IgG Activity Research Articles

Network Model of Immune Responses Reveals Key Effectors to Single and Co-infection Dynamics by a Respiratory Bacterium and a Gastrointestinal Helminth

Co-infections alter the host immune response but how the systemic and local processes at the site of infection interact is still unclear. The majority of studies on co-infections concentrate on one of the infecting species, an immune function or group of cells and often focus on the initial phase of the infection. Here, we used a combination of experiments and mathematical modelling to investigate the network of immune responses against single and co-infections with the respiratory bacterium Bordetella bronchiseptica and the gastrointestinal helminth Trichostrongylus retortaeformis. Our goal was to identify representative mediators and functions that could capture the essence of the host immune response as a whole, and to assess how their relative contribution dynamically changed over time and between single and co-infected individuals. Network-based discrete dynamic models of single infections were built using current knowledge of bacterial and helminth immunology; the two single infection models were combined into a co-infection model that was then verified by our empirical findings. Simulations showed that a T helper cell mediated antibody and neutrophil response led to phagocytosis and clearance of B. bronchiseptica from the lungs. This was consistent in single and co-infection with no significant delay induced by the helminth. In contrast, T. retortaeformis intensity decreased faster when co-infected with the bacterium. Simulations suggested that the robust recruitment of neutrophils in the co-infection, added to the activation of IgG and eosinophil driven reduction of larvae, which also played an important role in single infection, contributed to this fast clearance. Perturbation analysis of the models, through the knockout of individual nodes (immune cells), identified the cells critical to parasite persistence and clearance both in single and co-infections. Our integrated approach captured the within-host immuno-dynamics of bacteria-helminth infection and identified key components that can be crucial for explaining individual variability between single and co-infections in natural populations.

Agonistic Autoantibodies as Vasodilators in Orthostatic Hypotension

Agonistic autoantibodies to the β-adrenergic and muscarinic receptors are a novel investigative and therapeutic target for certain orthostatic disorders. We have identified the presence of autoantibodies to β2-adrenergic and/or M3 muscarinic receptors by ELISA in 75% (15 of 20) of patients with significant orthostatic hypotension. Purified serum IgG from all 20 of the patients and 10 healthy control subjects were examined in a receptor-transfected cell-based cAMP assay for β2 receptor activation and β-arrestin assay for M3 receptor activation. There was a significant increase in IgG-induced activation of β2 and M3 receptors in the patient group compared with controls. A dose response was observed for both IgG activation of β2 and M3 receptors and inhibition of their activation with the nonselective β blocker propranolol and muscarinic blocker atropine. The antibody effects on β2 and/or M3 (via production of NO) receptor-mediated vasodilation were studied in a rat cremaster resistance arteriole assay. Infusion of IgG from patients with documented β2 and/or M3 receptor agonistic activity produced a dose-dependent vasodilation. Sequential addition of the β-blocker propranolol and the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester partially inhibited IgG-induced vasodilation (percentage of maximal dilatory response: from 57.7±10.4 to 35.3±4.6 and 24.3±5.8, respectively; P<0.01; n=3), indicating that antibody activation of vascular β2 and/or M3 receptors may contribute to systemic vasodilation. These data support the concept that circulating agonistic autoantibodies serve as vasodilators and may cause or exacerbate orthostatic hypotension.

Human Lipooligosaccharide IGG That Prevents Endemic Meningococcal Disease Recognizes an Internal Lacto-N-neotetraose Structure

Antibodies that initiate complement-mediated killing of Neisseria meningitidis as they enter the bloodstream from the oropharynx protect against disseminated disease. Human IgGs that bind the neisserial L7 lipooligosaccharide (LOS) are bactericidal for L3,7 and L2,4 meningococci in the presence of human complement. These strains share a lacto-N-neotetraose (nLc4) LOS α chain. We used a set of mutants that have successive saccharide deletions from the nLc4 α chain to characterize further the binding and bactericidal activity of nLc4 LOS IgG. We found that the nLc4 α chain conforms at least four different antigens. We separately purified IgG that required the nLc4 (non-reducing) terminal galactose (Gal) for binding and IgG that bound the truncated nLc3 α chain that lacks this Gal residue. IgG that bound the internal nLc3 α chain killed both L3,7 and L2,4 strains, whereas IgG that required the nLc4 terminal Gal residue for binding killed L2,4 stains but not L3,7 strains. These results show that the diversity of LOS antibodies in human serum is as much a function of the conformation of multiple antigens by a single glycoform as of the production of multiple glycoforms. Differences in sensitivity to killing by human nLc4 LOS IgG may account for the fact that fully two-thirds of endemic group B meningococcal disease in infants and children is caused by L3,7 strains, but only 20% is caused by L2,4 stains.

Autoantibody activation of beta-adrenergic and muscarinic receptors contributes to an “autoimmune” orthostatic hypotension

Background Orthostatic hypotension (OH) is characterized by an abnormal autonomic response to upright posture. Activating autoantibodies to β1/2-adrenergic (AAβ1/2AR) and M2/3 muscarinic receptors (AAM2/3R) produce vasodilative changes in the vasculature that may contribute to OH. Methods Immunoglobulin (Ig)G from 6 patients with idiopathic OH harboring autoantibodies and from 10 healthy control subjects were examined for: 1) β1AR and M2R activity with a perfused Purkinje fiber assay and PKA assay in H9c2 cells and 2) vasodilator β2AR and M3R activity using a pressurized cremaster resistance arteriole assay. Changes in IgG activity with and without propranolol, atropine, and L-NAME were used to estimate AAβAR, AAM2R, and AAM3R activation of their respective functions. Results All six patients had elevated enzyme-linked immunosorbent assay titers to at least one of the receptors compared with controls. βAR-mediated contractility activity and M2R activity were increased in five of the six patients. IgG from all six patients produced a direct vasodilator effect on cremaster arterioles. βAR and nitric oxide synthase blockade led to near normalization of IgG-induced vasodilation. Conclusion AAβ1/2AR and AAM2/3R are present in some patients with idiopathic OH compatible with an in vivo effect. These autoantibodies and their cardiovascular effects provide new mechanistic insights into the pathophysiology of OH.

Presence and Evolution of a Catalytic Activity in Patients with Severe, Mild or Moderate Hemophilia A

Abstract 1211The development of neutralizing anti-Factor VIII (FVIII) antibodies is the major complication of the treatment of patients with hemophilia A (HA). Several mechanisms of inhibition have been described: steric hindrance, immune complex formation and catalytic antibodies. Anti-FVIII catalytic antibodies act like enzymes and lead to the hydrolysis of FVIII. Lacroix-Desmazes et al. had shown the presence of catalytic antibodies in the plasma of patients with severe HA who had developed inhibitors (13/24) (Lacroix-Desmazes et al, NEJM, 2002). Previous studies on catalytic antibodies reported results for patients with severe HA at one time point, exclusively. Thus, we proposed to extend the analysis of catalytic antibodies to patients with a mild or moderate HA and to follow over their lifetime patients who develop inhibitor.We studied plasma samples from 33 patients with HA. Sixteen were patients with severe HA, including 8 patients with inhibitor (Inh+) and 8 patients without inhibitor (Inh-), and 17 were mild or moderate HA patients (7 Inh+ and 10 Inh-). Among Inh+ patients, 6 were treated on-demand (3 severe and 3 moderate HA patients) and 9 were submitted to an immune tolerance induction (ITI) protocol (4 severe and 5 mild or moderate patients with HA). A therapeutic preparation of pooled normal IgG (IVIg) from healthy donors was used as a source of normal IgG. We also used plasma from 13 male healthy donors. As described previously [2], IgG were purified from plasma by affinity-chromatography on protein G followed by a size-exclusion chromatography in presence of urea. Then, catalytic activity was evaluated by the hydrolysis of FVIII after incubation with purified IgG (Lacroix-Desmazes et al, Nature, 1999). Inhibitor titer is measured by modified Bethesda test.Mean FVIII-hydrolyzing rates were determined for healthy donors, HA patients and IVIg, used as control. Catalytic activity of HA patients IgG was significantly higher than those of healthy donors or IVIg (p<0.01 and p<0.001, respectively). Sixty four per cent of patients with HA had catalytic antibodies, regardless the phenotype nor inhibitor presence. In addition, prevalence of FVIII-hydrolyzing antibodies for Inh+ and Inh- HA patients was 94% and 47%, respectively. However, the mean FVIII-hydrolysis rate was comparable for both groups (0.23 ± 0.06 mmol/min/mol). Surprisingly, we showed that the mean catalytic activity of mild or moderate HA patients were significantly lower than those of severe HA patients (0.17 ± 0.05 versus 0.31 ± 0.07). Interestingly, we were able to study the evolution of both catalytic and inhibitory activities for patients who developed inhibitor. We observed 2 profiles: in the first case, FVIII-hydrolysis rate and inhibitor titer followed the same trend, but in the second case, these two parameters showed a dissociated evolution. These results were independent of the type of treatment (on-demand or ITI).For the first time, we studied catalytic activity for patients with mild or moderate HA. In comparison with patients with severe HA, catalytic activity is much lower for patients with mild or moderate HA. In addition, most of patients with severe HA had FVIII-hydrolytic antibodies (88 %), in contrast with previous studies (50%) (Lacroix-Desmazes et al, NEJM, 2002). In the same way, we showed the presence of catalytic antibodies in patients without inhibitor. Moreover, we studied the evolution of catalytic activity and inhibitor titer over the time for patients with inhibitor and showed that catalytic activity did not necessarily follow the same trend as inhibitory activity. The results suggested that catalytic antibodies could not act like neutralizing antibodies. Disclosures:No relevant conflicts of interest to declare.

Enhanced humoral and mucosal immune responses after intranasal immunization with chimeric multiple antigen peptide of LcrV antigen epitopes of Yersinia pestis coupled to palmitate in mice

Yersinia pestis is the causative agent of the most deadly disease plague. F1 and V antigens are the major vaccine candidates. Six protective epitopes of V antigen of varying length (15-25aa) were assembled on a lysine backbone as multiple antigen peptide (MAP) using standard Fmoc chemistry. Palmitate was coupled at amino terminus end. Amino acid analysis, SDS-PAGE, immunoblot and immunoreactivity proved the authenticity of MAP. MAP was immunized intranasally encapsulated in PLGA (polylactide-co-glycolide) microspheres and with/without/adjuvants murabutide and CpG ODN 1826 (CpG), in three strains of mice. Humoral and mucosal immune responses were studied till day 120 and memory response was checked after immunization with native V antigen on day 120. Epitope specific serum and mucosal washes IgG, IgA, IgG subclasses and specific activity were measured by indirect ELISA and sandwich ELISA, respectively. IgG and IgA peak antibody titers of all the MAP construct formulations in sera were ranging from 71,944 to 360,578 and 4493 to 28,644, respectively. MAP with CpG showed significantly high (p<0.0001) antibody titers ranging from 101,690 to 360,578 for IgG and 28,644 for IgA. Mucosal peak IgG and IgA titers were ranging from 1425 to 8072 and 1425 to 7183, respectively in intestinal washes and 799-4528 and 566-4027, respectively in lung washes. MAP with CpG showed significantly high (p<0.001) SIgA titers of 8000 in lung and 16,000 in intestinal washes. IgG isotyping revealed IgG2a/IgG1 ratio>1 with CpG. Serum and mucosal antipeptide IgG and IgA specific activities correlated well with antibody titers. All the constituent peptides contributed towards immune response. Structural analysis of MAP revealed little or no interaction between the peptides. Present study showed MAP to be highly immunogenic with high and long lasting antibody titers in serum and mucosal washes with good recall response with/without CpG as an adjuvant which can be used for vaccine development for plague.

Chemical composition and immune status of dairy goat colostrum fractions during the first 10 h after partum

The objective of this study was to examine the chemical and immunological quality of goat colostrum following delivery. Twenty dairy goats of the Majorera breed were milked 1h postpartum and then every hour for 10h after the first milking. Residual colostrum (RC) was also obtained at the time of the first milking after i.v. injection of 2I.U. of oxytocin. Colostrum yield, approximate composition, SCC, IgG, IgM, IgA and chitotriosidase activity were measured in milking colostrum (MC), RC, and colostrums in each hour sample. A PROC MIXED procedure was performed and a Tukey's test was done to determine the statistical significance of differences in the composition of the colostrum fractions and the colostrum obtained over time. At the first milking, MC and RC weighed 2506 and 237g, respectively. At 1h and 10h after the first milking, the colostrum yield was 174 and 120g, respectively, with a continuous drop in yield over the course of the experiment. A drop in protein production was also seen over time. Colostrum protein percentages were 10.4 and 10.2 in MC and RC. Colostrum protein percentages at 1 and 10h after first milking were 9.7% and 4.5%, respectively. The percentage of colostrum fat increased 1h after the first milking and then decreased to 6.1% at 10h after the first milking. The lactose colostrum percentage displayed an increase during the experimental period. Colostrum SCC was not affected by colostrum fractioning or time, with a range of 4.2–5.8×106cells/mL. IgG, IgM, IgA levels and chitotriosidase activity did not present differences between colostrum fractions at the first milking but displayed a drastic drop in subsequent milkings. In sum, the chemical and immunological quality of colostrum dropped quickly after the first milking, and thus goat keepers need only recover the first milked colostrum when they rear goat kids separately from dams.

Effect of dietary γ‐aminobutyric acid on laying performance, egg quality, immune activity and endocrine hormone in heat‐stressed Roman hens

This study was conducted to evaluate the effect of γ-aminobutyric acid (GABA) on laying performance, egg quality, digestive enzyme activity, hormone level and immune activities in Roman hens under heat stress. Roman hens (320 days old) were fed with 0, 25, 50, 75 and 100 mg/kg GABA, respectively during a 60-day experiment. Compared with control, supplementation of 50 mg/kg GABA improved the laying performance and egg quality by significantly increasing egg production, average egg weight and shell strength (P < 0.05), while decreasing the feed-egg ratio and cholesterol level. Anti-oxidation activity was improved by significantly increasing the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), but decreasing malondialdehyde level in serum (P < 0.05), while significantly increasing the glucose and total protein (TP) level, follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E(2) ), insulin, triiodothyronine (T(3) ) and free triiodothyronine (FT(3) ) levels, and IgG, IgA and complement (C3)activity in serum (P < 0.05). The results indicated that oral GABA improved laying performance and physical condition mainly by modulating hormone secretion, enhancing anti-oxidation and immune activity, and maintaining electrolyte balance. Fifty mg/kg was the optimum level for laying hens under heat stress in the present study.

2-Hydroxyethyl methacrylate (HEMA) promotes IgG but not IgM antibody production in vivo in mice

Individuals working in a dental clinic are exposed to 2-hydroxyethyl methacrylate (HEMA). HEMA has been found to have several effects on the immune system, including acting as an adjuvant in mice and stimulating the production of human IgG1 in vitro. In this study we continued to explore the immunomodulatory properties of HEMA in mice. Mice were co-injected subcutaneously with the following: HEMA + ovalbumin (OVA) in bicarbonate buffer, OVA in bicarbonate buffer, HEMA in bicarbonate buffer, or bicarbonate buffer alone. Mice immunized with OVA were killed 2 wk after a booster injection. Mice exposed to HEMA only were killed 6 d after the last injection with HEMA. Serum and spleens were collected. The activities of anti-OVA IgG and anti-OVA IgM were determined using ELISAs, as was the in vitro production of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by splenocytes after 2 d of incubation. Splenocyte proliferation was analyzed using [(3) H]thymidine decomposition. Mice exposed twice to HEMA in vivo had a higher baseline and a higher concanavalin A-stimulated proliferation of splenocytes, and produced less TNF-α in relation to IL-6, compared with controls. Immunization of mice with OVA/HEMA resulted in a higher anti-OVA IgG activity, relative to anti-OVA IgM activity, compared with controls. In conclusion, HEMA has selective effects on cytokine and antibody production in mice.

Immunization of Teenagers with a Fifth Dose of Reduced DTaP-IPV Induces High Levels of Pertussis Antibodies with a Significant Increase in Opsonophagocytic Activity

Waning vaccine-induced immunity against Bordetella pertussis is observed among adolescents and adults. A high incidence of pertussis has been reported in this population, which serves as a reservoir for B. pertussis. A fifth dose of reduced antigen of diphtheria-tetanus-acellular-pertussis and inactivated polio vaccine was given as a booster dose to healthy teenagers. The antibody activity against B. pertussis antigens was measured prior to and 4 to 8 weeks after the booster by different assays: enzyme-linked immunosorbent assays (ELISAs) of IgG and IgA against pertussis toxin (PT) and filamentous hemagglutinin (FHA), IgG against pertactin (PRN), opsonophagocytic activity (OPA), and IgG binding to live B. pertussis. There was a significant increase in the IgG activity against PT, FHA, and PRN following the booster immunization (P < 0.001). The prebooster sera showed a geometric mean OPA titer of 65.1 and IgG binding to live bacteria at a geometric mean concentration of 164.9 arbitrary units (AU)/ml. Following the fifth dose, the OPA increased to a titer of 360.4, and the IgG concentration against live bacteria increased to 833.4 AU/ml (P < 0.001 for both). The correlation analyses between the different assays suggest that antibodies against FHA and PRN contribute the most to the OPA and IgG binding.

Non-Apical Membrane Antigen 1 (AMA1) IgGs from Malian Children Interfere with Functional Activity of AMA1 IgGs as Judged by Growth Inhibition Assay

BackgroundApical membrane antigen 1 (AMA1) is one of the best-studied blood-stage malaria vaccine candidates. When an AMA1 vaccine was tested in a malaria naïve population, it induced functionally active antibodies judged by Growth Inhibition Assay (GIA). However, the same vaccine failed to induce higher growth-inhibitory activity in adults living in a malaria endemic area. Vaccination did induce functionally active antibodies in malaria-exposed children with less than 20% inhibition in GIA at baseline, but not in children with more than that level of baseline inhibition.MethodsTotal IgGs were purified from plasmas collected from the pediatric trial before and after immunization and pools of total IgGs were made. Another set of total IgGs was purified from U.S. adults immunized with AMA1 (US-total IgG). From these total IgGs, AMA1-specific and non-AMA1 IgGs were affinity purified and the functional activity of these IgGs was evaluated by GIA. Competition ELISA was performed with the U.S.-total IgG and non-AMA1 IgGs from malaria-exposed children.ResultsAMA1-specific IgGs from malaria-exposed children and U.S. vaccinees showed similar growth-inhibitory activity at the same concentrations. When mixed with U.S.-total IgG, non-AMA1 IgGs from children showed an interference effect in GIA. Interestingly, the interference effect was higher with non-AMA1 IgGs from higher titer pools. The non-AMA1 IgGs did not compete with anti-AMA1 antibody in U.S.-total IgG in the competition ELISA.ConclusionChildren living in a malaria endemic area have a fraction of IgGs that interferes with the biological activity of anti-AMA1 antibody as judged by GIA. While the mechanism of interference is not resolved in this study, these results suggest it is not caused by direct competition between non-AMA1 IgG and AMA1 protein. This study indicates that anti-malaria IgGs induced by natural exposure may interfere with the biological effect of antibody induced by an AMA1-based vaccine in the target population.

Monomeric and Dimeric IgG Fractions Show Differential Reactivity Against Pathogen-Derived Antigens

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

Effects on mouse immunity of long-term exposure in vivo to minute amounts of HEMA

2-Hydroxyethyl methacrylate (HEMA) leaks from cured restorations over time. Hence, HEMA can come into contact with cells of the immune system that are present in the oral mucosa and in the dental pulp. In this study, our aim was to develop a model of long-term exposure to minute amounts of HEMA and to record the immunological effects in mice. Osmotic pumps filled with either HEMA (8.2 M or 183 μM) or 0.9% NaCl (control) were implanted subcutaneously into the backs of mice and left in situ for 40 d, during which time the animals were immunized with ovalbumin (OVA). After 40 d, spleens and serum were collected. Splenocyte proliferation in vitro was analyzed by measuring the decomposition of [(3)H]thymidine. Splenocyte cytokine production and serum anti-OVA IgG, IgM and IgA activity were analyzed using ELISAs. Mice exposed to both the higher and the lower HEMA concentrations gained significantly less weight and produced significantly reduced amounts of interleukin-2 (IL-2) in vitro compared with control mice. Mice exposed to the lower HEMA concentration had a significantly reduced concanavalin A-stimulated splenocyte proliferation in vitro and blood anti-OVA IgA activity. In conclusion, long-term exposure to minute amounts of HEMA in vivo affects the general health of mice and suppresses certain immunological functions.

Fc-Glycosylation of IgG1 is Modulated by B-cell Stimuli

We have recently shown that IgG1 directed against antigens thought to be involved in the pathogenesis of rheumatoid arthritis harbor different glycan moieties on their Fc-tail, as compared with total sera IgG1. Given the crucial roles of Fc-linked N-glycans for the structure and biological activity of IgG, Fc-glycosylation of antibodies is receiving considerable interest. However, so far little is known about the signals and factors that could influence the composition of these carbohydrate structures on secreted IgG produced by B lymphocytes. Here we show that both "environmental" factors, such as all-trans retinoic acid (a natural metabolite of vitamin A), as well as factors stimulating the innate immune system (i.e. CpG oligodeoxynucleotide, a ligand for toll-like receptor 9) or coming from the adaptive immune system (i.e. interleukin-21, a T-cell derived cytokine) can modulate IgG1 Fc-glycosylation. These factors affect Fc-glycan profiles in different ways. CpG oligodeoxynucleotide and interleukin-21 increase Fc-linked galactosylation and reduce bisecting N-acetylglucosamine levels, whereas all-trans retinoic acid significantly decreases galactosylation and sialylation levels. Moreover, these effects appeared to be stable and specific for secreted IgG1 as no parallel changes of the corresponding glycans in the cellular glycan pool were observed. Interestingly, several other cytokines and molecules known to affect B-cell biology and antibody production did not have an impact on IgG1 Fc-coupled glycan profiles. Together, these data indicate that different stimuli received by B cells during their activation and differentiation can modulate the Fc-linked glycosylation of secreted IgG1 without affecting the general cellular glycosylation machinery. Our study, therefore, furthers our understanding of the regulation of IgG1 glycosylation at the cellular level.

Regulation of antinucleoprotein IgG by systemic vaccination and its effect on influenza virus clearance.

Seasonal influenza epidemics recur due to antigenic drift of envelope glycoprotein antigens and immune evasion of circulating viruses. Additionally, antigenic shift can lead to influenza pandemics. Thus, a universal vaccine that protects against multiple influenza virus strains could alleviate the continuing impact of this virus on human health. In mice, accelerated clearance of a new viral strain (cross-protection) can be elicited by prior infection (heterosubtypic immunity) or by immunization with the highly conserved internal nucleoprotein (NP). Both heterosubtypic immunity and NP-immune protection require antibody production. Here, we show that systemic immunization with NP readily accelerated clearance of a 2009 pandemic H1N1 influenza virus isolate in an antibody-dependent manner. However, human immunization with trivalent inactivated influenza virus vaccine (TIV) only rarely and modestly boosted existing levels of anti-NP IgG. Similar results were observed in mice, although the reaction could be enhanced with adjuvants, by adjusting the stoichiometry among NP and other vaccine components, and by increasing the interval between TIV prime and boost. Importantly, mouse heterosubtypic immunity that had waned over several months could be enhanced by injecting purified anti-NP IgG or by boosting with NP protein, correlating with a long-lived increase in anti-NP antibody titers. Thus, current immunization strategies poorly induce NP-immune antibody that is nonetheless capable of contributing to long-lived cross-protection. The high conservation of NP antigen and the known longevity of antibody responses suggest that the antiviral activity of anti-NP IgG may provide a critically needed component of a universal influenza vaccine.

Fc-glycosylation of IgG1 is modulated by B cell Stimuli

Anticitrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA) and have been implicated in disease pathogenesis. We have recently shown that IgG1 ACPA of RA patients harbour different...

Catabolism of pemphigus foliaceus autoantibodies by high-dose IVIg therapy

High-dose intravenous immunoglobulin (HD-IVIg) has several distinguishing therapeutic characteristics. However, a certain number of pemphigus cases have been experienced, which did not respond to HD-IVIg. This is the first case report that the serum level of anti-desmoglein (Dsg)1 antibody rebounded, critically associated with IgG serum level. We describe a patient with pemphigus foliaceus (PF), unresponsive to oral prednisolone followed by pulse therapy and double-filtration plasmapheresis, in whom clinical remission was induced by 4 courses of HD-IVIg. Anti-Dsg1 antibody levels, serum IgG and disease activity were monitored. Anti-Dsg1 antibody titers rapidly decreased after IVIg treatment when total IgG levels were high; however, the serum level of anti-Dsg1 antibody rebounded as the total IgG level returned to normal. The levels of anti-Dsg1 antibody were decreased for an average of 13.7 days after treatment. The therapeutic effect of IVIg treatment was associated with an increased serum level of total IgG. IVIg therapy reduced the titers of autoantibody by accelerating the catabolism of immunoglobulin induced by high IgG serum levels. IVIg itself appears to accelerate IgG degradation rather than suppress IgG production. Sufficient suppression of antibody production is critical for successful treatment with IVIg.

Effects of induced parturition in goats on immunoglobulin G and chitotriosidase activity in colostrum and plasma and on plasma concentrations of prolactin

The effect of induction of parturition with a PGF 2α analog on plasma concentration of prolactin (PRL) and its effects on colostrum concentration of IgG and chitotriosidase (ChT) activity were studied in 16 pregnant Majorera goats. Treated goats, those in which parturition was induced, had greater concentrations of PRL than control goats 24 h before parturition ( P < 0.05) and 48 h after parturition ( P < 0.05). Control goats had greater concentrations of PRL than treated goats 96 h after parturition ( P < 0.05). Plasma concentration of IgG did not differ between groups during the experimental period, but colostrum concentrations of IgG were greater in control goats than in treated goats at parturition ( P < 0.05). Plasma ChT activity decreased during the period 72 h before parturition to 24 h after parturition in control and treated goats. Time evolution after partum affected the colostrum ChT activity, being greater at parturition than after parturition in both groups ( P < 0.05). In summary, concentration of IgG in colostrum is slightly diminished if parturition is induced. Induction of parturition causes an early increase in PRL, which is most likely responsible for preterm suppression of IgG transport into mammary secretions.

Plasmablast-derived polyclonal antibody response after influenza vaccination

Conventional measurement of antibody responses to vaccines largely relies on serum antibodies, which are primarily produced by bone marrow plasma cells and may not represent the entire vaccine-induced B cell repertoire, including important functional components such as those targeted to mucosal sites. After immunization or infection, activated B cells differentiate into plasmablasts in local lymphoid organs, then traffic through circulation to the target sites where they further develop into plasma cells. On day 7 after influenza vaccination, a burst of plasmablasts, highly enriched for vaccine-specific antibody secreting cells, appears in the peripheral blood. This provides a unique window to the overall B cell response to the vaccine, without interference of pre-existing cross-reactive serum antibody. In this study we isolated B cells from volunteers on day 7 after immunization with the inactivated influenza vaccine and cultured them ex vivo to collect plasmablast-derived polyclonal antibodies (PPAb). The PPAb contained secreted IgG and IgA, which was approximately 0.2ng per antibody secreting cell. Influenza-specific IgG and IgA binding activity was detected in PPAb at dilutions up to 105 by ELISA. The ratio of the titers of influenza-specific IgA to IgG by ELISA was 4-fold higher in PPAb than in day 28 post-vaccination sera, suggesting that vaccine-induced IgA is enriched in PPAb compared to sera. Functional activity was also detected in PPAb as determined by microneutralization and hemagglutination inhibition assays. In addition to bulk B cell cultures, we also cultured plasmablast subsets sorted by cell surface markers to generate PPAb. These results suggest that PPAb better reflects the mucosal IgA response than serum samples. Since PPAb are exclusively produced by recently activated B cells, it allows assessing vaccine-induced antibody response without interference from pre-existing cross-reactive serum antibodies and permits an assessment of antibody avidity based on antigen specific binding and antibody quantity. Therefore this assay is particularly useful for studying vaccine/infection-induced antibodies against antigens that might have previously circulated, such as antibody responses to rotavirus, dengue or influenza viruses in which cross-reactive antibodies against different virus serotypes/subtypes play a critical role in immunity and/or pathogenesis.

Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.