Antibody Subclasses Research Articles

Formation of humoral immunity in calves in health and disease

It is known that the health and productivity of young animals are determined in many respects by the development of the humoral link of the immune sys� tem most objectively reflecting the effectiveness of the interaction of T and B cells and antigenpresenting cells during the immune response. During postnatal development of animals, a successive change of natu� ral passive humoral protection to active immunity occurs in animals, the level of which, with consider� ation of age and individual characteristics, allows assessing both the degree of resistance of the organism due to lactogenic immunity and subsequently the ade� quacy of the reaction of the immune system to the action of environmental factors. In early postnatal ontogeny, it is impossible to dif� ferentiate maternal isotypes (classes, subclasses) of immunoglobulins or specific antibodies and those produced by own immune system present in new� borns, and the revelation, for example, of allotypic variants is difficult. Therefore, the most accessible is a determination of the level of immunoglobulins in dynamics, which makes it possible with high probabil� ity to judge the formation of humoral immunity in young animals. The purpose of the present investigation was to study humoral immunity in calves with a different level of passive protection in health and disease under con� dition of an industrial complex.

Alterations in Antibody Subclass Immune Reactivity to Trophoblast‐derived Fetal Fibronectin and α2‐Macroglobulin in Women with Recurrent Pregnancy Loss

Increasing evidence supports the involvement of complex antibody-mediated immunologic events at the decidua-trophoblast interface. Our objective is to define the humoral immune responses of pregnant women with a history of recurrent pregnancy loss (RPL) compared with gestation-age-matched and non-pregnant controls in terms of trophoblast-derived antigenic targets and IgG subclasses. Immunoprecipitation and Western immunoblotting were performed to characterize IgG subclass reactivity to Sw.71 trophoblast-derived fetal fibronectin and alpha-2-macroglobulin, using serum obtained from first-trimester pregnant RPL subjects, gestation-age-matched controls, and non-pregnant controls. Using a generalized linear model, sera from women with a history of RPL exhibited increased IgG(3) immunoreactivity to trophoblast-derived fetal fibronectin and alpha-2-macroglobulin compared with controls (P < 0.001 and P < 0.001, respectively). IgG(3) reactivity in women with RPL may play a significant role in aberrant immune-regulatory mechanisms in early pregnancy. Further investigations into the role of autoantibodies against trophoblast-derived proteins in implantation and pregnancy are warranted.

Vitamin D deficiency in patients with Graves’ disease: probably something more than a casual association

In addition to its established role in calcium metabolism and skeletal homeostasis, vitamin D was recently identified as a factor playing a role in both innate and adaptive immunity (reviewed in Ref. [1]). The vitamin D receptor is expressed on monocytes and activated lymphocytes and the biologically active metabolite of vitamin D, i.e., 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], does modulate T cell responses [1]. Studies in animal models of autoimmune diseases consistently showed that the administration of 1,25(OH)2D3 produces selective immunosuppressant effects, as assessed by its ability to either prevent or markedly suppress experimental autoimmune encephalomyelitis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes mellitus, and inflammatory bowel disease [2]. Currently available clinical studies report a high prevalence of vitamin D deficiency in patients with several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, type 1 diabetes mellitus, and Crohn’s disease [3]. With specific regard to autoimmune thyroid diseases (AITD), available data remain controversial. The administration of 1,25(OH)2D3 had little effect in reducing the severity of experimental autoimmune thyroiditis induced by immunizing CBA mice with thyroglobulin [4]. In a model of experimental Graves’ disease in which mice were immunized with adenovirus encoding for the TSH-receptor gene, BALB/cJ and C57BL/6J mice receiving either a sufficient or a deficient vitamin D diet developed comparable levels of TSH binding inhibitory and thyroid stimulating antibodies [5]. The IgG subclasses of antibodies directed to the TSH-receptor were also similar in mice of either strain, irrespective of the treatment with a vitamin D-deficient or vitamin D-sufficient diet [5]. However, as argued by the authors, it should be considered that in this experiment mice fed a vitamin D deficient diet, in spite of having nearly undetectable serum levels of 25(OH)D, displayed low but detectable concentrations of 1,25(OH)2D. When human studies are taken into account, a high prevalence of vitamin D deficiency was reported both in patients with chronic autoimmune thyroiditis and in those with Graves’ disease [6, 7]. In this issue of Endocrine, Yasuda et al. [8] measured the serum levels of 25(OH) vitamin D in 26 female patients with newly onset Graves’ disease and in 46 healthy female controls. Vitamin D deficiency was diagnosed when laboratory results indicated a serum level below 15 ng/ml. The authors reported significantly lower levels of vitamin D and a greater prevalence of vitamin D deficiency in Graves’ patients as opposed to healthy controls. A significant inverse relationship was found between the circulating concentrations of vitamin D and thyroid volume. This observation would be in line with the finding by Vilarrasa et al. [9] who reported an inverse association between body mass index (which is positively associated with thyroid volume) and circulating 25(OH) vitamin D. On the other hand, Yasuda et al. [8] found no association between the serum levels of 25(OH)D3 and TSH-, FT3-, FT4-, or TSHreceptor antibodies. Although the results of this study are limited by the small number of investigated patients and by its cross-sectional design, some specific comments can be drawn. First, a large Graves’ goiter is associated with a higher rate of relapsing hyperthyroidism after stopping treatment with anti-thyroid drugs [10]. Thus, Graves’ patients bearing large goiters and being less prone to be successfully cured with anti-thyroid drugs, would more M. Rotondi L. Chiovato (&) Unit of Internal Medicine and Endocrinology, Fondazione Salvatore Maugeri I.R.C.C.S., Laboratory for Endocrine Disruptors and Chair of Endocrinology, University of Pavia, Via S. Maugeri 10, 27100 Pavia, Italy e-mail: luca.chiovato@fsm.it

Donor-specific human leukocyte antigen antibodies of the immunoglobulin G3 subclass are associated with Chronic rejection and graft loss after liver transplantation

In a previous study, we found that 92% of patients with chronic rejection had donor-specific human leukocyte antigen antibodies (DSAs), but surprisingly, 61% of comparator patients without rejection also had DSAs. We hypothesized that immunoglobulin G (IgG) subclasses were differentially distributed between the 2 groups. A modified single-antigen bead assay was used to detect the presence of individual IgG subclasses against human leukocyte antigen in 39 chronic rejection patients and 66 comparator patients. DSAs of the IgG1 subclass were most common and were found in 45% of all patients; they were followed by IgG3 DSAs (21%), IgG4 DSAs (14%), and IgG2 DSAs (13%). The percentage of patients with multiple IgG subclasses was significantly higher in the chronic rejection group versus the comparator group (50% versus 14%, P < 0.001). Patients with normal graft function in the presence of DSAs mostly had isolated IgG1, whereas patients with chronic rejection had a combination of IgG subclasses. Patients who developed DSAs of the IgG3 subclass showed an increased risk of graft loss (hazard ratio = 3.35, 95% confidence interval = 1.39-8.05) in comparison with patients with DSAs of other IgG subclasses or without DSAs. Although further study is needed, the determination of the IgG subclass in DSA-positive patients may help us to identify patients with a higher risk of chronic rejection and graft loss.

ISCOMATRIX: a novel adjuvant for use in prophylactic and therapeutic vaccines against infectious diseases

The ISCOMATRIX adjuvant has antigen delivery and presentation properties as well as immunomodulatory capabilities, which combine to provide enhanced and accelerated immune responses. The responses are broad, including a range of subclasses of antibodies as well as CD4(+) and CD8(+) T-cells. A range of ISCOMATRIX vaccines (ISCOMATRIX adjuvant combined with antigen) have now been tested in clinical trials and have been shown to be generally safe and well tolerated as well as immunogenic, generating both antibody (Ab) and T-cell responses. The mechanisms by which ISCOMATRIX adjuvant facilitates its immune effects are the scope of significant study and indicate that ISCOMATRIX adjuvant (i) rapidly traffics antigen into the cytosol of multiple dendritic cell subsets, (ii) induces the induction of an array of cytokines and chemokines and (iii) links the innate and adaptive immune responses in vivo in a Toll-like-receptor-independent but MyD88-dependent manner. These data highlight the clinical utility of ISCOMATRIX adjuvant in the development of prophylactic and therapeutic vaccines for infectious disease.

IgG Subclasses Against Helicobacter pylori Isolates: An important Tool for Disease Characterization

Helicobacter pylori infects around 50% of the world's population and is associated with diverse pathologies. In the most severe cases, the bacterium causes peptic ulcers and gastric cancer. The interplay between H. pylori and the host's immune response may help to determine the specific outcome of the infection. To study the relationship between antibody subclasses and variation in immune recognition, we determined the immunoglobulin G1 and 2 (IgG1 and IgG2) titres of sera obtained from patients with different H. pylori-associated pathologies. IgG1 and IgG2 titres were determined by ELISA in 44 sera of patients with different H. pylori-associated diseases (peptic ulcer, bleeding peptic ulcers, gastric cancer and dyspepsia). Soluble proteins from lysates were obtained from 12 different clinical isolates from similar associated diseases. We found that soluble proteins from lysates of H. pylori strains (SPLHP) recognition patterns in these sera were highly variable. Overall, IgG2 titres were higher than the IgG1 titres in the infected patients. In particular, those with peptic ulcers showed marked elevation in IgG2/IgG1 ratios, while SPLHPs from dyspeptic patients resulted in high IgG1 titres. Our results reveal that correlation of antibody subclass titres with Th1/Th2 markers may aid pathology characterization and show a potential diagnosis that could be formally evaluated in other studies.

Nucleoprotein Nanostructures Combined with Adjuvants Adapted to the Neonatal Immune Context: A Candidate Mucosal RSV Vaccine

BackgroundThe human respiratory syncytial virus (hRSV) is the leading cause of severe bronchiolitis in infants worldwide. The most severe RSV diseases occur between 2 and 6 months-of-age, so pediatric vaccination will have to be started within the first weeks after birth, when the immune system is prone to Th2 responses that may turn deleterious upon exposure to the virus. So far, the high risk to prime for immunopathological responses in infants has hampered the development of vaccine. In the present study we investigated the safety and efficacy of ring-nanostructures formed by the recombinant nucleoprotein N of hRSV (NSRS) as a mucosal vaccine candidate against RSV in BALB/c neonates, which are highly sensitive to immunopathological Th2 imprinting.Methodology and Principal FindingsA single intranasal administration of NSRS with detoxified E.coli enterotoxin LT(R192G) to 5–7 day old neonates provided a significant reduction of the viral load after an RSV challenge at five weeks of age. However, neonatal vaccination also generated an enhanced lung infiltration by neutrophils and eosinophils following the RSV challenge. Analysis of antibody subclasses and cytokines produced after an RSV challenge or a boost administration of the vaccine suggested that neonatal vaccination induced a Th2 biased local immune memory. This Th2 bias and the eosinophilic reaction could be prevented by adding CpG to the vaccine formulation, which, however did not prevent pulmonary inflammation and neutrophil infiltration upon viral challenge.Conclusions/SignificanceIn conclusion, protective vaccination against RSV can be achieved in neonates but requires an appropriate combination of adjuvants to prevent harmful Th2 imprinting.

Development and characterization of a human antibody reference panel against erythropoietin suitable for the standardization of ESA immunogenicity testing

Recombinant human erythropoietin (EPO) has been used therapeutically for more than two decades in the treatment of anemia. Although EPO is generally well tolerated, in rare cases, patients have developed anti-EPO antibodies that can negatively impact safety and efficacy. Therefore, the detection of antibodies against EPO is a regulatory requirement during clinical development and post-approval. Although it is a rare phenomenon, antibody-mediated pure red cell aplasia (PRCA) is a serious complication than can result from antibodies that develop and neutralize EPO as well as endogenous erythropoietin.Currently, there are no universally accepted analytical methods to detect the full repertoire of binding and neutralizing anti-EPO antibodies. A number of different methods that differ in terms of antibodies detected and assay sensitivities are used by different manufacturers. There is also a lack of antibody reference reagents, and therefore no consistent basis for detecting and measuring anti-EPO antibodies. Reference reagents, with established ranges, are essential to monitor the safety and efficacy of all erythropoiesis-stimulating agents (ESAs) structurally related to human erythropoietin.This is the first report of the development and characterization of a panel of fully human antibodies against EPO suitable as reference reagents. The characteristics of antibodies within the panel were selected based on the prevalence of non-neutralizing IgG and IgM antibodies in non-PRCA patients and neutralizing IgG antibodies, including IgG1 and IgG4, in antibody-mediated PRCA subjects. The reference panel includes antibodies of high- and low-affinity with binding specificity to neutralizing and non-neutralizing erythropoietin epitopes. The subclass of human antibodies in this reference panel includes an IgG1, IgG2, and IgG4, as well as an IgM isotype. This antibody panel could help select appropriate immunogenicity assays, guide validation, and monitor assay performance. Further, this human anti-ESA antibody panel may help set the limits of each assay platform in terms of the full repertoire of the anti-ESA antibodies, and may facilitate standardization of ESA immunogenicity reporting across assay platforms.

Early diagnosis of congenital toxoplasmosis in newborn infants using IgG subclasses against two Toxoplasma gondii recombinant proteins

The aim of this work was to evaluate the utility of ELISA-based testing of total IgG (IgGt) antibodies and its subclasses (IgG1, IgG2, IgG3 and IgG4) against soluble (STAg) and recombinant (rSAG1 and rMIC3) antigens of Toxoplasma gondii for diagnosing congenital toxoplasmosis. Sera from 217 newborns initially testing positive for specific IgM in filter paper dried blood spots were tested for specific IgM and IgG by ELFA-VIDAS. Congenital toxoplasmosis was confirmed in 175 and ruled out in 42 infants. The validity of the ELISA tests was determined using the persistence of IgG antibodies (ELFA-VIDAS kit) at the end of 12 months, which is considered the reference test for the diagnosis of congenital toxoplasmosis. The frequency of positivity with IgGt against STAg, rSAG1 and rMIC3 was found in 97.2%, 96.3% and 80.2%, respectively, of the newborns with confirmed congenital toxoplasmosis. IgG1 reacted with all three antigens, while IgG3 and IgG4 reacted preferentially with rMIC3. Higher mean values of reactivity (sample optical density/cut-off) were found for all subclasses when using rMIC3. All of the antigens showed high sensitivity and low specificity in detecting anti-T. gondii IgGt and IgG1 and low sensitivity and high specificity in detecting IgG3 and IgG4. In conclusion, the combined detection of IgG antibody subclasses against recombinant toxoplasmic antigens may be useful for the early diagnosis of congenital toxoplasmosis.

Comparative Evaluation of Two Hemagglutinating Encephalomyelitis Coronavirus Vaccine Candidates in Mice

Porcine hemagglutinating encephalomyelitis (PHE) is caused by the coronavirus hemagglutinating encephalomyelitis virus (PHE-CoV), and the recent, rapid spread of PHE-CoV in piglets from many countries emphasizes the urgent need for a PHE-CoV vaccine. Here we use a murine model for evaluation of the induction of humoral and cellular immune responses by inactivated and PHE-CoV DNA vaccines in order to define the immune correlates for protection against PHE-CoV. The inactivated vaccine was composed of purified PHE-CoV and aluminum hydroxide gel (alum), which was chosen as an adjuvant because of its long history of safety for human use. The PHE-CoV DNA vaccine was constructed by subcloning the S1 gene of PHE-CoV into the pVAX1 vector to create the recombinant plasmid pV-S1. Our results showed that the inactivated PHE-CoV vaccine (IPV) elicited a high level of humoral immunity, resulting in good protection efficacy against PHE-CoV challenge. The IPV induced the IgG1 subclass of serum antibodies and expression of the cytokine interleukin-4 (IL-4), suggesting that the IPV generated a predominantly Th2-type immune response. The DNA vaccine was found to mediate primarily a cellular immune response with high levels of IgG2a and the cytokines IL-2 and gamma interferon (IFN-γ). However, mice that were vaccinated twice with the DNA vaccine and boosted with the IPV could mount a sufficient neutralizing antibody response against live PHE-CoV, with little variation in IgG1 and IgG2a levels, and showed high levels of IL-2 and IL-4. This response may activate both B and T cells to mount a specific humoral and cellular immune response that could, in turn, elicit a phagocyte-mediated defense against PHE-CoV infections to achieve viral clearance.

DETECCIÓN DE ANTICUERPOS Y ANTÍGENOS PARA EL DIAGNÓSTICO DE FASCIOLA HEPATICA EN ALPACAS NATURALMENTE INFECTADAS.

The aim of this study was to standardize the indirect ELISA test for the detection of antibodies and to evaluate a coproantigen detection test (ES-78 sandwich ELISA) for the Fasciola hepatica diagnoses in alpacas. For the indirect ELISA, the excretion–secretion products (ES) were used as antigens and a peroxidasa anti IgG immunoconjugate of alpaca was obtained through protein A affinity cromatography. The detection ELISA was tested with the FASCIDIG diagnostic kit, which detects metabolics antigens using an AcM-ES78 monoclonal antibody of the IgG 2a sub class. Optical Density (OD) values between 0.075 and 1.435 were obtained using the indirect ELISA, where the cut-off point was 0.226. The positive control group animals were positive to the test with values between 0.331 and 1.435. The negative control group animals were negative to the test with values between 0.075 and 0.226. OD values between 0.060 and 1.532 were obtained using the coproantigen detection ELISA, where the cut-off point was 0.240. The positive control group animals were positive to the test with OD values between 0.244 and 1.532 and the negative control group animals were negative to the test with values between 0.060 and 0.240. The sensitivity, specificity and positive and negative predictive values were 100% for both ELISA tests. Both test can replace the coproparasitologic test. They are suitable for individual diagnosis and for herd diagnosis infections with F. hepatica. The indirect ELISA has demonstrated to be a useful and sensitive tool for the pasive fasciolosis diagnosis in alpacas. The coproantigen detection ELISA is a simple, fast and efficient method in the detection of active F. hepatica infection. Both diagnostic methods would be the most suitables techniques for sero-epidemiologic surveys in large herds under intensive grazing systems.

Second External Quality Assurance Study for the Serological Diagnosis of Hantaviruses in Europe

Hantaviruses are endemic throughout the world and hosted by rodents and insectivores. Two human zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), are caused by hantaviruses and case fatality rates have reached 12% for HFRS and 50% for HPS in some outbreaks. Symptomatic hantavirus infections in Europe are summarised as HFRS mainly due to Puumala, Dobrava-Belgrade and Saaremaa virus. While HFRS has an overall low incidence in Europe, the number of cases varies from 100 per year in all Eastern and Southern Europe up to 1,000 per year only in Finland. To assess the quality of hantavirus diagnostics, the European Network for the Diagnostics of “Imported” Viral Diseases (ENIVD) organised a first external quality assurance (EQA) in 2002. The purpose of this second EQA study is to collect updated information on the efficiency and accurateness of hantavirus serological methods applied by expert laboratories. A serum panel of 14 samples was sent to 28 participants in Europe of which 27 sent results. Performance in hantavirus diagnosis varied not only on the method used but also on the laboratories and the subclass of antibodies tested. Commercial and in-house assays performed almost equally. Enzyme immunoassays were mainly used but did not show the best performances while immunoblot assays were the less employed and showed overall better performances. IgM antibodies were not detected in 61% of the positive IgM samples and IgM detection was not performed by 7% of the laboratories indicating a risk of overlooking acute infections in patients. Uneven performances using the same method is indicating that there is still a need for improving testing conditions and standardizing protocols.

CXCR3 promotes the production of IgG1 autoantibodies but is not essential for the development of lupus nephritis in NZB/NZW mice

Autoantibody immune complexes and cellular infiltrates drive nephritis in patients with systemic lupus erythematosus (SLE) and in murine lupus. The chemokine receptor CXCR3 is assumed to promote cellular infiltration of inflamed tissues. Moreover, CXCR3 deficiency ameliorates lupus nephritis in the MRL/MpJ-Fas(lpr) (MRL/lpr) mouse model of SLE. Hence, CXCR3 blockade has been suggested as a novel therapeutic strategy for the treatment of lupus nephritis. We undertook this study to test the effect of CXCR3 in the (NZB × NZW)F(1) (NZB/NZW) mouse model of SLE. CXCR3(-/-) NZB/NZW mice were generated and monitored for survival, proteinuria, and kidney infiltration. Anti-double-stranded DNA (anti-dsDNA) and total IgG1, IgG2a, and IgG2b antibody levels were determined by enzyme-linked immunosorbent assay. T cell and plasma cell infiltrates in the kidneys and interferon-γ production were determined by flow cytometry. Plasma cell infiltrates were measured using enzyme-linked immunospot assay. Kidney tissue was evaluated for pathologic changes. CXCR3(-/-) NZB/NZW mice exhibited reduced production of total and anti-dsDNA antibodies of the IgG1 subclass, but had normal titers of IgG2a and IgG2b antibodies compared to CXCR3(+/+) NZB/NZW mice. Cellular infiltrates and glomerulonephritis were not reduced in CXCR3(-/-) mice. CXCR3 has an effect on (auto)antibody production but is not essential for lupus pathogenesis in NZB/NZW mice, indicating that the effect of CXCR3 on the development of kidney disease varies between MRL/lpr and NZB/NZW mice. These results suggest that CXCR3-dependent and -independent mechanisms can mediate lupus nephritis. Hence, therapeutic CXCR3 blockade could be beneficial for only a subgroup of patients with SLE.

Evaluation of pneumonia virus of mice as a possible human pathogen.

Pneumonia virus of mice (PVM), a relative of human respiratory syncytial virus (RSV), causes respiratory disease in mice. There is serologic evidence suggesting widespread exposure of humans to PVM. To investigate replication in primates, African green monkeys (AGM) and rhesus macaques (n = 4) were inoculated with PVM by the respiratory route. Virus was shed intermittently at low levels by a subset of animals, suggesting poor permissiveness. PVM efficiently replicated in cultured human cells and inhibited the type I interferon (IFN) response in these cells. This suggests that poor replication in nonhuman primates was not due to a general nonpermissiveness of primate cells or poor control of the IFN response. Seroprevalence in humans was examined by screening sera from 30 adults and 17 young children for PVM-neutralizing activity. Sera from a single child (6%) and 40% of adults had low neutralizing activity against PVM, which could be consistent with increasing incidence of exposure following early childhood. There was no cross-reaction of human or AGM sera between RSV and PVM and no cross-protection in the mouse model. In native Western blots, human sera reacted with RSV but not PVM proteins under conditions in which AGM immune sera reacted strongly. Serum reactivity was further evaluated by flow cytometry using unfixed Vero cells infected with PVM or RSV expressing green fluorescent protein (GFP) as a measure of viral gene expression. The reactivity of human sera against RSV-infected cells correlated with GFP expression, whereas reactivity against PVM-infected cells was low and uncorrelated with GFP expression. Thus, PVM specificity was not evident. Our results indicate that the PVM-neutralizing activity of human sera is not due to RSV- or PVM-specific antibodies but may be due to low-affinity, polyreactive natural antibodies of the IgG subclass. The absence of PVM-specific antibodies and restriction in nonhuman primates makes PVM unlikely to be a human pathogen.

Stochastic sensing of proteins with receptor-modified solid-state nanopores

Solid-state nanopores are capable of the label-free analysis of single molecules. It is possible to add biochemical selectivity by anchoring a molecular receptor inside the nanopore, but it is difficult to maintain single-molecule sensitivity in these modified nanopores. Here, we show that metallized silicon nitride nanopores chemically modified with nitrilotriacetic acid receptors can be used for the stochastic sensing of proteins. The reversible binding and unbinding of the proteins to the receptors is observed in real time, and the interaction parameters are statistically analysed from single-molecule binding events. To demonstrate the versatile nature of this approach, we detect His-tagged proteins and discriminate between the subclasses of rodent IgG antibodies.

Immunoglobulin G subclass levels and antibody responses to the 2009 influenza A (H1N1) monovalent vaccine among human immunodeficiency virus (HIV)-infected and HIV-uninfected adults

Immunoglobulin (Ig)G levels are important for antibody vaccine responses and IgG subclass deficiencies have been associated with severe 2009 influenza A (H1N1) infections. Studies have demonstrated variations in immune responses to the H1N1 vaccine, but the aetiology of this is unknown. We determined the associations between pre-vaccination overall and influenza-specific IgG subclass levels and 2009 H1N1-specific antibody responses post-vaccination (robust versus poor at day 28) stratified by human immunodeficiency virus (HIV) status. Logistic regression models were utilized to evaluate whether pre-vaccination IgG subclass levels were associated with the antibody response generated post-vaccination. We evaluated 48 participants as part of a clinical study who were stratified by robust versus poor post-vaccination immune responses. Participants had a median age of 35 years; 92% were male and 44% were Caucasian. HIV-infected adults had a median CD4 count of 669 cells/mm(3) , and 79% were receiving highly active anti-retroviral therapy. HIV-infected participants were more likely to have IgG2 deficiency (<240 mg/dl) than HIV-uninfected individuals (62% versus 4%, P < 0·001). No association of pre-vaccination IgG subclass levels (total or influenza-specific) and the antibody response generated by HIN1 vaccination in either group was found. In summary, pre-vaccination IgG subclass levels did not correlate with the ability to develop robust antibody responses to the 2009 influenza A (H1N1) monovalent vaccine. IgG2 deficiencies were common among HIV-infected individuals but did not correlate with poor influenza vaccine responses. Further investigations into the aetiology of disparate vaccine responses are needed.

Monoclonal antibodies directed against human FcRn and their applications

The MHC class I-like Fc receptor (FcRn) is an intracellular trafficking Fc receptor that is uniquely responsible for the extended serum half-life of antibodies of the IgG subclass and their ability to transport across cellular barriers. By performing these functions, FcRn affects numerous facets of antibody biology and pathobiology. Its critical role in controlling IgG pharmacokinetics has been leveraged for the design of therapeutic antibodies and related biologics. FcRn also traffics serum albumin and is responsible for the enhanced pharmacokinetic properties of albumin-conjugated therapeutics. The understanding of FcRn and its therapeutic applications has been limited by a paucity of reliable serological reagents against human FcRn. Here, we describe the properties of a new panel of highly specific monoclonal antibodies (mAbs) directed against human FcRn with diverse epitope specificities. We show that this antibody panel can be used to study the tissue expression pattern of human FcRn, to selectively block IgG and serum albumin binding to human FcRn in vitro and to inhibit FcRn function in vivo. This mAb panel provides a powerful resource for probing the biology of human FcRn and for the evaluation of therapeutic FcRn blockade strategies.

Pulmonary events in antiphospholipid syndrome: influence of antiphospholipid antibody type and levels

Objective: This prospective clinical study examined the association between subclasses of antiphospholipid antibodies (aPL) and pulmonary manifestations in antiphospholipid syndrome (APS).Methods: The cohort involved 329 patients: 214 patients with primary APS (PAPS) and 115 patients with secondary APS (SAPS). aPL analysis included detection of serum anticardiolipin antibodies [aCL (IgG/IgM)], β2 glycoprotein I [β2GPI (IgG/IgM)], and lupus anticoagulant (LA).Results: In SAPS, high aCL IgG levels (> 100 PLU/mL) were more common in major pulmonary arterial thrombosis (p = 0.006) and medium aCL IgG levels (41–99 PLU/mL) in adult respiratory distress syndrome (ARDS; p = 0.047) and fibrosing alveolitis (p = 0.002). aCL IgG antibodies were more common in SAPS (p = 0.037). In PAPS, fibrosing alveolitis was more common in patients with medium β2GPI IgM levels (p = 0.0001). LA correlated with pulmonary embolism (p = 0.03) and microthrombosis (p = 0.03) in SAPS, and with pulmonary microthrombosis (p = 0.03) in PAPS. Males were more likely to develop secondary pulmonary hypertension when diagnosed with PAPS (p = 0.019).Conclusion: Certain classes of aPL are associated with distinct pulmonary manifestation, indicating their predictive role and importance in diagnosis and treatment of APS.

Characterization and Application of Monoclonal Antibody Against Hepatitis C Virus Nonstructual Protein Three

Developing assays for detecting HCV antigens could be beneficial because viral proteins appear earlier than antibodies and are more stable than RNA in the serum. Monoclonal antibody was prepared by immunization and cell fusion. Subclass, specificity, and efficiency of monoclonal antibodies were determined by ELISA. Epitope specificity of monoclonal antibodies was analyzed by ELISA additivity test. HCV antigen in serum of hepatitis patients was examined by double monoclonal antibody sandwich ELISA. Five hybridoma cell lines were screened and named HCV(1), HCV(2), HCV(3), HCV(4), and HCV(5). These five monoclonal antibodies had high specificity and efficiency. The additivity test showed that HCV(2), HCV(4), and HCV(5) recognized different epitopes, which can be matched in ELISA. Of 173 anti-HCV positive patients, 37 (21.4%) were positive for HCV antigen. Of 1498 anti-HCV negative patients, 10 (0.67%) were positive for HCV antigen. Fifty normal controls were negative for HCV antigen. HCV antigen detection had moderate agreement and correlation with HCV RNA detection (kappa=0.577, p<0.01; r=0.59, p<0.01). This result indicates that the monoclonal antibody against HCV NS(3) may be a potential diagnostic reagent, which would provide a foundation for developing a sandwich ELISA of HCV antigen detection.

Abstract 3452: Antiphosphatidylserine-prothrombin Antibodies Are Associated With Outcome After TIA

Background and Purpose: Newly defined subclasses of antiphospholipid antibodies (aPLs) have been associated with thrombotic and atherosclerotic vascular events. We aimed to determine if they predict outcome after TIA. Methods: Detailed clinical data and blood samples were obtained prospectively from subjects with TIA hospitalized &lt;48 hours from symptom onset. In a retrospective analysis standard aPLs, anti-cardiolipin (ACL) and β-2-glycoprotein-1 (β2GP1), and newer aPLs, anti-phosphatidylserine-prothrombin (aPS-PT), β2GP1 D4/5 subunit and β2GP1 D1 subunit, were measured by ELISA. The primary outcome was a composite endpoint of stroke or death within 90 days or identification of a high-risk stroke mechanism (defined as &gt;50% stenosis in a vessel referable to symptoms or cardioembolism warranting anticoagulation). A secondary outcome was the presence of clinical or sub-clinical atherosclerosis. Results: Over 4.5 years, 167 patients were enrolled. Mean age was 62±14 years; 55% were female. Mean time from symptom onset to blood sampling was 26.2 ± 12.7 hours. Overall, 41 patients (25%) had the composite endpoint: 25 (15%) with a &gt;50% stenosis, 14 (8%) with cardioembolism and 8 (5%) with clinical events (5 strokes, 3 deaths). The results of aPL testing using predefined cut-points to define positivity are summarized in the table . Using a cutoff of 30 units aPS-PT IgG antibodies were significantly associated with stroke/death (OR 17.3 95% CI 2.4-123.8 p=0.004) with a trend towards an association with the composite endpoint (OR 4.9 95% CI 0.8-30.4 p=0.09). In multivariate analysis adjusting for ABCD2 score, aPS-PT IgG remained associated with stroke/death (OR 16.6 95% CI 2.1-133.7 p=0.008). Using a data derived cut-off of 20 units (∼95% percentile within study population) aPS-PT IgG antibodies were significantly associated with stroke/death (OR 7.2 95% CI 1.2-42.5 p=0.03) and the composite endpoint (OR 4.2 95% CI 1.1-16.6 p=0.04). After adjusting for ABCD2 score, the association with stroke/death remained significant (OR 6.1 95% CI 1.0-38.5 p=0.05) and the composite endpoint approached significance (OR 3.8 95% CI 0.9-15.4 p=0.06). Elevated aPS-PT IgG levels occurred more frequently in subjects with clinical or sub-clinical atherosclerosis than those without (9% vs 0%, p=0.012). Other aPLs were not associated with atherosclerosis. Conclusion: In contrast to other aPLs, anti-phosphatidylserine-prothrombin IgG antibodies are elevated in high-risk TIA patients.