Genome-Wide Analyses Suggest Mechanisms Involving Early B-Cell Development in Canine IgA Deficiency.

Immunoglobulin A deficiency (IgAD) is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS) to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei) identified 35 genomic loci suggestively associated (p <0.0005) to IgA levels. In German shepherd, three genomic regions (candidate genes include KIRREL3 and SERPINA9) were genome-wide significantly associated (p <0.0002) with IgA levels. A ~20kb long haplotype on CFA28, significantly associated (p = 0.0005) to IgA levels in Shar-Pei, was positioned within the first intron of the gene SLIT1. Both KIRREL3 and SLIT1 are highly expressed in the central nervous system and in bone marrow and are potentially important during B-cell development. SERPINA9 expression is restricted to B-cells and peaks at the time-point when B-cells proliferate into antibody-producing plasma cells. The suggestively associated regions were enriched for genes in Gene Ontology gene sets involving inflammation and early immune cell development.

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs.

BACKGROUND Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.

Antibody-Drug Conjugates for Tumor Targeting-Novel Conjugation Chemistries and the Promise of non-IgG Binding Proteins

Antibody-drug conjugates (ADCs) have emerged as a promising class of anticancer agents, combining the specificity of antibodies for tumor targeting and the destructive potential of highly potent drugs as payload. An essential component of these immunoconjugates is a bifunctional linker capable of reacting with the antibody and the payload to assemble a functional entity. Linker design is fundamental, as it must provide high stability in the circulation to prevent premature drug release, but be capable of releasing the active drug inside the target cell upon receptor-mediated endocytosis. Although ADCs have demonstrated an increased therapeutic window, compared to conventional chemotherapy in recent clinical trials, therapeutic success rates are still far from optimal. To explore other regimes of half-life variation and drug conjugation stoichiometries, it is necessary to investigate additional binding proteins which offer access to a wide range of formats, all with molecularly defined drug conjugation. Here, we delineate recent progress with site-specific and biorthogonal conjugation chemistries, and discuss alternative, biophysically more stable protein scaffolds like Designed Ankyrin Repeat Proteins (DARPins), which may provide such additional engineering opportunities for drug conjugates with improved pharmacological performance.

On the Dark Side of Therapies with Immunoglobulin Concentrates: The Adverse Events

Therapy by human immunoglobulin G (IgG) concentrates is a success story ongoing for decades with an ever increasing demand for this plasma product. The success of IgG concentrates on a clinical level is documented by the slowly increasing number of registered indication and the more rapid increase of the off-label uses, a topic dealt with in another contribution to this special issue of Frontiers in Immunology. A part of the success is the adverse event (AE) profile of IgG concentrates which is, even at life-long need for therapy, excellent. Transmission of pathogens in the last decade could be entirely controlled through the antecedent introduction by authorities of a regulatory network and installing quality standards by the plasma fractionation industry. The cornerstone of the regulatory network is current good manufacturing practice. Non-infectious AEs occur rarely and mainly are mild to moderate. However, in recent times, the increase in frequency of hemolytic and thrombotic AEs raised worrying questions on the possible background for these AEs. Below, we review elements of non-infectious AEs, and particularly focus on hemolysis and thrombosis. We discuss how the introduction of plasma fractionation by ion-exchange chromatography and polishing by immunoaffinity chromatographic steps might alter repertoire of specificities and influence AE profiles and efficacy of IgG concentrates.

Diagnostic value of immunoassays for heparin-induced thrombocytopenia: a systematic review and meta-analysis.

Immunoassays are essential in the workup of patients with suspected heparin-induced thrombocytopenia. However, the diagnostic accuracy is uncertain with regard to different classes of assays, antibody specificities, thresholds, test variations, and manufacturers. We aimed to assess diagnostic accuracy measures of available immunoassays and to explore sources of heterogeneity. We performed comprehensive literature searches and applied strict inclusion criteria. Finally, 49 publications comprising 128 test evaluations in 15 199 patients were included in the analysis. Methodological quality according to the revised tool for quality assessment of diagnostic accuracy studies was moderate. Diagnostic accuracy measures were calculated with the unified model (comprising a bivariate random-effects model and a hierarchical summary receiver operating characteristics model). Important differences were observed between classes of immunoassays, type of antibody specificity, thresholds, application of confirmation step, and manufacturers. Combination of high sensitivity (>95%) and high specificity (>90%) was found in 5 tests only: polyspecific enzyme-linked immunosorbent assay (ELISA) with intermediate threshold (Genetic Testing Institute, Asserachrom), particle gel immunoassay, lateral flow immunoassay, polyspecific chemiluminescent immunoassay (CLIA) with a high threshold, and immunoglobulin G (IgG)-specific CLIA with low threshold. Borderline results (sensitivity, 99.6%; specificity, 89.9%) were observed for IgG-specific Genetic Testing Institute-ELISA with low threshold. Diagnostic accuracy appears to be inadequate in tests with high thresholds (ELISA; IgG-specific CLIA), combination of IgG specificity and intermediate thresholds (ELISA, CLIA), high-dose heparin confirmation step (ELISA), and particle immunofiltration assay. When making treatment decisions, clinicians should be a aware of diagnostic characteristics of the tests used and it is recommended they estimate posttest probabilities according to likelihood ratios as well as pretest probabilities using clinical scoring tools.

The growing spectrum of antibody-associated inflammatory brain diseases in children.

OBJECTIVE To describe the clinical spectrum, diagnostic evaluation, current management, and neurologic outcome of pediatric antibody-associated inflammatory brain diseases (AB-associated IBrainD). METHODS We performed a single-center retrospective cohort study of consecutive patients aged ≤18 years diagnosed with an AB-associated IBrainD at The Hospital for Sick Children, Toronto, Ontario, Canada, between January 2005 and June 2013. Standardized clinical data, laboratory test results, neuroimaging features, and treatment regimens were captured. RESULTS Of 169 children (93 female, 55%) diagnosed with an IBrainD, 16 (10%) had an AB-associated IBrainD. Median age at presentation was 13.3 years (range 3.1-17.9); 11 (69%) were female. Nine patients (56%) had anti-NMDA receptor encephalitis, 4 (25%) had aquaporin-4 autoimmunity, 2 (13%) had Hashimoto encephalitis, and 1 (6%) had anti-glutamic acid decarboxylase 65 (GAD65) encephalitis. The key presenting features in children with anti-NMDA receptor encephalitis, Hashimoto encephalopathy, and anti-GAD65 encephalitis included encephalopathy, behavioral symptoms, and seizures; patients with aquaporin-4 autoimmunity showed characteristic focal neurologic deficits. Six patients (38%) required intensive care unit admission at presentation. Median time from symptom onset to diagnosis was 55 days (range 6-358). All but 1 patient received immunosuppressive therapy. One child with anti-NMDA receptor encephalitis died due to multiorgan failure. At last follow-up, after a median follow-up time of 1.7 years (range 0.8-3.7), 27% of the children had function-limiting neurologic sequelae. CONCLUSIONS Children with AB-associated IBrainD represent an increasing subgroup among IBrainD; 1 in 4 children has function-limiting residual neurologic deficits. Awareness of the different clinical patterns is important in order to facilitate timely diagnosis and initiate immunosuppressive treatment.

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes.

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single pro-inflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could well be explained by the widespread gene expression dysregulation known as "genomic storm" in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this "storm". Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some pro-inflammatory molecules, complement components and endogenous "danger" signals. The improved survival in endotoxemia was associated with serum levels of pro-inflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.

Protective effect of a germline, IL-17-neutralizing antibody in murine models of autoimmune inflammatory disease.

Monoclonal antibodies (mAbs) inhibiting cytokines have recently emerged as new drug modalities for the treatment of chronic inflammatory diseases. Interleukin-17 (IL-17) is a T-cell-derived central mediator of autoimmunity. Immunization with Qβ-IL-17, a virus-like particle based vaccine, has been shown to produce autoantibodies in mice and was effective in ameliorating disease symptoms in animal models of autoimmunity. To characterize autoantibodies induced by vaccination at the molecular level, we generated mouse mAbs specific for IL-17 and compared them to germline Ig sequences. The variable regions of a selected hypermutated high-affinity anti-IL-17 antibody differed in only three amino acid residues compared to the likely germline progenitor. An antibody, which was backmutated to germline, maintained a surprisingly high affinity (0.5 nM). The ability of the parental hypermutated antibody and the derived germline antibody to block inflammation was subsequently tested in murine models of multiple sclerosis (experimental autoimmune encephalomyelitis), arthritis (collagen-induced arthritis), and psoriasis (imiquimod-induced skin inflammation). Both antibodies were able to delay disease onset and significantly reduced disease severity. Thus, the mouse genome unexpectedly encodes for antibodies with the ability to functionally neutralize IL-17 in vivo.

Organisation of the equine immunoglobulin constant heavy chain genes. II. Equine cgamma genes.

The number of immunoglobulin G constant heavy chain genes (cgamma genes) varies broadly among mammalian species, reflecting structural and functional differences between expressed immunoglobulin G (IgG) isotypes and allotypes. Up to now equine IgG isotypes have been defined only at the biochemical and serological level. It is still not clear how many IgG isotypes exist in horses and whether there are any allotypes. Here, we describe the isolation and characterisation of equine cgamma genes. An equine genomic lambda phage library was screened with a human cgamma4 probe. Cross-hybridising equine cgamma sequences were cloned twice and characterised by restriction mapping with the human cgamma4 and a murine sgamma1 probe. Genomic equine DNA probes for both, cgamma genes and corresponding switch regions (sgamma), were isolated and used for a more detailed BamHI restriction analysis, comparing genomic DNA of various horses. This analysis reveals the existence of at least five, or probably six cgamma genes in the equine haploid genome. Beside the porcine system, this is the highest number of cgamma genes described for any mammalian species. Moreover, for two of these cgamma genes, BamHI restriction fragment length polymorphism became evident.

Organization of the equine immunoglobulin heavy chain constant region genes; III. Alignment of c mu, c gamma, c epsilon and c alpha genes.

Previous restriction analysis of cloned equine DNA and genomic DNA of equine peripheral blood mononuclear cells had indicated the existence of one c epsilon, one c alpha and up to six c gamma genes in the haploid equine genome. The c epsilon and c alpha genes have been aligned on a 30 kb DNA fragment in the order 5' c epsilon-c alpha 3'. Here we describe the alignment of the equine c mu and c gamma genes by deletion analysis of one IgM, four IgG and two equine light chain expressing heterohybridomas. This analysis establishes the existence of six c gamma genes per haploid genome. The genomic alignment of the cH-genes is 5' c mu/(/) c gamma 1/(/) c gamma 2/(/) c gamma 3/(/) c gamma 4/(/) c gamma 5/(/) c gamma 6/(/) c epsilon-c alpha 3', naming the c gamma genes according to their position relative to c mu. For three of the c gamma genes the corresponding IgG isotypes could be identified as IgGa for c gamma 1, IgG(T) for c gamma 3 and IgGb for c gamma 4.

Delivery of the p67 sporozoite antigen of Theileria parva by using recombinant Salmonella dublin: secretion of the product enhances specific antibody responses in cattle

The p67 sporozoite antigen of Theileria parva has been fused to the C-terminal secretion signal of Escherichia coli hemolysin and expressed in secreted form by attenuated Salmonella dublin aroA strain SL5631. The recombinant p67 antigen was detected in the supernatant of transformed bacterial cultures. Immunization trials in cattle revealed that SL5631 secreting the antigen provoked a 10-fold-higher antibody response to p67 than recombinant SL5631 expressing but not secreting p67. Immunized calves were challenged with a 80% lethal dose of T. parva sporozoites and monitored for the development of infection. Two of three calves immunized intramuscularly with the p67-secreting SL5631 strain were found to be protected, whereas only one of three animals immunized with the nonsecreting p67-expressing SL5631 strain was protected. This is the first demonstration that complete eukaryotic antigens fused to the C-terminal portion of E. coli hemolysin can be exported from attenuated Salmonella strains and that such exported antigens can protect cattle against subsequent parasite challenge.

ANTIBODY-DEPENDENT AND ANTIBODY-INDEPENDENT CELLULAR CYTOTOXICITY AGAINST SHIGELLA

Diarrhea is a major cause of morbidity and mortality worldwide. Shigella causes up to 20% of all diarrhea. Gut-level immunity and breast-feeding of infants are important factors in protection against shigellosis. The lumen of the gut is lined with lymphocytes which mediate natural killer cytotoxicity, NKC, and antibody-dependent cellular cytotoxicity, ADCC. NKC and ADCC are extracellular, nonphagocytic leukocyte killing mechanisms, which occur in the absence of complement, without prior antigen stimulation, and without regard to the major histocompatibility complex. In this study, virulent and avirulent shigellae were used as the target cells. Leukocytes from peripheral blood, breast milk, and guinea pig gut-associated tissues were used as effector cells. Adult human peripheral blood mononuclear cells and lymphocytes, but not macrophages or polymorphonuclear leukocytes, mediated NKC and ADCC at an optimal effector to target cell ratio of 100:1 in a 60 minute bactericidal assay. An antiserum dilution of 1:10 was optimal for ADCC. Whole, viable lymphocytes were necessary for cytotoxicity. Lymphocyte NKC, but not ADCC, was greatly enhanced by interferon. Lymphocyte NKC occurred against several virulent strains of S. sonnei and a virulent strain of S. flexneri. ADCC (using immune serum directed against S. sonnei) occurred against virulent S. sonnei, but not against avirulent S. sonnei or virulent S. flexneri. Lymphocyte ADCC was not inhibited by the presence of phenylbutazone or by pretreatment of lymphocytes with anti-HNK serum plus complement. Both adherent and non-adherent breast milk leukocytes mediated NKC and ADCC. Mononuclear cells from young children demonstrated normal ADCC, when compared to ADCC of adult cells. Neonatal cord blood and a CGD patient's peripheral blood mononuclear and ploymorphonuclear cells demonstrated high ADCC compared to adult cells. Intraepithelial lymphocytes, spleen cells, and peritoneal cells from normal guinea pigs demonstrated NKC and ADCC. Animals which had been starved and opiated were made susceptible to infection by Shigella. The susceptible animals demonstrated deficient NKC and ADCC with all three leukocyte populations. High NKC and ADCC activity of gut-associated leukocytes from human breast milk and guinea pig tissues may correlate with resistance to infection. ^

THE ROLE OF B CELLS IN THE T CELL DEPENDENT RELEASE OF HUMAN B CELL GROWTH FACTOR

Quiescent human B cells are postulated to go through activation and proliferation phases before undergoing differentiative phase for immunoglobulin secretion. The present studies address some of the aspects of activation and proliferation phase of normal human B cells. The definitions of signals responsible for B cell activation and proliferation resulted in the development of a highly specific, reproducible B cell growth factor (BCGF) assay. This BCGF bioassay utilizes activation by rabbit anti-human IgM-antibody. The functional specificity of this assay for measuring BCGF activity was demonstrated by the finding that target B cells proliferated but did not differentiate. The factor specificity was determined by specific absorption of BCGF by anti-IgM activated B cells. This assay was utilized for the studies of T-B cell collaboration and the essential function of monocytes in the production and/or release of B cell growth factor in a syngeneic in vitro system. It is apparent that highly purified T cells are poor producers of BCGF by themselves and require monocytes to secrete significant quantities of BCGF upon PHA stimulation. Macrophage soluble factor, Interleukin 1, is capable of replacing monocyte function for the release of BCGF by activated T cells. In our studies, B cells are incapable to function as accessory cells to replace monocyte function. Normal B cells are also not capable of producing BCGF under our experimental observations. However, the addition of these B cells at an optimum cell density (T:B ratio 1:1) doubles the monocyte dependent release of BCGF by syngeneic T cells. The augmentative role of B cells is expanded to understand the mechanism of BCGF release by T cells. It is observed from our studies that DR antigen of B cell surface is involved in the release of BCGF. The functional difference between DR of B cells and monocytes is observed as IL-1 could replace DR-treated monocytes whereas failed to replace DR-treated B cells for the release of BCGF by T cells. This functional difference may be attributed to the reported microheterogeneity in DR of B cells and monocytes. The addition of irradiated B cells increased the monocyte dependent T cell proliferation, suggesting the increase of T cell pool for BCGF release. In summary, the development of a biological assay specific for B cell growth factor led to the delineation of an interesting role of B cells in the release of its own growth factor by T cells. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody in HIV-1 infected adults

Context: Despite tremendous strides in HIV treatment over the past decade, resistance remains a major problem. A growing number of patients develop resistance and require new therapies to suppress viral replication. ^ Objective: To assess the safety of multiple administrations of the anti-CD4 receptor (anti-CD4) monoclonal antibody ibalizumab given as intravenous (IV) infusions, in three dosage regimens, in subjects infected with human immunodeficiency virus (HIV-1). ^ Design: Phase 1, multi-center, open-label, randomized clinical trial comparing the safety, pharmacokinetics and antiviral activity of three dosages of ibalizumab. ^ Setting: Six clinical trial sites in the United States. ^ Participants: A total of twenty-two HIV-positive patients on no anti-retroviral therapy or a stable failing regimen. ^ Intervention: Randomized to one of two treatment groups in Arms A and B followed by non-randomized enrollment in Arm C. Patients randomized to Arm A received 10 mg/kg of ibalizumab every 7 days, for a total of 10 doses; patients randomized to Arm B received a total of six doses of ibalizumab; a single loading dose of 10 mg/kg on Day 1 followed by five maintenance doses of 6 mg/kg every 14 days, starting at Week 1. Patients assigned to Arm C received 25 mg/kg of ibalizumab every 14 days for a total of 5 doses. All patients were followed for safety for an additional 7 to 8 weeks. ^ Main Outcome Measures: Clinical and laboratory assessments of safety and tolerability of multiple administrations of ibalizumab in HIV-infected patients. Secondary measures of efficacy include HIV-1 RNA (viral load) measurements. ^ Results: 21 patients were treatment-experienced and 1 was naïve to HIV therapy. Six patients were failing despite therapy and 15 were on no current HIV treatment. Mean baseline viral load (4.78 log 10; range 3.7-5.9) and CD4+ cell counts (332/μL; range 89-494) were similar across cohorts. Mean peak decreases in viral load from baseline of 0.99 log10(1.11 log10, and 0.96 log 10 occurred by Wk 2 in Cohorts A, B and C, respectively. Viral loads decreased by >1.0 log10 in 64%; 4 patients viral loads were suppressed to < 400 copies/mL. Viral loads returned towards baseline by Week 9 with reduced susceptibility to ibalizumab. CD4+ cell counts rose transiently and returned toward baseline. Maximum median elevations above BL in CD4+ cell counts for Cohorts A, B and C were +257, +198 and +103 cells/μL, respectively and occurred within 3 Wks in 16 of 22 subjects. The half-life of ibalizumab was 3-3.5 days and elimination was characteristic of capacity-limited kinetics. Administration of ibalizumab was well tolerated. Four serious adverse events were reported during the study. None of these events were related to study drug. Headache, nausea and cough were the most frequently reported treatment emergent adverse events and there were no laboratory abnormalities related to study drug. ^ Conclusions: Ibalizumab administered either weekly or bi-weekly was safe, well tolerated, and demonstrated antiviral activity. Further studies with ibalizumab in combination with standard antiretroviral treatments are warranted.^