Dimeric immunoglobulin A for serodiagnosis of mucosal infections

The absence or limited effectiveness of vaccines for hepatitis C virus (HCV) infections and tuberculosis (TB) places greater onus on early detection and treatment as primary prevention strategies. In anticipation of new antivirals for HCV and better regimens of anti-TB drugs promising higher cure rates, improved diagnostics is critical to ensure effective treatment and global control of viral hepatitis and TB. This thesis presents 1) dimeric immunoglobulin (Ig) A (dIgA) as a promising biomarker of acute viral hepatitis, and 2) unique strategies in improving TB serodiagnosis, such as biomarker combinations and iterative assay development in efforts to satisfy critical gaps in TB case finding. DIgA is abundantly produced in a mucosal immune response towards communicable diseases such as pulmonary TB, HCV and hepatitis A and E virus (HAV, HEV) infections. Historically, dIgA has been neglected in diagnostic research since it contributes only 10% of total IgA in blood, and standard assays for IgA fail to reflect this dynamic biomarker. This thesis describes a simple approach to measuring dIgA using a recombinant chimeric secretory component (cSC), innovated based on the inherent properties of the polymeric Ig receptor. Novel cSC-based ELISAs were used to detect antigen-specific dIgA in serum of patients infected with HAV, HCV, HEV, active TB and appropriate controls. The diagnostic performance of antigen-specific dIgA was compared to antigen-specific IgG, IgA and IgM. Since optimal antigens for antibody-based TB diagnostics have not been identified, candidate M. tuberculosis complex (MTB) antigens and markers of host innate immunity were screened before further work proceeded with antigen 60 (A60), first using commercial Bacille Calmete Guerin (BCG) –derived A60, then using in-house MTB strain CDC 1551-derived A60 for comparison. Virus-specific dIgA were detectable in serum of individuals infected with HAV, HEV and HCV. Anti-HAV and anti-HEV dIgA discriminate acutely infected from uninfected subjects with 100% accuracy, with higher specificity compared to anti-HAV and anti-HEV IgM assays. Anti-HCV dIgA levels peaked early and declined to undetectable levels after four weeks post-primary bleed, even in ongoing infections, while serum anti-HCV monomeric IgA, IgG and IgM persisted. Subjects with early acute HCV infection had significantly higher levels of anti-HCV dIgA compared to those with chronic infections. For diagnosing active TB, MTB-specific dIgA was specific (sp: 94-98%) although insufficiently sensitive (sn: 7-35%). The heterogeneous antibody response across patients, and low correlation between antibody classes observed necessitates a combination of biomarkers. An algorithm of A60-specific IgG and IgA with a C-reactive Protein “rule out” discriminated active TB from non-TB patients with 82% sensitivity and 90% specificity, but was not reproducible in a blinded panel, especially in human immunodeficiency virus (HIV)-positive individuals. Data from a subset of the samples indicated that MTB A60 might be a better antigenic preparation compared to BCG A60. However, the divergent antigenic composition of BCG and MTB A60, and how these data can be manipulated into translation of better diagnostic assays requires further investigation. In summary, the work presented highlights unique serological patterns of different antibody classes in patients with TB and viral hepatitis. Essentially, dominant anti-MTB IgA or IgG responses among active TB patients, and the transient production of anti-HCV dIgA have important implications on development of diagnostics and sero-epidemiological tools, which merits intensified research.