Kathryn H. Ching

Anti-cytokine autoantibodies are associated with opportunistic infection in patients with thymic neoplasia

Patients with thymic malignancy have high rates of autoimmunity leading to a variety of autoimmune diseases, most commonly myasthenia gravis caused by anti-acetylcholine receptor autoantibodies. High rates of autoantibodies to cytokines have also been described, although prevalence, spectrum, and functionality of these anti-cytokine autoantibodies are poorly defined. To better understand the presence and function of anti-cytokine autoantibodies, we created a luciferase immunoprecipitation system panel to search for autoantibodies against 39 different cytokines and examined plasma from controls (n = 30) and patients with thymic neoplasia (n = 17). In this screen, our patients showed statistically elevated, but highly heterogeneous immunoreactivity against 16 of the 39 cytokines. Some patients showed autoantibodies to multiple cytokines. Functional testing proved that autoantibodies directed against interferon-α, interferon-β, interleukin-1α (IL-1α), IL-12p35, IL-12p40, and IL-17A had biologic blocking activity in vitro. All patients with opportunistic infection showed multiple anti-cytokine autoantibodies (range 3-11), suggesting that anti-cytokine autoantibodies may be important in the pathogenesis of opportunistic infections in patients with thymic malignancy. This study was registered at http://clinicaltrials.gov as NCT00001355.

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log(10) for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Two major autoantibody clusters in systemic lupus erythematosus

Systemic lupus erythematosus is a chronic autoimmune disease of complex clinical presentation and etiology and is likely influenced by numerous genetic and environmental factors. While a large number of susceptibility genes have been identified, the production of antibodies against a distinct subset of nuclear proteins remains a primary distinguishing characteristic in disease diagnosis. However, the utility of autoantibody biomarkers for disease sub-classification and grouping remains elusive, in part, because of the difficulty in large scale profiling using a uniform, quantitative platform. In the present study serological profiles of several known SLE antigens, including Sm-D3, RNP-A, RNP-70k, Ro52, Ro60, and La, as well as other cytokine and neuronal antigens were obtained using the luciferase immunoprecipitation systems (LIPS) approach. The resulting autoantibody profiles revealed that 88% of a pilot cohort and 98% of a second independent cohort segregated into one of two distinct clusters defined by autoantibodies against Sm/anti-RNP or Ro/La autoantigens, proteins often involved in RNA binding activities. The Sm/RNP cluster was associated with a higher prevalence of serositis in comparison to the Ro/La cluster (P = 0.0022). However, from the available clinical information, no other clinical characteristics were associated with either cluster. In contrast, evaluation of autoantibodies on an individual basis revealed an association between anti-Sm (P = 0.006), RNP-A (P = 0.018) and RNP-70k (P = 0.010) autoantibodies and mucocutaneous symptoms and between anti-RNP-70k and musculoskeletal manifestations (P = 0.059). Serologically active, but clinically quiescent disease also had a higher prevalence of anti-IFN-α autoantibodies. Based on our findings that most SLE patients belong to either a Sm/RNP or Ro/La autoantigen cluster, these results suggest the possibility that alterations in RNA-RNA-binding protein interactions may play a critical role in triggering and/or the pathogenesis of SLE.

Antibody-profiling technologies for studying humoral responses to infectious agents

Analyses of humoral responses against different infectious agents are critical for infectious disease diagnostics, understanding pathogenic mechanisms, and the development and monitoring of vaccines. While ELISAs are often used to measure antibody responses to one or several targets, new antibody-profiling technologies, such as protein microarrays, can now evaluate antibody responses to hundreds, or even thousands, of recombinant antigens at one time. These large-scale studies have uncovered new antigenic targets, provided new insights into vaccine research and yielded an overview of immunoreactivity against almost the entire proteome of certain pathogens. However, solid-phase antigen arrays also have drawbacks that limit the type of information obtained, including suboptimal detection of conformational epitopes, high backgrounds due to impure antigens and a narrow dynamic range of detection. We have developed a solution-phase antibody-profiling technology, luciferase immunoprecipitation systems (LIPS), which harnesses light-emitting recombinant antigen fusion proteins to quantitatively measure patient antibody titers. Owing to the highly linear light output of the luciferase reporter, some antibodies can be detected without serum dilution in a dynamic range of detection often spanning seven orders of magnitude. When LIPS is applied iteratively with multiple target antigens, a high-definition antibody profile is obtained. Here, we discuss the application of these different antibody-profiling technologies and their associated limitations with particular emphasis on protein microarrays. We also describe LIPS in detail and discuss several clinically relevant uses of the technology. Together, these new technologies offer new tools for understanding humoral responses to known and emerging infectious agents.

Rapid, Simple, Quantitative, and Highly Sensitive Antibody Detection for Lyme Disease

ABSTRACT There is currently a need for improved serological tests for the diagnosis and monitoring of Lyme disease, an infection caused by Borrelia burgdorferi. In the present study, we evaluated luciferase immunoprecipitation systems (LIPSs) for use for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. Initially, serum samples from a cohort of patients and controls (n = 46) were used for training and were profiled by the use of 15 different B. burgdorferi antigen constructs. For the patient sera, the antibody responses to several B. burgdorferi antigens, including VlsE, flagellin (FlaB), BmpA, DbpA, and DbpB, indicated that the antigens had high levels of immunoreactivity. However, the best diagnostic performance was achieved with a synthetic protein, designated VOVO, consisting of a repeated antigenic peptide sequence, VlsE-OspC-VlsE-OspC, Analysis of an independent set of serum samples (n = 139) used for validation showed that the VOVO LIPS test had 98% sensitivity (95% confidence interval [CI], 93% to 100%; P < 0.0001) and 100% specificity (95% CI, 94% to 100%; P < 0.0001). Similarly, the C6 peptide enzyme-linked immunosorbent assay (ELISA) also had 98% sensitivity (95% CI, 93% to 100%; P < 0.0001) and 98% specificity (95% CI, 90% to 100%; P < 0.0001). Receiver operating characteristic analysis revealed that the rates of detection of Lyme disease by the LIPS test and the C6 ELISA were not statistically different. However, the VOVO LIPS test displayed a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution. These results suggest that screening by the LIPS test with VOVO and other B. burgdorferi antigens offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.

Serological Studies Confirm the Novel Astrovirus HMOAstV-C as a Highly Prevalent Human Infectious Agent

Molecular identification of a microbe is the first step in determining its prevalence of infection and pathogenic potential. Detection of specific adaptive immune responses can provide insights into whether a microbe is a human infectious agent and its epidemiology. Here we characterized human anti-IgG antibody responses by luciferase immunoprecipitation systems (LIPS) against two protein fragments derived from the capsid protein of the novel HMOAstV-C astrovirus. While antibodies to the N-terminal fragment were not informative, the C-terminal capsid fragment of HMOAstV-C showed a high frequency of immunoreactivity with serum from healthy blood donors. In contrast, a similar C-terminal capsid fragment from the related HMOAstV-A astrovirus failed to show immunoreactivity. Detailed analysis of adult serum from the United Sates using a standardized threshold demonstrated HMOAstV-C seropositivity in approximately 65% of the samples. Evaluation of serum samples from different pediatric age groups revealed that the prevalence of antibodies in 6–12 month, 1–2 year, 2–5 year and 5–10 year olds was 20%, 23%, 32% and 36%, respectively, indicating rising seroprevalence with age. Additionally, 50% (11/22) of the 0–6 month old children showed anti-HMOAstV-C antibody responses, likely reflecting maternal antibodies. Together these results document human humoral responses to HMOAstV-C and validate LIPS as a facile and effective approach for identifying humoral responses to novel infectious agents.

Distinct Profiles of Antibodies to Kaposi Sarcoma-Associated Herpesvirus Antigens in Patients with Kaposi Sarcoma, Multicentric Castleman Disease, and Primary Effusion Lymphoma

Antibody responses against lytic and latent Kaposi sarcoma (KS)-associated herpesvirus antigens were investigated in patients with KS, multicentric Castleman disease (MCD), and primary effusion lymphoma. Antibodies against the lytic antigen K8.1 were 5-fold higher in patients with MCD than those with KS, whereas antibodies to the sum of latent antigens v-cyclin and LANA were 27-fold higher in patients with KS, compared with patients with MCD (P < 001). The sum of anti-v-cyclin and anti-LANA antibody titers discriminated patients with KS from those with MCD and KS with 93% sensitivity and 83% specificity. These results suggest that antibody responses to lytic and latent KS-associated herpesvirus antigens differ in these diseases.

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.

Salivary anti-Ro60 and anti-Ro52 Antibody Profiles to Diagnose Sjögren’s Syndrome

Simple and non-invasive saliva-based diagnostics may be useful for the identification, understanding, and monitoring of autoimmune and infectious diseases. Previously, Luciferase Immunoprecipitation Systems (LIPS) were used for sensitive detection of patient serum autoantibodies in Sjögren’s Syndrome (SjS), a chronic autoimmune disease affecting the salivary and lacrimal glands. Here we explored the ability of LIPS to diagnose SjS based on IgG autoantibodies in patient saliva. From LIPS testing, anti-Ro60 autoantibodies were detected in the saliva of 70% (19/27) of SjS patients with 96% specificity. Positive anti-Ro60 autoantibodies were also found in 70% of the matched serum samples (96% specificity). LIPS detected Ro52 autoantibodies in the saliva and serum of 67% of SjS patients with 100% specificity. Overall, the autoantibody titers in saliva were approximately 4000-fold lower by volume than serum, but still distinguished seropositive patients from controls. These results suggest that LIPS salivary-based testing for SjS autoantibodies is a practical alternative to serum and compatible with point-of-care testing.

Primary Sjögren's syndrome is characterized by distinct phenotypic and transcriptional profiles of IgD+ unswitched memory B cells.

The significance of distinct B cell abnormalities in primary Sjögrens syndrome (SS) remains to be established. We undertook this study to analyze the phenotype and messenger RNA (mRNA) transcript profiles of B cell subsets in patients with primary SS and to compare them with those in sicca syndrome patients and healthy controls.