Antibody Subclasses Research Articles

Frequent IgG subclass and mannose binding lectin deficiency in patients with chronic fatigue syndrome

Chronic fatigue syndrome (CFS) is a severe disease characterized by various symptoms of immune dysfunction. CFS onset is typically with an infection and many patients suffer from frequently recurrent viral or bacterial infections. Immunoglobulin and mannose binding lectin (MBL) deficiency are frequent causes for increased susceptibility to infections. In this study we retrospectively analysed 300 patients with CFS for immunoglobulin and MBL levels, and B-cell subset frequencies. 25% of the CFS patients had decreased serum levels of at least one antibody class or subclass with IgG3 and IgG4 subclass deficiencies as most common phenotypes. However, we found elevated immunoglobulin levels with an excess of IgM and IgG2 in particular in another 25% of patients. No major alteration in numbers of B cells and B-cell subsets was seen. Deficiency of MBL was found in 15% of the CFS patients in contrast to 6% in a historical control group. In a 2nd cohort of 168 patients similar frequencies of IgG subclass and MBL deficiency were found. Thus, humoral immune defects are frequent in CFS patients and are associated with infections of the respiratory tract.

ID: 205: A new RNA-based adjuvant enhances virus-specific vaccine responses by locally activating DC and inducing IFN-I responses TLR- and RLH-dependently

Among new adjuvants conferring a TH1-shift, RNAdjuvant® consisting of non-coding RNA complexed with a cationic carrier peptide is a promising candidate. Upon RNAdjuvant® treatment, bone marrow-derived myeloid dendritic cells (BM-mDC) and plasmacytoid DC (BM-pDC) showed Toll-like receptor (TLR)7-dependent IFN-I induction and upregulation of CD69 and CD86. This result was verified by the ex vivo analysis of splenic DC. Immunization of mice with seasonal influenza vaccines plus RNAdjuvant® significantly enhanced vaccine-induced influenza-specific IgG responses. In particular, IgG2b and IgG2c antibody subclasses were elevated, indicating a TH1-biased immune response. RNAdjuvant® mediated enhancement of antibody responses was conferred by concomitant TLR- and RIG-I-like helicase (RLH)-dependent signaling. Thus, upon administration of RNAdjuvant® two different signaling pathways were induced, explaining the strong immunoenhancing effect of RNAdjuvant®. Notably, upon intramuscular (i.m.) injection of RNAdjuvant® IFN-I induction and CD69 upregulation on DC were only detectable in draining, but not in non-draining lymph nodes. Furthermore, transient lymphopenia as a sensitive readout of serum cytokine responses was only detected upon intravenous (i.v.), but not upon i.m., injection of RNAdjuvant®. These observations indicated that i.m. administration of RNAdjuvant® induced primarily local effects. In conclusion, RNAdjuvant® is a novel efficacious immune enhancer that augments vaccine-induced TH1 responses in a MyD88- and Cardif-dependent manner by triggering strictly local effects. Thus, RNAdjuvant® holds promise to be a particularly well tolerable new adjuvant also in humans.

Ageing of the B-cell repertoire.

Older people are more susceptible to infection, less responsive to vaccination and have a more inflammatory immune environment. Using spectratype analysis, we have previously shown that the B-cell repertoire of older people shows evidence of inappropriate clonal expansions in the absence of challenge, and that this loss of B-cell diversity correlates with poor health. Studies on response to vaccination, using both spectratyping and high-throughput sequencing of the repertoire, indicate that older responses to challenge are lacking in magnitude and/or delayed significantly. Also that some of the biologically significant differences may be in different classes of antibody. We have also previously shown that normal young B-cell repertoires can vary between different phenotypic subsets of B cells. In this paper, we present an analysis of immunoglobulin repertoire in different subclasses of antibody in five different populations of B cell, and show how the repertoire in these different groups changes with age. Although some age-related repertoire differences occur in naive cells, before exogenous antigen exposure, we see indications that there is a general dysregulation of the selective forces that shape memory B-cell populations in older people.

Bortezomib Inhibits Osteoclastogenesis and Bone Resorption Through Modulation of CHIT1 and YKL40 Expression

Background: VSIG4 acts as a co-inhibitory ligand to negatively regulate T cell proliferation and cytokine production by arresting cell cycle at G0/G1 phase and upregulating tolerance-inducing p27KIP-1. VSIG4 expression is restricted to macrophages. VSIG4 fusion protein also inhibits T cell production of Th1, Th2, and Th17 cytokines. Thus, we hypothesized that endogenous VSIG4 impairs helper T cell functions and then inhibits the subsequent antibody response. Materials and Methods: Mice were immunized subcutaneously with 100 g ovalumin (OVA) in a 1:1 emulsion with phosphate-buffered saline (PBS) and CFA in 100 l PBS. OVAspecific IgM or IgG subclasses were determined using culture supernatants or sera from immunized mice with OVA-coated plates and horseradish peroxidase-conjugated anti-mouse IgG1, -IgG2a, -IgG2b, and -IgG3. To study the VSIG4 role in antibody class switching via regulation of helper T cells, BALB/c mice were immunized intraperitoneally with 500 g of 2,4,6-Trinitrophenyl hapten (TNP)Keyhole Limpet Hemocyanin (KLH) for 14 days. Isolated B cells from TNP-KLH-immunized mice and purified CD4+ T cells from BALB/c wild-type or VSIG4 KO mice immunized s.c. with OVA/CFA were cocultured in the presence of TNPOVA (50 ng/ml) for 5 days. Culture supernatants were collected, and ELISA was performed on a TNP-CGG (100 g/ml)-coated plate to measure anti-TNP IgM and IgG titers. Results: Isotype switching of OVA-specific antibody subclasses to IgG1, IgG2a, IgG2b, and IgG3 was enhanced in OVA-immunized VSIG4 KO mice. 2,4,6-TNPe KLH-primed B cells cocultured with OVAprimed CD4+ T cells from OVA-immunized VSIG4 KO mice in the presence of TNP-OVA showed enhanced isotype switching to IgG subclasses compared to those cocultured with cells isolated from OVA-immunized wild-type (WT) mice. Furthermore, the levels of CD40L expression, the frequency of memory CD4+ T cells, and the production of isotype switching-inducing cytokines increased significantly in OVA-primed CD4+ T cells from VSIG4 knockout (KO) mice. T cells from OVA-specific T cell receptor transgenic mice produced more IFNwhen cocultured with macrophages from VSIG4 KO mice compared to WT mice. Conclusion: Our results demonstrate that macrophage-associated VSIG4 plays a negative role in helper T cell-dependent isotype switching by inhibiting helper T cell activation and differentiation, and suppressing the isotype switching-inducing cytokine production in antigen-primed CD4+ helper T cells.

Principles of antibody-mediated TNF receptor activation.

From the beginning of research on receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF), agonistic antibodies have been used to stimulate TNFRSF receptors in vitro and in vivo. Indeed, CD95, one of the first cloned TNFRSF receptors, was solely identified as the target of cell death-inducing antibodies. Early on, it became evident from in vitro studies that valency and Fcγ receptor (FcγR) binding of antibodies targeting TNFRSF receptors can be of crucial relevance for agonistic activity. TNFRSF receptor-specific antibodies of the IgM subclass and secondary cross-linked or aggregation prone dimeric antibodies typically display superior agonistic activity compared with dimeric antibodies. Likewise, anchoring of antibodies to cell surface-expressed FcγRs potentiate their ability to trigger TNFRSF receptor signaling. However, only recently has the relevance of oligomerization and FcγR binding for the in vivo activity of antibody-induced TNFRSF receptor activation been straightforwardly demonstrated in vivo. This review discusses the crucial role of oligomerization and/or FcγR binding for antibody-mediated TNFRSF receptor stimulation in light of current models of TNFRSF receptor activation and especially the overwhelming relevance of these issues for the rational development of therapeutic TNFRSF receptor-targeting antibodies.

IgG Donor-Specific Anti-Human HLA Antibody Subclasses and Kidney Allograft Antibody-Mediated Injury.

Antibodies may have different pathogenicities according to IgG subclass. We investigated the association between IgG subclasses of circulating anti-human HLA antibodies and antibody-mediated kidney allograft injury. Among 635 consecutive kidney transplantations performed between 2008 and 2010, we enrolled 125 patients with donor-specific anti-human HLA antibodies (DSA) detected in the first year post-transplant. We assessed DSA characteristics, including specificity, HLA class specificity, mean fluorescence intensity (MFI), C1q-binding, and IgG subclass, and graft injury phenotype at the time of sera evaluation. Overall, 51 (40.8%) patients had acute antibody-mediated rejection (aABMR), 36 (28.8%) patients had subclinical ABMR (sABMR), and 38 (30.4%) patients were ABMR-free. The MFI of the immunodominant DSA (iDSA, the DSA with the highest MFI level) was 6724±464, and 41.6% of patients had iDSA showing C1q positivity. The distribution of iDSA IgG1-4 subclasses among the population was 75.2%, 44.0%, 28.0%, and 26.4%, respectively. An unsupervised principal component analysis integrating iDSA IgG subclasses revealed aABMR was mainly driven by IgG3 iDSA, whereas sABMR was driven by IgG4 iDSA. IgG3 iDSA was associated with a shorter time to rejection (P<0.001), increased microcirculation injury (P=0.002), and C4d capillary deposition (P<0.001). IgG4 iDSA was associated with later allograft injury with increased allograft glomerulopathy and interstitial fibrosis/tubular atrophy lesions (P<0.001 for all comparisons). Integrating iDSA HLA class specificity, MFI level, C1q-binding status, and IgG subclasses in a Cox survival model revealed IgG3 iDSA and C1q-binding iDSA were strongly and independently associated with allograft failure. These results suggest IgG iDSA subclasses identify distinct phenotypes of kidney allograft antibody-mediated injury.

Measles elimination in the World Health Organization European Region in light of recent outbreaks: Spotlight on the Balkans

No: 1446 Presentation at ESCV 2015: Oral 42 Subclass specific antibody responses to HCMV in transplant recipients and their association with constant heavy IgG chain polymorphism and virus replication S. Benedikt ∗, L. Weseslindtner, I. Gorzer, K. Pollak, E. Puchhammer-Stockl Department of Virology, Medical University Vienna, Austria Background: Human cytomegalovirus (HCMV) causes severe infections in transplant recipients. The significance of the HCMVspecific antibody (AB) response in limiting HCMV replication is so far not entirely clear. In the present study we therefore analysed the HCMV-specific subclass AB profile in lung transplant recipients (LTRs) and its association with HCMV-DNAemia. In addition, we have analyzed its association with the patients allotypes GM3 and GM17, which are expressed at the CH1 region of the constant y1 heavy chain, and differ by single genetic variations, mainly by the single nucleotide polymorphism (SNP) rs1071803 (359 a/g nucleotide variation). Methods: We determined HCMV-specific total IgG, IgG1 and IgG3 AB levels by ELISA and HCMV-DNAemia by quantitative PCR during the post-transplant follow-up in 57 LTRs, and in 44 of these the GM 359a/g variant (GM3/17) was determined by genotyping. Results: In seropositive LTRs HCMV ABs increased with HCMV viremia (p=0.0005), and this was significantly associated with development of low level DNAemia ( 1000 copies/ml: p=0.0516). Only IgG3 but no IgG1 increase was observed with viremia (IgG3: p=0.0004). IgG1 levels were significantly lower in patients with the 359g/g (GM3/3) than in those with the 359a/g (GM3/17) variant (p<0.0001). The IgG3 increase with viremia was significant especially in patients carrying the IgG1 low level 359g/g variant (p<0.0002). Conclusions: The present data suggest that the HCMV specific AB response significantly contributes to limit HCMV replication after transplantation and provide evidence that the patients GM 3/17 variant is significantly associated with their HCMV IgG subclass profile. http://dx.doi.org/10.1016/j.jcv.2015.07.052 Abstract No: 1477 Presentation at ESCV 2015: Oral 43 Measles elimination in the World Health Organization European Region in light of recent outbreaks: Spotlight on the Balkans J.M. Hubschen1,∗, I. Salimovic-Besic2,1, A. Dedeic-Ljubovic2,1, S. Rakic Adrovic3,1, A. Sausy1, C.P. Muller1 1 Department of Immunology, Luxembourg Institute of Health/Laboratoire National de Sante, Esch-Sur-Alzette, France 2 University Clinical Centre – Sarajevo, Department for Clinical Microbiology, Sarajevo, BiH, Bosnia and Herzegovina 3 Institute of Virology, Vaccine and Sera “Torlak”, Belgrade, Serbia Background: The World Health Organization European Region (WHO-EURO) has adopted the goal to eliminate measles by 2015, but in light of current case numbers and epidemics it is clear that this target will not bemet. Elimination is defined on a country level as the absence of endemic cases for at least 12months. To interrupt endemic transmission, a very high vaccination coverage overall in the country but also in the different regions and cohorts is required. TheWHOEURO region is very heterogeneous and has a multiplicity of challenges and problems. In 2014, more than 16,000 measles cases were registered in WHO-EURO and outbreaks were reported from many different countries including Bosnia and Herzegovina and Serbia. Methods: The currently ongoing outbreaks in Bosnia and Herzegovina and in Serbia are investigated combining clinical, epidemiological and laboratory data. The mainly affected cohorts are identified and special emphasis is placed on differences in outbreak causes and patterns and virus exportations from the Balkans to other countries within and outside of WHO-EURO. Results: The results of the outbreak investigations are used as a basis to discuss current challenges and threats to the WHOEURO measles elimination goal such as migrant populations, anti-vaccination groups, vaccination gaps resulting from political instability or inadequate planning, insufficient routine immunization coverage, lack of political and financial commitment, etc. Conclusion: To achievemeasles elimination in theWHOEURO region, each country has to address its specific problems and challenges, identify and immunize susceptible population groups, react quickly and efficiently in outbreak situations, and renew and reenforce its commitment to attain the elimination goal. http://dx.doi.org/10.1016/j.jcv.2015.07.053

Abstract 1324: A translational platform to design antibodies targeting triple negative breast cancer-specific antigens for cancer immunotherapy

Abstract Triple negative breast cancers (TNBCs) are characterised by the lack of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER2) rendering them insensitive to current endocrine and anti-HER2 therapies, therefore novel treatment options are required. Breast cancers have been shown to be immunogenic and patient tumors feature infiltrating immune cells. Since monoclonal antibodies are an important therapeutic modality in the clinical management of many cancers, engineering antibodies against triple negative breast cancer specific antigens that may activate immune effector cells within tumors and in the circulation may lead to tumour clearance and may provide new treatment strategies for this patient group. Here we describe a platform for selection of TNBC-associated antigens, engineering of novel antibodies against these antigens and functional studies to elucidate antibody mechanisms of action and therapeutic potential in vitro and vivo. Our approach involves identification of tumour-associated antigens from patient tumor specimens using a transcriptomics approach and validation and evaluation of biological functions in breast cancer cells using cell culture models. Antibody heavy (VH) and light (VL) sequences are cloned in a dual expression vector system for recombinant expression in HEK293 cells to rapidly produce antibodies with reactivity to specific tumor antigens. The system is optimised for the seamless expression of V genes of any species including human sequences, along with human constant (C) genes of different antibody classes and subclasses. The latter permits the study of different class/subclass antibodies in order to identify those most effective in triggering immune cell activation and tumor clearance. Engineered monoclonal antibodies are subsequently tested for their antigen binding properties and capacity to activate immune effector cells against cancer cells in vitro. The therapeutic potential of selected candidates is evaluated in vivo using xenograft TNBC mouse models engrafted with components of the human immune system using adoptive transfer of healthy volunteer and patient derived peripheral blood cells. Xenograft models featuring human immune components serve to evaluate the therapeutic potential of monoclonal antibodies in the context of their capacity to redirect immune effector functions against cancer cells. Our findings demonstrate a translational pathway designed to facilitate the discovery and evaluation of novel antibody therapeutic options for triple negative breast cancer immunotherapy. Citation Format: Kristina M. Ilieva, Rebecca Marlow, Anthony Cheung, Erika Francesch, Panagiotis Karagiannis, Matthew Fittall, Silvia Crescioli, Andrew Tutt, Sophia Karagiannis. A translational platform to design antibodies targeting triple negative breast cancer-specific antigens for cancer immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1324. doi:10.1158/1538-7445.AM2015-1324

General Gastroenterological Surgery: Spleen.

General Gastroenterological Surgery: Spleen.

Six host-range restricted poxviruses from three genera induce distinct gene expression profiles in an in vivo mouse model.

BackgroundHost-range restricted poxviruses make promising vaccine vectors due to their safety profile and immunogenicity. An understanding of the host innate immune responses produced by different poxvirus vectors would aid in the assessment, selection and rational design of improved vaccines for human and veterinary applications. Novel avipoxviruses are being assessed to determine if they are different from other poxvirus vectors. Analysis of the transcriptome induced in a mouse model would aid in determining if there were significant differences between different poxvirus vectors which may reflect different adjuvant potential as well as establish if they should be further evaluated as vaccine vectors.ResultsWe compared host transcript abundance in the spleens of BALB/c mice twenty four hours after intravenous infection (105 pfu/mouse) with six host-restricted poxvirus species from three genera, namely Lumpy Skin Disease virus (LSDV), Canarypox virus (CNPV), Fowlpox virus (FWPV), modified vaccinia Ankara (MVA) and two novel South African avipoxviruses, Feral Pigeonpox virus (FeP2) and Penguinpox virus (PEPV). These six viruses produced qualitatively and quantitatively distinct host responses with LSDV, followed by MVA, inducing the greatest interferon (IFN) response. FeP2 and PEPV caused very little change to host transcript abundance compared to the other 4 viruses tested. CNPV and FWPV induced the up regulation of two immunoglobulin genes (Ighg and Ighg3 (IgG3)) with CNPV inducing a third, Ighm (IgM). HIV-1–specific IgG3 antibodies have been correlated with decreased risk of HIV-1 infection in the RV144 trial, which included a CNPV-based vector (Yates et al. (Sci Transl Med, 6(228) p228, 2014). Up regulation of IgG3 by CNPV and FWPV but not the other poxviruses tested in vivo, implies that these two avipoxvirus-vector backbones may be involved in stimulation of the clinically important IgG3 antibody subclass. Differential transcript abundance associated with the different poxviruses is further discussed with particular emphasis on responses related to immune responses.ConclusionSix, genetically diverse host-restricted poxviruses produce different responses in a mouse model early after infection. These differences may affect the immune response induced to vaccine antigen in vectors based on these viruses. The two novel avipoxviruses were clearly distinguishable from the other viruses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1659-1) contains supplementary material, which is available to authorized users.

Immunization with 3-oxododecanoyl-L-homoserine lactone-r-PcrV conjugate enhances survival of mice against lethal burn infections caused by Pseudomonas aeruginosa.

Quorum Sensing and type III secretion system play an important role in the virulence of Pseudomonas (P.) aeruginosa in burn wound infections. We aimed to explore the feasibility of using 3-oxo-C₁₂-HSL-r-PcrV conjugate as a candidate vaccine against P. aeruginosa caused infections. 3-oxo-C₁₂-HSL-r-PcrV conjugate was prepared and used for immunization of mice (10 μg, subcutaneous, three times, at 2-week intervals). Mice were divided into five groups: I: PcrV; II: 3-oxo-C₁₂-HSL-r-PcrV (10 μg); III: 3-oxo-C₁₂-HSL-r-PcrV (20 μg); IV: 3-oxo-C₁₂-HSL; and V: PBS receiving groups. After each shot of immunization, total and isotype antibody responses against corresponding antigen were measured to determine the immunization efficacy. One month after the last immunization, all groups were burned and challenged subeschar with P. aeruginosa PAO1. Survival rate and bacterial quantity in the skin and internal organs (liver and spleen) were evaluated 25-hr after burn infection. Immunization with 3-oxo-C₁₂-HSL-r-PcrV significantly increased total IgG and specific subclass antibodies (IgG₁, IgG₂a, IgG₂b, and IgM) in the serum of the groups II and III compared to the control group (p<0.001). While all the control mice (PBS injected group) died within 2 days after bacterial challenge, 64% of the group I, 78% of group II, and 86% of group III, survived within 14 days after challenge. Interestingly, bacterial burden in the liver and spleen of 3-oxo-C₁₂-HSL-r-PcrV injected group (III) was significantly lower than the control group (p<0.001). The present study proposed two-component vaccine to inhibit Pseudomonas infections in burned mouse.

Endogenous VSIG4 negatively regulates the helper T cell-mediated antibody response

VSIG4 acts as a co-inhibitory ligand to negatively regulate T cell proliferation and cytokine production, and its expression is restricted to macrophages. We hypothesized that endogenous VSIG4 impairs helper T cell functions and then inhibits the subsequent antibody response. Isotype switching of ovalbumin (OVA)-specific antibody subclasses to IgG1, IgG2a, IgG2b, and IgG3 was enhanced in OVA-immunized VSIG4 knockout (KO) mice. 2,4,6-Trinitrophenyl hapten (TNP) – Keyhole Limpet Hemocyanin (KLH)-primed B cells cocultured with OVA-primed CD4+ T cells from OVA-immunized VSIG4 KO mice in the presence of TNP-OVA showed enhanced isotype switching to IgG subclasses compared to those cocultured with cells isolated from OVA-immunized wild-type (WT) mice. Furthermore, the levels of CD40L expression, the frequency of memory CD4+ T cells, and the production of isotype switching-inducing cytokines increased significantly in OVA-primed CD4+ T cells from VSIG4 KO mice. T cells from OVA-specific T cell receptor (TCR) transgenic mice produced more IFN-γ when cocultured with macrophages from VSIG4 KO mice compared to WT mice. Thus, our results demonstrate that macrophage-associated VSIG4 plays a negative role in helper T cell-dependent isotype switching by inhibiting helper T cell activation and differentiation, and suppressing the isotype switching-inducing cytokine production in antigen-primed CD4+ helper T cells.

Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases

Monoclonal antibodies are being developed as therapeutics to complement drugs and vaccines or to fill the gap where no drugs or vaccines exist. These therapeutic antibodies (ThAb) may be especially important for infectious diseases in which there is antibiotic resistance, toxin-mediated pathogenesis, or for emerging pathogens. The unique structure of antibodies determines the specific nature of the effector function, so when developing ThAb, the desired effector functions need to be considered and integrated into the design and development processes to ensure maximum efficacy and safety. Antibody subclass is a critical consideration, but it is noteworthy that almost all ThAb that are licenced or currently in development utilise an IgG1 backbone. This review outlines the major structural properties that vary across subclasses, how these properties affect functional immunity, and discusses the various approaches used to study subclass responses to infectious diseases. We also review the factors associated with the selection of antibody subclasses when designing ThAb and highlight circumstances where different subclass properties might be beneficial when applied to particular infectious diseases. These approaches are critical to the future design of ThAb and to the study of naturally-acquired and vaccine-induced immunity.

Detection of specific IgG, IgM, IgE and IgG subclass antibodies for serological diagnosis of human cystic echinococcosis

Summary An enzyme linked immunosorbent assay (ELISA), based on sheep hydatid cyst fluid antigen was used for the detection of specific antibodies of IgG, IgM, IgE and IgG subclass in the serum samples of 62 clinically and radiologically diagnosed cystic echinococcosis (CE) patients, 8 clinically suspected cases of CE, 25 other parasitic disease controls and 25 healthy controls. The diagnostic sensitivity in the clinically and radiologically suggestive cases (n = 62) for IgG antibody detection was highest (93 %), followed by IgE, IgG4, IgG1, IgG2, IgM and IgG3 with 89 %, 87 %, 85 %, 76 %, 70 % and 55 % respectively. The detection of specific IgE, IgG1 and IgG4 antibody showed the higher diagnostic sensitivity and specificity to the extent that they can be safely used as better substitute to IgG. Even though, the diagnostic sensitivity of IgG was highest (93%) but was less specific (88 %) due to the frequent non-specific reactions in the sera of patients with other parasitic infections and healthy controls. None of the sera samples from healthy controls gave non-specific reaction with IgE, IgG1 and IgG4 and there was a considerably reduced cross-reaction with these antibodies. The most discriminatory and specific antibodies found in this study belonged to IgE, IgG1 and IgG4; therefore, these antibodies may serve as useful diagnostic markers for CE.

Reappraisal of PLA2R1 in membranous nephropathy: immunostaining method influence and association with IgG4-dominant phenotype.

The M-type phospholipase A2 receptor (PLA2R1) was identified recently as a specific target antigen in idiopathic membranous nephropathy. However, the influence of different sample preparation techniques on the immunostaining of PLA2R1 is unclear. Previous studies have identified IgG4 as the dominant subclass of PLA2R1 antibodies. However, it remains unclear whether the IgG subclass profiles of the glomerular immune complexes of PLA2R1-positive and -negative idiopathic membranous nephropathy cases are similar. To address these questions, we conducted the present study of 58 idiopathic membranous nephropathy cases. The PLA2R1 positivity rate for the paraffin-embedded sections was 61%, whereas that for the frozen sections was 65%. Nonspecific background staining was observed in the frozen sections. Discrepancies between different sample preparations occurred in three cases (6%); two cases were PLA2R1-positive in paraffin sections and PLA2R1-negative in frozen sections and one case was PLA2R1-negative in paraffin sections and PLA2R1-positive in frozen sections. Regarding the IgG subclass profile, 89% of the PLA2R1-positive cases demonstrated the IgG4-dominant/codominant phenotype versus 31% of the PLA2R1-negative cases (p < 0.001). Clinical characteristics and pathological findings did not significantly differ between PLA2R1-positive and -negative cases. In summary, the PLA2R1 immunofluorescence results were not affected by the different sample preparation techniques, although paraffin-embedded sections were preferred for the histological detection of PLA2R1 because of the nonspecific background staining observed in frozen sections. The observed lower frequency of the non-IgG4-dominant/codominant phenotype in PLA2R1-negative idiopathic membranous nephropathy cases may suggest that there are heterogeneous subgroups of this disease.

The Solution Structures of Two Human IgG1 Antibodies Show Conformational Stability and Accommodate Their C1q and FcγR Ligands

The human IgG1 antibody subclass shows distinct properties compared with the IgG2, IgG3, and IgG4 subclasses and is the most exploited subclass in therapeutic antibodies. It is the most abundant subclass, has a half-life as long as that of IgG2 and IgG4, binds the FcγR receptor, and activates complement. There is limited structural information on full-length human IgG1 because of the challenges of crystallization. To rectify this, we have studied the solution structures of two human IgG1 6a and 19a monoclonal antibodies in different buffers at different temperatures. Analytical ultracentrifugation showed that both antibodies were predominantly monomeric, with sedimentation coefficients s20,w (0) of 6.3-6.4 S. Only a minor dimer peak was observed, and the amount was not dependent on buffer conditions. Solution scattering showed that the x-ray radius of gyration Rg increased with salt concentration, whereas the neutron Rg values remained unchanged with temperature. The x-ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2, whose positions were unchanged in different buffers to indicate conformational stability. Constrained atomistic scattering modeling revealed predominantly asymmetric solution structures for both antibodies with extended hinge structures. Both structures were similar to the only known crystal structure of full-length human IgG1. The Fab conformations in both structures were suitably positioned to permit the Fc region to bind readily to its FcγR and C1q ligands without steric clashes, unlike human IgG4. Our molecular models for human IgG1 explain its immune activities, and we discuss its stability and function for therapeutic applications.

Affinity of FVIII-specific antibodies reveals major differences between neutralizing and nonneutralizing antibodies in humans

AbstractRecently, we reported that distinct immunoglobulin (Ig) isotypes and IgG subclasses of factor VIII (FVIII)-specific antibodies are found in different cohorts of patients with hemophilia A and in healthy individuals. Prompted by these findings, we further investigated the distinguishing properties among the different populations of FVIII-specific antibodies. We hypothesized that the affinity of antibodies would discriminate between the neutralizing and nonneutralizing antibodies found in different study cohorts. To test this idea, we established a competition-based enzyme-linked immunosorbent assay technology to assess the apparent affinities for each isotype and IgG subclass of FVIII-specific antibodies without the need for antibody purification. We present a unique data set of apparent affinities of FVIII-specific antibodies found in healthy individuals, patients with congenital hemophilia A with and without FVIII inhibitors, and patients with acquired hemophilia A. Our data indicate that FVIII-specific antibodies found in patients with FVIII inhibitors have an up to 100-fold higher apparent affinity than that of antibodies found in patients without inhibitors and in healthy individuals. High-affinity FVIII-specific antibodies could be retrospectively detected in longitudinal samples of an individual patient with FVIII inhibitors 543 days before the first positive Bethesda assay. This finding suggests that these antibodies might serve as potential biomarkers for evolving FVIII inhibitor responses.

Distinct features of immunoglobulin G2 aquaporin‐4 antibody carriers with neuromyelitis optica

AbstractObjectiveNeuromyelitis optica (NMO) is an inflammatory disease in which NMO‐immunoglobulin G (IgG) targeting aquaporin‐4 (AQP4) is specifically detected. Although the main subclass of AQP4 antibody was reported to be in the IgG1 subclass, other subclasses have also been described, including IgG2 AQP4 antibody, as a second common subclass. NMO patients were analyzed to clarify the clinical features of NMO patients with IgG2 AQP4 antibody.MethodsSerum samples from 58 NMO patients, who met the revised 2006 criteria for NMO, were analyzed for AQP4 antibody subclass expression using an established flow cytometric assay, and clinical features were compared according to the main AQP4 antibody subclasses.ResultsA total of 50 patients (86.2%) had IgG1 AQP4 antibodies, while eight (13.8%) expressed IgG2 AQP4 antibody as the main subclass. Those eight individuals exhibited younger age of onset (P = 0.0089), lower AQP4 antibody titers (P = 0.0024) and a more common fulfillment of Barkhof's criteria (P = 0.0466) than patients with IgG1 AQP4 antibody expression.ConclusionsResults from the present study suggest that the characteristics of individuals with IgG2 AQP4 antibody as a main subclass are more similar to multiple sclerosis and somewhat distinct from NMO patients with IgG1 AQP4 antibody.

Purification of monoclonal antibodies by chemical affinity mixed mode chromatography

The application of several pyridine-based compounds as immobilised ligands has been investigated for the purification of monoclonal antibodies via mixed mode chromatography. The ligands employed were 4′-terpyridinysulfanylethylamine (4′-TerPSEA), 5-bromo-2-pyridinylsulfanylethylamine (5-Br-2-PSEA), 2-quinolinylsulfanylethylamine (2-QSEA) and 4-pyridinylsulfanylethylamine (4-PSEA). The performance attributes of adsorbents, derived from the immobilisation of these different ligands onto Sepharose 6 Fast Flow™, was evaluated from batch adsorption studies and from chromatographic experiments with humanised IgG1, IgG2 and IgG4 monoclonal antibodies produced by stable CHO cell lines cultured in chemical defined media. These results demonstrated that monoclonal antibodies of different subclasses can be efficiently purified from crude CHO cell culture supernatants using these new chemical affinity chromatographic systems. Moreover, the majority of the CHO host proteins could be eliminated during the chromatographic purification step with these resins, as monitored by a specific ELISA assay.

Obinutuzumab: a review of its use in patients with chronic lymphocytic leukaemia.

Obinutuzumab (Gazyva(®); Gazyvaro(®)) is an intravenously administered, glycoengineered, humanized, type II, anti-CD20 monoclonal antibody of the IgG1 subclass. It is available in the EU and the USA as combination therapy with oral chlorambucil in adults with previously untreated chronic lymphocytic leukaemia (CLL). In a multinational phase III study in this patient population, obinutuzumab plus chlorambucil significantly prolonged progression-free survival compared with oral chlorambucil alone and intravenous rituximab plus oral chlorambucil. Significant advantages with obinutuzumab plus chlorambucil over chlorambucil alone and rituximab plus chlorambucil were also observed in event-free survival, the time to a new anti-leukaemia treatment and overall response. The overall survival benefit with obinutuzumab plus chlorambucil is as yet unclear, although the most recent analysis suggests a benefit over chlorambucil alone. In the phase III study, obinutuzumab plus chlorambucil had a manageable tolerability profile in accordance with what would be expected for an anti-CD20 antibody. Neutropenia and infusion-related reactions were the most frequently reported grade 3 or higher treatment-emergent adverse events. In the majority of patients, infusion-related reactions were mild to moderate in severity and occurred predominantly during the first infusion and were managed by slowing or temporarily halting the infusion. Thus, current evidence suggests that obinutuzumab plus chlorambucil is a welcome addition to the treatment options currently available for adults with previously untreated CLL and is recommended by the National Comprehensive Cancer Network guidelines as the preferred first option for some, including those with comorbidities.