Use of Protein Antigens for Early Serological Diagnosis of Leprosy

Abstract

Leprosy is a chronic and debilitating human disease caused by infection with the Mycobacterium leprae bacillus. Despite the marked reduction in the number of registered worldwide leprosy cases as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains relatively stable. This indicates that M. leprae is still being transmitted and that, without earlier diagnosis, M. leprae infection will continue to pose a health problem. Current diagnostic techniques, based on the appearance of clinical symptoms or of immunoglobulin M (IgM) antibodies that recognize the bacterial phenolic glycolipid I, are unable to reliably identify early-stage leprosy. In this study we examine the ability of IgG within leprosy patient sera to bind several M. leprae protein antigens. As expected, multibacillary leprosy patients provided stronger responses than paucibacillary leprosy patients. We demonstrate that the geographic locations of the patients can influence the antigens they recognize but that ML0405 and ML2331 are recognized by sera from diverse regions (the Philippines, coastal and central Brazil, and Japan). A fusion construct of these two proteins (designated leprosy IDRI diagnostic 1 [LID-1]) retained the diagnostic activity of the component antigens. Upon testing against a panel of prospective sera from individuals who developed leprosy, we determined that LID-1 was capable of diagnosing leprosy 6 to 8 months before the onset of clinical symptoms. A serological diagnostic test capable of identifying and allowing treatment of early-stage leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.Cases in which Mycobacterium leprae infection manifests to cause leprosy present as a bacteriologic, clinical, immunologic, and pathological spectrum ranging from the extremes observed in paucibacillary (PB) and multibacillary (MB) patients (21,24). PB patients have one or a few skin lesions and a low or absent bacterial index (BI; a measure of the number of acidfast bacilli in the dermis, expressed on a logarithmic scale) and demonstrate specific cell-mediated immunity against M. leprae, but they have low or absent titers of M. leprae-specific antibodies and a granulomatous dermatopathology. In marked contrast, MB patients have multiple symmetric skin lesions and a high BI and demonstrate high titers of anti-M. leprae antibodies but an absence of specific cell-mediated immunity and a dermatopathology largely devoid of functional lymphocytes (21). Despite the implementation of a WHO-directed eradication program over the last 20 years, the worldwide annual rate of new case detection for leprosy remains stable at approxi-

Identification of serological biomarkers of infection, disease progression and treatment efficacy for leprosy

“…The risk factors identified in this study include contact with cases of PB leprosy with two-five lesions or MB disease, physical closeness, such as residing within the sponses to LID-1 up to one year prior to the development of clinical symptoms. These antibody titres were higher and occurred much sooner than the increases in the anti-PGL-I IgM responses observed in the same individuals (Duthie et al 2007). It was also shown that a heightened IgG titre against LID-1 was a good indicator of early infection and active disease, as titres remained low or undetectable in contacts who did not progress to disease.…”

“…The reactivities of the antibodies in the baseline samples against three key antigens, including LID-1 (which had been shown in a previous study to elicit a positive antibody response in individuals who were diagnosed with clinical leprosy up to one year prior to the onset of disease symptoms) (Duthie et al 2007), ML2028 (Ag85B) and native MLSA, which was examined to assess reactivity against LAM, were determined by Western blot analysis, as shown in Fig. 4.…”

“…At the time of submission, the disease status of these individuals remained unchanged. Further analysis of HHC 12-2 and HHC 18-2 over a two-year period -Because antibody titres to LID-1 have previously been shown to increase prior to clinical diagnosis (Duthie et al 2007) we were especially interested in those HHCs who showed stronger reactivities against this antigen. HHC 12-2 and HHC 18-2 showed the strongest responses against LID-1 and ML2028 at baseline.…”

“…The titres of IgG antibodies to protein antigens generally correlate with the bacterial load and strong responses in MB patients can rival anti-PGL-I responses. The best protein candidates to assess the IgG response include ML0405, ML2331, ML2055 (Reece et al 2006, Duthie et al 2007), ML2028 (Launois et al 1994) and ML2038 , Kai et al 2008. A bioinformatics analysis predicted ML0405 to be one of the most promiscuous M. leprae proteins studied to date and to contain T cell epitopes predicted to be recognised by 50 out of 51 human leukocyte antigen-DR alleles along with seven potential T cell epitopes and 39 potential B cell epitopes, whereas ML2331 contained 24 potential B cell epitopes (Sampaio et al 2011).…”

Defining mycobacteria: Shared and specific genome features for different lifestyles

“…The work of Duthie et al [173], focused on the search for potential serologically reactive protein antigens prior to testing for the rigorous requirement for leprosy specifi city when tested in various endemic populations. Such approaches lead to the identifi cation of new antigens (ML0405, ML 2331) from which novel fusion proteins were designed.…”