PR3 peptides. Sera of both WG patients and healthy controls recognized a restricted number of PR3 peptides. Four of these peptide areas were recognized significantly more strongly by WG sera than by control sera. Sera drawn at the initial presentation of WG mainly recognized these peptides. Two of the recognized peptide areas were located near the active center of PR3. A restricted number of epitope areas of PR3 are recognized both by WG patient sera and control sera. Four peptide areas were bound stronger by sera of WG patients at initial presentation than by healthy controls.

Identification of murine B-cell and T-cell epitopes of Escherichia coli outer membrane protein F with synthetic polypeptides.

The major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains. Because Escherichia coli OmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed. Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer. T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined. For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants. Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified. Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein. Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice. Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B-cell responses have been investigated based on the assumption that differences in epitope specificity could influence protective or pathologic host reactions. Because of the high level of structural homology of OmpF to porins isolated from other enteric pathogens, the identification of T- and B-cell-stimulatory determinants of E. coli OmpF may have broader application.

Characterization of Antibodies Induced by Human Factor VIII in a Murine Knockout Model of Hemophilia A

To investigate the usefulness of factor VIII (FVIII) knockout mice as an animal model of hemophilia A, we characterized the antibody response in FVIII knockout mice to recombinant human FVIII, administered intravenously or subcutaneously with or without adjuvant, and compared results to those in normal mice. Anti-factor VIII antibodies were detected after both intravenous and subcutaneous administration, with the highest titers after subcutaneous administration plus adjuvant. Depending on the administration strategy. knockout mice formed antibodies more rapidly and developed higher titers of inhibitory antibodies (Bethesda) than normal mice, suggesting differences in epitope specificity. Blotting thrombin cleavage products separated by gel electrophoresis showed that both strains developed antibodies against the nonfunctional B domain as well as against functional domains of factor VIII. The antibodies were mainly of the IgG1 subclass and resembled type I antibodies in hemophilia A.