IgG Subtype Research Articles

Coordinated Fc-effector and neutralization functions in HIV-infected children define a window of opportunity for HIV vaccination.

Objectives:Antibody function has been extensively studied in HIV-infected adults but is relatively understudied in children. Emerging data suggests enhanced development of broadly neutralizing antibodies (bNAbs) in children but Fc effector functions in this group are less well defined. Here, we profiled overall antibody function in HIV-infected children.Design:Plasma samples from a cross-sectional study of 50 antiretroviral therapy-naive children (aged 1–11 years) vertically infected with HIV-1 clade A were screened for HIV-specific binding antibody levels and neutralizing and Fc-mediated functions.Methods:Neutralization breadth was determined against a globally representative panel of 12 viruses. HIV-specific antibody levels were determined using a multiplex assay. Fc-mediated antibody functions measured were antibody-dependent: cellular phagocytosis (ADCP); neutrophil phagocytosis (ADNP); complement deposition (ADCD) and natural killer function (ADNK).Results:All children had HIV gp120-specific antibodies, largely of the IgG1 subtype. Fifty-four percent of the children exhibited more than 50% neutralization breadth, with older children showing significantly broader neutralization activity. Apart from ADCC, observed only in 16% children, other Fc-mediated functions were common (>58% children). Neutralization breadth correlated with Fc-mediated functions suggesting shared determinants of enhanced antibody function exist.Conclusions:These results are consistent with previous observations that children may develop high levels of neutralization breadth. Furthermore, the striking association between neutralization breadth and Fc effector function suggests that HIV vaccination in children could yield multifunctional antibodies. Paediatric populations may therefore provide an ideal window of opportunity for HIV vaccination strategies.

Naturally induced humoral response against Plasmodium vivax reticulocyte binding protein 2P1

BackgroundPlasmodium vivax is the most prevalent malaria parasite in many countries. A better understanding of human immunity to this parasite can provide new insights for vaccine development. Plasmodium vivax Reticulocyte Binding Proteins (RBPs) are key parasite proteins that interact with human proteins during erythrocyte invasion and are targets of the human immune response. The aim of this study is to characterize the human antibody response to RBP2P1, the most recently described member of the RBP family.MethodsThe levels of total IgG and IgM against RBP2P1 were measured using plasmas from 68 P. vivax malaria patients and 525 villagers in a malarious village of western Thailand. The latter group comprises asymptomatic carriers and healthy uninfected individuals. Subsets of plasma samples were evaluated for anti-RBP2P1 IgG subtypes and complement-fixing activity.ResultsAs age increased, it was found that the level of anti-RBP2P1 IgG increased while the level of IgM decreased. The main anti-RBP2P1 IgG subtypes were IgG1 and IgG3. The IgG3-seropositive rate was higher in asymptomatic carriers than in patients. The higher level of IgG3 was correlated with higher in vitro RBP2P1-mediated complement fixing activity.ConclusionsIn natural infection, the primary IgG response to RBP2P1 was IgG1 and IgG3. The predominance of these cytophilic subtypes and the elevated level of IgG3 correlating with complement fixing activity, suggest a possible role of anti-RBP2P1 antibodies in immunity against P. vivax.

A phase 1/2, open-label, dose-escalation, safety and tolerability study of NC410 in subjects with advanced or metastatic solid tumors.

TPS2659 Background: Leukocyte-Associated Immunoglobulin-like Receptor (LAIR)-1 and LAIR-2 are members of the Leukocyte Receptor Complex (LRC) (An & Brodsky, 2016). LAIR-1 is a co-inhibitory receptor expressed on several subsets of immune cells, and functions to delimit immune responses (Meyaard et al., 1997). LAIR-2 is a secreted protein with homology to the transmembrane protein LAIR-1 (Lebbink et al., 2008). In cancer, it is hypothesized that LAIR-1 expression on several subsets of leukocytes prevents optimal immune responses by limiting both innate and adaptive immune functionality. LAIR-1 serves to suppress anti-tumor immunity through the inhibition of stimulatory signaling pathways. Specifically, LAIR-1 is a checkpoint and adhesion receptor on T cells that limits T cell activation and increases adhesion to collagens (Meyaard, 2008). LAIR-2 is capable of blocking LAIR-1 functional interactions with ligands, resulting in improved immune function on multiple immune cell subsets. Dysregulation of LAIR-1 ligands in the tumor microenvironment results in excessive production of collagens and complement C1q as well as altered forms of collagens, that leads to immune inhibition through binding to LAIR-1+ immune cells. NC410 is a dimeric form of the LAIR-2 protein fused to a human Fc domain of the immunoglobulin (Ig) subtype IgG1. The rationale for developing NC410 as a cancer therapeutic is based on nonclinical data demonstrating LAIR-1 signaling blockade can improve the immune response. Because LAIR-2 binds to ligands shared with LAIR-1 with increased affinity, NC410 acts as a decoy receptor for LAIR-1 ligands releasing suppression from myeloid cells and T cells and promoting anti-tumor immunity. NC410 may also mediate remodeling of the tumor extracellular matrix, further contributing to anti-tumor activity. Methods: This is a multi-center, first in human, phase 1/2, open-label, single-armed study to determine the safety and tolerability, define maximum tolerated dose (MTD) and/or pharmacologically active dose, assess preliminary efficacy, and explore predictive and pharmacodynamic biomarkers of NC410 in subjects with advanced or metastatic solid tumors. Key eligibility criteria include measurable disease based on RECIST v1.1 and being able to consent for collection of biopsies at screening and on treatment. Phase 1 is a classic 3+3 dose escalation design to determine the safety, tolerability, DLT, MTD and recommended phase 2 dose (RP2D) (NCT04408599). Ongoing exploratory analyses include the assessment of predictive biomarkers associated with treatment benefit, and pharmacodynamic markers associated with study drug activity. Phase 2 is going to enroll ovarian, colorectal, NSCLC, H&N, and gastric carcinomas and other tumors depending on biomarker data available from the Phase 1 part of the study. Clinical trial information: 04408599.

Neural Epidermal Growth Factor-Like 1 Protein-Positive Membranous Nephropathy in Chinese Patients.

The neural EGF-like 1 (NELL-1) protein is a novel antigen in primary membranous nephropathy. The prevalence and clinical characteristics of NELL-1-positive membranous nephropathy in Chinese individuals with primary membranous nephropathy are unclear. A total of 832 consecutive patients with biopsy-proven primary membranous nephropathy were enrolled. The glomerular expression of phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A (THSD7A) was screened. Glomerular immunohistochemistry staining for NELL-1 was performed in 43 patients with PLA2R- and THSD7A-negative membranous nephropathy, 31 patients with PLA2R-positive membranous nephropathy, and two patients with PLA2R and THSD7A double positivity. The NELL-1 antibody was also detected in the sera of patients with NELL-1-positive membranous nephropathy by western blot. Clinical and pathologic features were comparable between patients with isolated NELL-1-positive, isolated PLA2R/THSD7A-positive, and triple antigen-negative membranous nephropathy. Among the 832 patients with primary membranous nephropathy, 11 of 54 (20%) patients with PLA2R-negative membranous nephropathy had THSD7A-positive membranous nephropathy. NELL-1-positive membranous nephropathy accounted for 35% (15 of 43) of all patients with PLA2R- and THSD7A-negative membranous nephropathy. One patient was double positive for NELL-1 and PLA2R in glomerular deposits and positive for only the PLA2R antibody in the serum. Most patients with NELL-1-positive membranous nephropathy were women. No tumors were found. There were significant differences in the prevalence of IgG subtypes between patients with different antigen positivity. Among patients with isolated NELL-1-positive membranous nephropathy, although 80% (12 of 15) were IgG4 staining positive, the proportion of IgG4 dominance was only 67% (ten of 15). About one third of patients who were PLA2R and THSD7A negative were NELL-1 positive in Chinese patients with primary membranous nephropathy. NELL-1-positive membranous nephropathy was more common than THSD7A-positive membranous nephropathy in PLA2R-negative membranous nephropathy.

The lysosomal endopeptidases Cathepsin D and L are selective and effective proteases for the middle-down characterization of antibodies.

Mass spectrometry is gaining momentum as a method of choice to de novo sequence antibodies (Abs). Adequate sequence coverage of the hypervariable regions remains one of the toughest identification challenges by either bottom‐up or top‐down workflows. Methods that efficiently generate mid‐size Ab fragments would further facilitate top‐down MS and decrease data complexity. Here, we explore the proteases Cathepsins L and D for forming protein fragments from three IgG1s, one IgG2, and one bispecific, knob‐and‐hole IgG1. We demonstrate that high‐resolution native MS provides a sensitive method for the detection of clipping sites. Both Cathepsins produced multiple, albeit specific cleavages. The Abs were cleaved immediately after the CDR3 region, yielding ~ 12 kDa fragments, that is, ideal sequencing‐sized. Cathepsin D, but not Cathepsin L, also cleaved directly below the Ab hinge, releasing the F(ab’)2. When constrained by the different disulfide bonds found in the IgG2 subtype or by the tertiary structure of the hole‐containing bispecific IgG1, the hinge region digest product was not produced. The Cathepsin L and Cathepsin D clipping motifs were related to sequences of neutral amino acids and the tertiary structure of the Ab. A single pot (L + D) digestion protocol was optimized to achieve 100% efficiency. Nine protein fragments, corresponding to the VL, VH, CL, CH1, CH2, CH3, CL + CH1, and F(ab')2, constituted ~ 70% of the summed intensities of all deconvolved proteolytic products. Cleavage sites were confirmed by the Edman degradation and validated with top‐down sequencing. The described work offers a complementary method for middle‐down analysis that may be applied to top‐down Ab sequencing.EnzymesCathepsin L—EC 3.4.22.15, Cathepsin D—EC 3.4.23.5.

Molecular and functional characterization of HtrA protein in Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae (A.pleuropneumoniae) causes serious economic loss for the swine industry. A high-temperature requirements A (HtrA)-like protease and its homologs have been reported to be involved in protein quality control and expression of important immunoprotective antigens in many pathogens. In this study, we showed that HtrA of A.pleuropneumoniae exhibited both chaperone and proteolytic activities. Moreover, Outer membrane protein P5 (OmpP5) in A.pleuropneumoniae and Heat shock protein 90 (Hsp90) in porcine lung tissues were first discovered and identified as specific proteolytic substrates for rHtrA. The maximum cleavage activity occurs at 50 ℃ in a time-dependent manner. In addition, rHtrA mainly induced IgG 2a subtype of IgG and Th1 (IFN-γ, IL-2) response in a mice model, and promoted a significant proliferation of spleen lymphocytes compare with negative control (P < 0.05). The survival rates of 37.5 % were observed against A.pleuropneumoniae strain. Together, these data demonstrate that rHtrA plays a multi-functional role in A.pleuropneumoniae.

Prevalence of SARS-CoV-2 Specific Antibodies in Asymptomatic Hemodialysis Patients

ABSTRACT Background Since the outbreak of the new coronavirus pandemic, the importance of carrying out an infection check to prevent acquisition and transmission among end-stage renal disease patients (ESRD) under maintenance hemodialysis (MHD) has become a major concern in the health care system. Applying serology screening tests could enlighten the view with regards to disease prevalence in dialysis wards. Methods We subjected 328 end-stage renal disease patients to maintenance hemodialysis. After dividing patients into suspicious and non-suspicious groups for COVID-19 infection based on their clinical manifestation, they were investigated for SARS-CoV-2 specific IgM and IgG screening against nucleoprotein (NP), spike protein (SP), and receptor-binding domain (RBD), utilizing our recently developed ELISA tests. Results We found that approximately 10.1% of asymptomatically tested cases were antibody positive. Although IgG positivity showed a higher prevalence than IgM across all three virus antigen subunits, there were no significant differences among mentioned immunoglobulins of the studied groups. The most prevalent antibody was from the IgG subtype against virus nucleoprotein (NP), while the lowest prevalence was attributed to receptor-binding domain (RBD) IgM. Conclusion High seropositive rate among asymptomatic end-stage renal disease patients, as a sample of high-risk population, reflected the importance of considering SARS-CoV-2 specific antibody screening for disease containment.

Antibody (IgA, IgG, and IgG Subtype) Responses to SARS-CoV-2 in Severe and Nonsevere COVID-19 Patients.

For the assessment of vaccine-induced immune response and to understand the role of antibodies in neutralization, it is necessary to assess dynamics of various antibodies in patients with different clinical manifestations. This study aims to quantitate circulating levels of IgA/IgG and IgG subtypes induced at different days postonset of symptoms, in severe and nonsevere patients. For this, serum or plasma samples (n = 146) collected from 79 COVID-19 patients were used. Indirect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific IgA, IgG, and IgG subtype specific enzyme-linked immunosorbent assays (ELISAs) were performed. Antibody titers between severe and nonsevere patients were compared at different times postonset of clinical symptoms. Titers in ELISA were compared to neutralizing antibody (Nab) titers determined by plaque reduction neutralization test (PRNT). Over 75% patients were positive for IgA/IgG antibodies in the first week. The ELISA titers did not differ during the first week; however, severe disease exhibited raised titers thereafter. Nab titers correlated with the ELISA titers in mild presentation but not in severe disease. IgA and IgG1 antibodies correlated stronger with Nabs. The findings highlighted that IgA together with IgG play an important in SARS-CoV-2 neutralization. These results will prove useful in assessing efficacy of vaccines and understanding disease pathogenesis.

Prognostic Significance and Prevalence of IgG Subtypes in Rh Haemolytic Disease of Fetus and Newborn

Many authors have reported poor prognostic value of anti-D antibody titer in the setting of Hemolytic Disease of Fetus and Newborn (HDFN). According to literature, HDFN cases with IgG1 and IgG3 have more severity compared to IgG2 and IgG4.Therefore, we planned this study to evaluate the prevalence and prognostic value of IgG subtypes in the setting of Rh HDFN. This was a retrospective study performed at a tertiary care center in north India from October 2015 to November 2017. Women with anti-D antibody were included in the study and categorized on the basis of presence of specific IgG subtype. "DAT IgG1/IgG3 ID" card (BIO-RAD) was used for determining the subclass of IgG. Various clinical, laboratory & interventional parameters were used to categorize fetal outcome in severe and non-severe cases. Perinatal outcome was then compared between women with different IgG subclass profile. Subclass distribution among 80 alloimmunized women was 26.2% for IgG1, 15% for IgG3, 46.2% for IgG1 + IgG3 and the rest had neither IgG1 nor IgG3. Severity of HDFN was significantly higher when IgG1 &/or IgG3 were present alone or in combination, compared to cases with absence of IgG1 or IgG3 (p value < 0.05). Risk of severe HDFN was significantly higher in the presence of IgG1 &/or IgG3 and the severity was highest when both IgG1 and IgG3 were present. We recommend that IgG subclass determination should be included in a multi-parameter protocol for more accurate prediction HDFN severity to ensure timely referral and intervention.

Immunization with SP_1992 (DiiA) Protein of Streptococcus pneumoniae Reduces Nasopharyngeal Colonization and Protects against Invasive Disease in Mice.

Knowledge-based vaccinology can reveal uncharacterized antigen candidates for a new generation of protein-based anti-pneumococcal vaccines. DiiA, encoded by the sp_1992 locus, is a surface protein containing either one or two repeats of a 37mer N-terminal motif that exhibits low interstrain variability. DiiA belongs to the core proteome, contains several conserved B-cell epitopes, and is associated with colonization and pathogenesis. Immunization with DiiA protein via the intraperitoneal route induced a strong IgG response, including different IgG subtypes. Vaccination with DiiA increased bacterial clearance and induced protection against sepsis, conferring 70% increased survival at 48 h post-infection when compared to the adjuvant control. The immunogenic response and survival rates in mice immunized with a truncated DiiA version lacking 119 N-terminal residues were remarkably lower, confirming the relevance of the repeat zone in the immunoprotection by DiiA. Intranasal immunization of mice with the entire recombinant protein elicited mucosal IgG and IgA responses that reduced bacterial colonization of the nasopharynx, confirming that this protein might be a vaccine candidate for reducing the carrier rate. DiiA constitutes an example of how functionally unannotated proteins may still represent promising candidates that can be used in prophylactic strategies against the pneumococcal carrier state and invasive disease.

Age-Dependent Evaluation of Immunoglobulin G Response after Chikungunya Virus Infection.

Current chikungunya antibody prevalence and titers are likely to differ based on the exposure rates before the 2006 reemergence in India. For vaccine usage, such data are of immense importance. This study addresses age-stratified IgG titers and its subtypes in Pune, India, endemic for the disease. 170 age-stratified serum pools from 791 individuals with prior chikungunya exposure, and 15 samples from acute disease phase were analyzed. An indirect ELISA based on inactivated chikungunya virus was used to determine anti-CHIKV-IgG and its subtypes. Neutralizing antibody titers (plaque reduction neutralization test [PRNT]) were compared with binding antibody titers (ELISA). Anti-CHIKV-IgG titers along with IgG1 and IgG4 increased till the age-group of until 11-15 years and remained comparable thereafter till > 65 years. IgG1 was the predominant IgG subtype detected in all the pools, whereas IgG4 was present in 151/170 pools. Strong positive correlation of IgG1 was obtained with CHIKV-PRNT50 titers. None of the sample had anti-CHIKV-IgG2, whereas five pools had IgG3 antibody. In the acute-phase serum sample, IgG1 was present in all the samples, whereas IgG4 was present in 8/15 samples. IgG4 was predominant in four samples. During acute phase and at different times postinfection, IgG1 circulated in high titers followed by IgG4. Higher antibody titers in adults reflect reexposures. The data will prove useful in assessing immune response to CHIKV vaccine in relation to IgG subtype.

Sequence and N-glycan diversity analysis of immunoglobulin G from buffalo milk using RP-UHPLC MS/MS.

Immunoglobulin G is the abundant antibody present in the colostrum and milk of major dairy animals. In the present study, buffalo milk IgG was characterized for its amino acid sequence and glycan diversity using reverse phase liquid chromatography coupled to ESI-Q-TOF MS in tandem mode. Amino acid sequence analysis of heavy chain constant region revealed the presence of two IgG subtypes namely IgG1 and IgG3, with IgG1 being the abundant. The complete light chain constant region sequence was also determined. N-glycan sequence analysis at a highly conserved site Asn-Ser-Thr revealed the presence of mainly biantennary complex type with core fucosylation (34%), bisecting GlcNAc (19%) and sialylation with both Neu5Ac and Neu5Gc (14%). The observed glycan diversity in buffalo milk IgG is in part comparable with bovine colostrum as well as human, bovine, goat serum counterparts.

Diagnostic Approach to Glomerulonephritis With Fibrillar IgG Deposits and Light Chain Restriction

IntroductionThe pathologic approach to glomerulonephritis (GN) with fibrillar IgG deposits and light chain restriction remains a diagnostic challenge.MethodAll GN with fibrillar deposits of IgG and apparent light chain restriction on standard immunofluorescence on frozen tissue (IF-F) accessioned at the Columbia Renal Pathology Laboratory from 2012 to 2019 were identified. Additional studies including staining for Congo red, DNAJB9, IgG subtypes, and immunofluorescence on pronase-digested paraffin sections (IF-P) were performed.ResultBased on the results, biopsy samples were reclassified as polytypic DNAJB9-positive fibrillary glomerulonephritis (pFGN, n = 14), monotypic DNAJB9-positive FGN (mFGN, n = 7), GN with polytypic DNAJB9-negative fibrillar IgG deposits (n = 2), and GN with monotypic DNAJB9-negative fibrillar IgG deposits (n = 6). Among DNAJB9-positive FGN samples, IgG subtype staining was able to exclude monotypic deposits by demonstrating reactivity for ≥2 IgG subtypes (usually IgG1 and IgG4) in 67% (14 of 21), including 9 that would have been misclassified as monotypic by IF-F and IF-P alone. Monotypic DNAJB9-positive fibrillary glomerulonephritis (FGN) was not associated with monoclonal gammopathy in 5 of 6 patients. GN with monotypic DNAJB9-negative fibrillar IgG deposits exhibited focal parallel fibril alignment and frequent association with chronic lymphocytic leukemia, but lacked the diagnostic microtubules of immunotactoid GN.ConclusionA systematic diagnostic approach with ancillary techniques is essential for proper classification and assignment of monoclonal gammopathy of renal significance status in cases of GN with fibrillary IgG deposits and light chain restriction by IF-F.

A chemokine-fusion vaccine targeting immature dendritic cells elicits elevated antibody responses to malaria sporozoites in infant macaques

Infants and young children are the groups at greatest risk for severe disease resulting from Plasmodium falciparum infection. We previously demonstrated in mice that a protein vaccine composed of the chemokine macrophage inflammatory protein 3α genetically fused to the minimally truncated circumsporozoite protein of P. falciparum (MCSP) elicits high concentrations of specific antibody and significant reduction of liver sporozoite load in a mouse model system. In the current study, a squalene based adjuvant (AddaVax, InvivoGen, San Diego, Ca) equivalent to the clinically approved MF59 (Seqiris, Maidenhead, UK) elicited greater antibody responses in mice than the previously employed adjuvant polyinosinic:polycytidylic acid, ((poly(I:C), InvivoGen, San Diego, Ca) and the clinically approved Aluminum hydroxide gel (Alum, Invivogen, San Diego, Ca) adjuvant. Use of the AddaVax adjuvant also expanded the range of IgG subtypes elicited by mouse vaccination. Sera passively transferred into mice from MCSP/AddaVax immunized 1 and 6 month old macaques significantly reduced liver sporozoite load upon sporozoite challenge. Protective antibody concentrations attained by passive transfer in the mice were equivalent to those observed in infant macaques 18 weeks after the final immunization. The efficacy of this vaccine in a relevant non-human primate model indicates its potential usefulness for the analogous high risk human population.

Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals.

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were composed of immunoglobulin heavy variable 3-53 (IGHV3-53) or IGHV3-66 and immunoglobulin heavy joining 6 (IGHJ6) genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different IGHV chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in 6 of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

Study of the Serum Immunoglobulin and Cell-Mediated Immunity in Patients with Congenital Severe Hemophilia.

It has been shown that a close relationship exists between the immune system and coagulation cascade. Hemophilia A is an X-linked, recessive bleeding disorder caused by deficiency of functional plasma clotting factor VIII that is classically treated with factor VIII replacement therapy. Despite this, some patients produce inhibitors or antibodies against epitopes of infused factor VIII, indicating the activation of the adaptive immune system. The aim of this study was to evaluate the change in the T cell frequency and serum immunoglobulin level in patients with congenital hemophilia A, especially those who produce inhibitors against factor VIII. This is a cross-sectional, case-control study on congenital hemophilia A patients with severe factor VIII deficiency. Twenty-eight hemophilia A male patients were randomly selected along with twenty age-matched healthy males, as the control group. Serum immunoglobulin concentration was measured by nephelometry (for IgG, IgA and IgM) and enzyme-linked immunosorbent assay (ELISA) (for IgE) and the frequency of CD4+ and CD8+ T cells were calculated using a flow cytometry method. Serum IgG was significantly higher in hemophilic patients compared to controls (14.35 ± 3.60, vs. 12.4 ± 1.72, p = 0.014). Among IgG subtypes, the IgG1 antibody was significantly higher in hemophilia patients than control group (p < 0.001). The frequency of CD4+ as well as CD8+ T cells did not significantly differ between patients and control group (p > 0.05). There was no significant difference between patients with and without inhibitors re-garding serum immunoglobulin level, different IgG subtypes, the frequency of CD4+ and CD8+ T cells as well as CD4/CD8 ratio (p > 0.05). Patients with hemophilia A have high levels of serum immunoglobulin especially IgG1. Therefore, further larger studies along with close observation and evaluation of the presence of serum inhibitor is recommended every 3 months.

Glycosylation of Plant-Produced Immunoglobulins.

Many economically important protein-based therapeutics like monoclonal antibodies are glycosylated. Due to the recognized importance of this type of posttranslational modification, glycoengineering of expression systems to obtain highly active and homogenous therapeutics is an emerging field. Although most of the monoclonal antibodies on the market are still produced in mammalian expression platforms, plants are emerging as an alternative cost-effective and scalable production platform that allows precise engineering of glycosylation to produce targeted human glycoforms at large homogeneity. Apart from producing more effective antibodies, pure glycoforms are required in efforts to link biological functions to specific glycan structures. Much is already known about the role of IgG1 glycosylation and this antibody class is the dominant recombinant format that has been expressed in plants. By contrast, little attention has been paid to the glycoengineering of recombinant IgG subtypes and the other four classes of human immunoglobulins (IgA, IgD, IgE, and IgM). Except for IgD, all these antibody classes have been expressed in plants and the glycosylation has been analyzed in a site-specific manner. Here, we summarize the current data on glycosylation of plant-produced monoclonal antibodies and discuss the findings in the light of known functions for these glycans.

Immunoglobulin subtyping and quantification in direct antiglobulin test: positive haemolysis in an HIV-prevalent setting

AimsPositive direct antiglobulin tests (DATs) are valuable in identifying the aetiology of autoimmune haemolysis and in guiding therapeutic intervention. However, in HIV-positive individuals with background polyclonal gammopathy, a positive DAT...

Immunoglobulin Sub-Typing and Quantification in Coombs Positive Hemolysis

Key words:immunoglobulin, coombs test, hemolysis, HIV, quantification Introduction A positive direct antiglobulin test (DAT, Coombs test) is valuable in identifying the etiology of autoimmune hemolysis and in guiding an immunomodulatory therapeutic intervention strategy. However, in HIV positive individuals with background polyclonal gammopathy, a false positive DAT is common. In this setting, immunoglobulin quantification and subtyping may be of value in characterizing the autoimmune hemolysis. There is paucity of published literature evaluating the diagnostic usefulness of IgG subtyping and quantification in HIV positive individuals who are investigated for possible autoimmune haemolysis (AIHA). The recently introduced gel card used for AIHA diagnosis can subtype and quantify the IgG antibody titre. This study evaluated the usefulness of IgG quantification and subtyping in the diagnostic workup of autoimmune hemolysis in patients who were DAT positive with and without HIV infection. Methods: This retrospective, cross sectional study was reviewed and approved by the institutional Ethics Committee. The study population included HIV positive and HIV negative patients who were investigated for autoimmune hemolysis in a quaternary care hospital as part of their diagnostic workup. Those with a positive DAT had their IgG subtyped and quantified using the ID -Card DAT IgG1/IgG3 and IgG-dilution cards (Bio-Rad©, Cressier, Switzerland). Results: From December 2019 to March 2020, 90 patients admitted in our hospital were investigated for AIHA. Forty four (49%) were found to be DAT positive. Of those who were DAT positive, 26 (59%) had biochemical evidence of haemolysis (raised LDH, raised unconjugated bilirubin). Of the total DAT positive population, 16 (36%) were HIV positive. Concurrent HIV and haemolysis was present in 8 patients. Four of the 8 had antibody titre≤1:30. Most patients had IgG 1 subtype. None of the HIV positive patients without features of haemolysis had IgG1 or IgG3 subtypes present and 2 of the 8 had antibody titres≤1:30. Conclusion: IgG quantification and subtyping was found to be of limited value in the diagnostic characterization of AIHA in HIV positive patients with false positive DAT. Disclosures Mahlangu: CSL Behring, Catalyst Biosciences, Freeline Therapeutics, Novo Nordisk, F. Hoffmann-La Roche Ltd, Sanofi, Spark and Takeda:Consultancy;CSL Behring, Catalyst Biosciences, Novo Nordisk, F. Hoffmann-La Roche Ltd, Sanofi, Spark and Takeda:Speakers Bureau;South Africa Medical Research Council, Wits Health Consortium, Colleges of Medicine of South Africa:Membership on an entity's Board of Directors or advisory committees;BioMarin, CSL Behring, Freeline Therapeutics, Novo Nordisk, Novartis, Pfizer, Sanofi, F. Hoffmann-La Roche Ltd, uniQure:Research Funding.

Chimeric Antigen Receptor T Cell Therapy in the Relapsed or Refractory Multiple Myeloma with Extramedullary Disease–a Single Institution Observation in China

Background: Extramedullary disease (EMD) is characterized as the presence of myeloma cells invasion outside the bone marrow in a patient with multiple myeloma (MM), it may be found 6%~8% in de novo patients, and 10% to 30% in relapsed or refractory (R/R) MM patients across the overall disease course (Joan Blade et al. 2011, Matthew Weinstock et al. 2013 and 2015, Cyrille Touzeau et al. 2015). EMD was considered as high-risk group of myeloma and is associated with adverse prognosis and the management is particularly challenging. Here we discuss and improve the understanding of the clinical characters and prognoses of the R/R MM with EMD in the treatment of CAR T therapy, illustrate our experience in a single institution. Methods: LCAR-B38M is a Chimeric antigen receptor (CAR) T cell therapy with 4-1BB as the co-stimulator, a dual epitope-binding CAR T cell therapy directed against 2 distinct B cell maturation antigen (BCMA) epitopes. In total of 57 patients were treated in China from The Second Affiliated Hospital of Xi'an Jiaotong University (LEGEND-2, NCT03090659), 17 patients with R/R MM with EMD were included and received the CAR T treatment. The study was initiated on March 30, 2016, and the analysis reported here is from a data cutoff date of July 31, 2019, the detailed treatment schema was presented in the former paper (Zhao et al, 2018). Results: Overall, 17 (29.8%) of 57 treated patients with EMD were included in our CAR T treatment. The median age of R/R MM patients with EMD (EMD group) and without EMD (non-EMD group) were 55 (range, 27-72) and 53.3 (range, 42-73) years old respectively, and the median number of prior therapy lines were 3 (range, 1-6 and 1-9) in both groups. There were no statistical difference in the proportion of male and female patients, median time from initial diagnosis and ISS stage in EMD group and non-EMD group. The Eastern Cooperative Oncology Group (ECOG) scores of patients receiving CAR T treatment were 0-2 points, the proportion of ECOG 1-2 points in EMD group were higher than non-EMD group (P &amp;lt; 0.05, R=0.1474, 95% CI, 0.03 to 0.73). IgG and IgA subtype were the main types in the non-EMD patients while the light chain type was more frequently seen in EMD group (P = 0.014, OR=0.1886, 95% CI, 0.05 to 0.66). The overall response rates (ORR) in the EMD group and non-EMM group were 82.4% and 90% respectively (Fig 1 A), and there were no significant difference in the median time to the first response and the best response between the EMD group and non-EMD group(P &amp;gt; 0.05). At cutoff, the median follow-up was 25 months, the median progression-free survival (PFS) in EMD group and non-EMD group were 8.1 months and 25 months (P &amp;lt; 0.001, R=0.32, 95% CI, -0.19 to 0.84), and the median overall survival (OS) were 13.9 months and not reached (P = 0.0019, R=4.833, 95% CI, 1.91 to 12.22), survival curves were shown in Fig 1 B, C . Conclusions: LCAR-B38M is a highly effective therapy in R/R MM patients with EMD and without EMD, and both groups had similar response time and reached a high ORR rate. R/R MM patients with EMD demonstrated a poor prognosis and they may lost their best response in a relatively shorter time compared with the patients without EMD. With a median follow-up of 25 months, LCARB38M manifested a relatively durable therapeutic effect in this high-risk R/R MM patients with EMD. Disclosures No relevant conflicts of interest to declare.