Anti-lipid antibody in M. tuberculosis infection-- basis for a new biomarker-based test to monitor treatment response

Tuberculosis (TB) faces difficult challenges for its treatment and control. Both the diagnosis of TB and Mycobacterium tuberculosis (M. tuberculosis) drug susceptibility testing take weeks and clinicians often do not know if the patient is taking an appropriate set of drugs until complications or even death occur. Consequently, early determination of a successful drug therapy response in individuals infected with M. tuberculosis is urgently needed. Since the M. tuberculosis cell wall is comprised of a diverse repertoire of lipids, we examined the possible role of these lipids as antigens for serologic response during M. tuberculosis infection. This dissertation is focused on the examination of lipid-antibody response as a potential biomarker used to monitor treatment response in M. tuberculosis infected hosts. Briefly, Chapter 1 describes the current pitfalls of monitoring tuberculosis treatment with current methods, including acid-fast bacilli (AFB) smear and culture conversion. Chapter 1 also covers the definition of biomarkers and the rationale to use M. tuberculosis cell wall lipids to develop an anti-lipid-antibody based test. Evidence from a similar biomarker-based test for syphilis is presented. The chapter also discusses the biological basis which guided the lipid-antibody biomarker search and discovery./nChapter 2 describes the use of a serum bank from patients with pulmonary TB provided by the World Health Organization-Tropical Diseases Research consortium (WHO-TDR) to identify M. tuberculosis lipid candidates as targets of antibody response. These samples were used to look for an antibody response to multiple mycobacterial lipids resolved by thin-layer chromatography immunoblot (TLC-I). This approach allowed us to identify M. tuberculosis cardiolipin by mass spectrometry and we determined that the IgM antibody response to cardiolipin can be used as a biomarker of infection./nIn Chapter 3, we investigate the biological evidence behind the production of anti-phospholipid IgM antibody during TB infection and anti-TB treatment. For this purpose, we used the Cornell mouse model of infection to monitor the change in IgM antibody response against four phospholipids including cardiolipin (CL), phosphatidyl choline (PTC), phosphatidyl ethanolamine (PE) and phosphatidyl inositol (PI) over the course of M. tuberculosis infection and treatment. We separated BALB/c mice into three groups including acute infection (AI), chronic infection (CI), and healthy control (HC). Both AI and CI groups were infected via the aerosol route with M. tuberculosis strain H37Rv at day 0. The AI group was treated from 4 to 12 weeks post-infection, while the CI group was treated from 20 to 28 weeks post-infection. We also measured the levels of pro-inflammatory cytokines, IL-5 and MCP-1. We observed that in treated AI mice, anti-phospholipid IgM antibody levels decreased compared to those of healthy mice at all time points. Anti-PTC IgM antibodies remained significantly higher in CI mice than in AI mice at all time points post-infection. The anti-PTC IgM antibody levels in CI mice decreased to levels similar to those of AI and HC mice at 32 weeks post-infection. The anti-phospholipid IgM antibody levels correlated with the bacterial load in the lungs, with treated mice showing fewer M. tuberculosis colony-forming units (CFU) after eight weeks of treatment. Furthermore, IL-5 was mainly produced by the site of infection in the lung and decreased with anti-tuberculosis treatment within the CI mice group./nFinally, in Chapter 4, we examine the use of anti-phospholipid IgM antibody changes as a biomarker for treatment response in patients with smear positive pulmonary TB. Serum samples were obtained from pulmonary TB patients at the start and end of the intensive phase of treatment (40 doses of anti-TB combination therapy) enrolled from Kampala, Uganda in a CDC-TB Trials Consortium randomized clinical trial. The samples were screened for IgM antibody levels against five commercially available phospholipids by an in-house ELISA assay. The lipid antigens included CL, PI, PE, PTC, and sphingolipid (SL). IgM antibody levels to CL, PE, PI, PTC and SL significantly decreased following anti-TB drug treatment in patients without lung cavities on their baseline chest radiograph. In contrast, patients with cavitary TB showed an overall increase in the anti-phospholipid IgM antibody response following anti-TB drug treatment, notably with a significant increase in anti-PE antibody levels. Thus, anti-lipid IgM response appears to be a useful biomarker for treatment response, especially in those with non-cavitary disease./nChapter 5 summarizes the conclusions in support of using the anti-phospholipid IgM antibody response as a useful biomarker for monitoring TB treatment response. This novel biomarker test would greatly facilitate TB management in resource-poor settings. The development of a point of care (POC) test based on anti-phospholipid IgM antibody will be an affordable and highly sensitive alternative to microscopy or culture testing for monitoring treatment response in individuals with TB. Chapter 5 also gives examples of three platforms that might be used for the development of such a POC test. However, we note that it is necessary to explore the specificity of this assay further by testing patients with HIV infection, latent TB infection, and non-TB pulmonary diseases.

Advisor: Riley, Lee W