i i Supervisor: Dr. Terry W. Pearson ABSTRACT Procyclic culture forms of Trypanosoma brucei species and antibodies to these parasites were used in developing antibody-detection and antigen-detection assays for diagnosis of African human sleeping sickness. An agglutination assay using live procyclic trypanosomes- the Procyclic Agglutination Trypanosomiasis Test (PATT) was developed for detecting anti-trypanosome antibodies in the sera of trypanosome-infected vervet monkeys and humans. Antibodies to procyclic surface antigens were detected by the PATT in sera of vervet monkeys as early as 7 days post-infection with T. b. rhodesiense. Positive agglutination titres were obtained with sera from monkeys with active, untreated infections and with sera taken soon after successful drag cure. Similar positive agglutination results were also observed using the PATT with sera from T. b.gambiense- infected patients from Cote d'Ivoire and Sudan and with documented sera from T. b. rhodesiense—infected patients from Kenya. No agglutination reactions were observed with preinfection sera from vervet monk, ys, with sera from uninfected Canadians or with sera from Americans working in endemic areas. Together these results confirm the diagnostic value of using procyclic trypanosomes to detect anti-trypanosome antibodies in. human African sleeping sickness. A double antibody sandwich ELISA using monoclonal antibodies and polyclonal rabbit antibodies to the surface membrane antigens of procyclic trypanosomes was developed. This assay detected circulating trypanosomal antigens in the sera of trypanosome-infected mice and in the sera from parasite-infected patients. However, limited success was obtained with this sandwich ELISA when tested on a larger repertoire of sera from infected humans. Rabbit antibodies made against whole lysates of T. b. rhodesiense procyclics were then employed in an antigen-trapping sandwich ELISA. The results demonstrated the effectiveness of this sandwich ELISA in revealing the infection status of vervet monkeys or humans infected with either T. b. rhodesiense or T. h. gambiense. Trypanosomal antigens were detected in the sera of parasitologically confirmed monkeys and patients but not in preinfection sera nor in control sera from uninfected North Americans. i i i The PATT and the sandwich ELISA exhibited higher sensitivities than the currently employed diagnostic assay for human sleeping sickness, the Card Agglutination Trypanosomiasis Test (CATT), when tested with sera of parasitologically-confirmed humans. The sandwich ELISA was superior to the antibody-detecting PATT and CATT in monitoring trypanocidal drug-treated patients. The overall sensitivity of the PATT and sandwich ELISA was 94.3% and 97.4% and the specificity was 84.5% and 95.5%, respectively. These results thus confirm the diagnostic value of these tests for the diagnosis of human African sleeping sickness. Identification of diagnostically useful antigens was attempted in order to facilitate the adaptation of these diagnostic assays to a simpler format for field application. Pooled sera obtained from trypanosome-infected patients was used as a probe to detect trypanosome antigens separated by high performance liquid chromatography, immunoaffinity and immunoblotting techniques. Most of the antigens were detected in the higher molecular weight range (>62 Kd). Immunization of mice with the target antigens yielded six trypanosome-specific monoclonal antibodies. In a double antibody sandwich ELISA, these antibodies were successful in trapping circulating parasite antigens in sera from trypanosome-infected mice as early as 3 days post-infection. Some of these antigens have been partially biochemically characterized. Trypanosomal antigens were also detected by these antibodies in the urine of infected mice. The antigen-capture sandwich ELISA using either the selected monoclonal antibodies or the rabbit anti-procyclic whole lysate antibodies gave similar results with sera from trypanosome-infected mice, human sleeping sickness patients and uninfected humans from North America and Kenya. The results showed that these MAbs and their antigens were useful in the diagnosis of African human sleeping sickness. Examiners: Dr. T erry^i Peaifs^, Supervisor lijche(Department of Biochemistrylijche(Department and Microbiology) Dr. William W.lCay, Departmental lum ber (Department of Biochemistry and Microbiology) L . Dr.7Ro'SertWTOl^sm^ E&partmental Member (Department of Biophemis^and Microbiology] Dr^Miphael J. Ashwood-Smith, Outside Member (Djepapment of Biology) Dr. Robot D. Burice, Outside Member (Department of Biology) Dr. Timothy Lee, External Examiner (University of Calgary) V TABLE OF CONTENTS ABSTRACT................................................................................................ ii TABLE OF CONTENTS .............................................................. v LIST OF TABLES .............................................................................. vii LIST OF FIGURES...................................................................................... ix ACKNOWLEDGEMENTS............................................................................ xiii FOREWORD.................................... ............................................................. xiv INTRODUCTION........................................................................................... 1 CHAPTER 1 Use of Procyclic trypanosomes in an antibody detection assay for African human sleeping sickness ................................... 1 Introduction ............................................................................ 40 Materials and Methods ........................................................ 42 R esults.,.............................. 46 Discussion ................................................................................ 60 CHAPTER 2 Detection of circulating trypanosomal antigens by double antibody sandwich ELISA using antibodies to procyclic trypanosomes ........................................................................... 62 Introduction .............................................................................. 62 Materials and Methods ........................................................ 65 Results....................... ............................................................... 73 Discussion ................................................................................ 92 CHAPTER 3 Serodiagnosis of human African sleeping sickness by detection of anti-procyclic antibodies and trypanosome antigens 96 Introduction .............................................................................. 96 Materials and Methods ................................................ 98 Results .......................................................................... 104 Discussion ............................................................ 143 CHAPTER 4 Identification of procyclic trypanosomal antigens that have serodiagnostic potential for human sleeping sickness .............. 149 Introduction .............................................................................. 149 Materials and Methods ........................................................ 150 Results....................................................................................... 166 Discussion ................................................. 236 V DISCUSSION............................................................................... 243 LITERATURE CITED.................................................................................. 255 VII LIST OF TABLES Table 1 Measurement of anti-procyclic surface antibodies in vervet monkey sera before and during infection with T. b. rhodesiense and at various times after treatment with trypanocidal drugs ................. 52 Table 2 Total IgM and IgG levels in vervet monkey sera before and during infection with T. b. rhodesiense and after drug treatment 56 Table 3 Cellular, biochemical, parasitological and serological measurements on sera from African sleeping sickness patients from Daloa, C6te d'Ivoire ...................................................... 57 Table 4 Competitive solid-phase radioimmunometric assay of anti- trypanosome monoclonal antibodies ....................................... 87 Table 5 Binding of biotin- or enzyme-labeled MAbs to trypanosomal antigens trapped by homologous or heterologous MAbs in double antibody sandwich ELISA ...................................................... 88 Table 6 Comparison of three different enzyme assay systems in indirect ELISA.......................................................................................... 89 Table 7 Stability of antibody coated microtiter plates and nitrocellulose paper in ELISA and dot-blot assays after storage at different time intervals and temperatures ..................................................... 90 Table 8 Detection of antigens or epitopes in bloodstream form and procyclic culture form trypanosomes by ELISA ....................... 91 Table 9 Measurement of anti-procyclic antibodies and circulating trypanosomal antigens in vervet monkey sera before and during infection with T. b. rhodesiense and at various times after treatment with trypanocidal drugs ........................................... 122 Table 10 Detection of anti-trypanosome antibodies and trypanosome antigens in sera of trypanosome-infected Kenyans before drug treatment and at the time of relapse .................................... 126 Table 11 Measurement of anti-procyclic