Analysis of Recombinant Antibody Libraries from Patients with Chronic Chagas Heart Disease and Selection of Human Recombinant Antibodies against Trypanosoma cruzi Ribosomal P Proteins

V. GRIPPO, M. EVELINA, K. GOMEZ, C. SCHMULSKI, V. LABOVSKY, G. LEVY, S. LONGHI, M.J. LEVIN

Mérida, México

Congreso; HHMI Meeting of International Research Scholars.; 2005

Howard Hughes Medical Institute

Patients with chronic Chagas heart disease (cChHD), the severest form of the human chronic infection by Trypanosoma cruzi, have circulating antibodies that may be involved in the pathogenesis of cardiac dysfunction. To better understand this antibody repertoire, we constructed two combinatorial libraries, an Fab (1  108 cfu/microgram) and an scFv (1  109 cfu/microgram) library. The variable regions of these antibodies were compared with the variable regions of antibodies obtained from single plasma cells. The phage libraries were panned against whole T. cruzi homogenates and recombinant T. cruzi ribosomal P proteins. Several methods, both classical and original, were used to screen for specific Fab- and scFv-bearing phages. Anti–T. cruzi and anti-P antibodies were obtained that reacted with different T. cruzi proteins and with the C-terminal ends of the ribosomal P proteins. Furthermore, the repertoire and distribution of 148 rearranged VH genes were analyzed and compared with available data of repertoires derived from healthy individuals, patients with autoimmune diseases, and patients with other infections. Preliminary results show that the VH family usage in the unselected libraries has an overrepresentation of VH5 family– and underrepresentation of VH3-23–related germline genes. Amongst the anti–T. cruzi clones, a VH4 overrepresentation was evident, contributed by VH4-59– and VH4- 39–related genes. By contrast, plasma cells from cardiac inflammatory sites showed a preference for the VH1 family. The composition of CDR3 regions from the antibodies of the phage libraries and those derived from plasma cells were different, pointing to a higher degree of affinity maturation among the latter, a finding that was confirmed by analysis of the somatic mutations. The characterization of anti-P antibodies from patients with Chagas disease allowed us to compare them with anti-P antibodies from patients with systemic lupus erythematosus (SLE). Molecular differences underscore the fact that antibodies againstTrypanosoma cruzi, have circulating antibodies that may be involved in the pathogenesis of cardiac dysfunction. To better understand this antibody repertoire, we constructed two combinatorial libraries, an Fab (1  108 cfu/microgram) and an scFv (1  109 cfu/microgram) library. The variable regions of these antibodies were compared with the variable regions of antibodies obtained from single plasma cells. The phage libraries were panned against whole T. cruzi homogenates and recombinant T. cruzi ribosomal P proteins. Several methods, both classical and original, were used to screen for specific Fab- and scFv-bearing phages. Anti–T. cruzi and anti-P antibodies were obtained that reacted with different T. cruzi proteins and with the C-terminal ends of the ribosomal P proteins. Furthermore, the repertoire and distribution of 148 rearranged VH genes were analyzed and compared with available data of repertoires derived from healthy individuals, patients with autoimmune diseases, and patients with other infections. Preliminary results show that the VH family usage in the unselected libraries has an overrepresentation of VH5 family– and underrepresentation of VH3-23–related germline genes. Amongst the anti–T. cruzi clones, a VH4 overrepresentation was evident, contributed by VH4-59– and VH4- 39–related genes. By contrast, plasma cells from cardiac inflammatory sites showed a preference for the VH1 family. The composition of CDR3 regions from the antibodies of the phage libraries and those derived from plasma cells were different, pointing to a higher degree of affinity maturation among the latter, a finding that was confirmed by analysis of the somatic mutations. The characterization of anti-P antibodies from patients with Chagas disease allowed us to compare them with anti-P antibodies from patients with systemic lupus erythematosus (SLE). Molecular differences underscore the fact that antibodies against 108 cfu/microgram) and an scFv (1  109 cfu/microgram) library. The variable regions of these antibodies were compared with the variable regions of antibodies obtained from single plasma cells. The phage libraries were panned against whole T. cruzi homogenates and recombinant T. cruzi ribosomal P proteins. Several methods, both classical and original, were used to screen for specific Fab- and scFv-bearing phages. Anti–T. cruzi and anti-P antibodies were obtained that reacted with different T. cruzi proteins and with the C-terminal ends of the ribosomal P proteins. Furthermore, the repertoire and distribution of 148 rearranged VH genes were analyzed and compared with available data of repertoires derived from healthy individuals, patients with autoimmune diseases, and patients with other infections. Preliminary results show that the VH family usage in the unselected libraries has an overrepresentation of VH5 family– and underrepresentation of VH3-23–related germline genes. Amongst the anti–T. cruzi clones, a VH4 overrepresentation was evident, contributed by VH4-59– and VH4- 39–related genes. By contrast, plasma cells from cardiac inflammatory sites showed a preference for the VH1 family. The composition of CDR3 regions from the antibodies of the phage libraries and those derived from plasma cells were different, pointing to a higher degree of affinity maturation among the latter, a finding that was confirmed by analysis of the somatic mutations. The characterization of anti-P antibodies from patients with Chagas disease allowed us to compare them with anti-P antibodies from patients with systemic lupus erythematosus (SLE). Molecular differences underscore the fact that antibodies against 109 cfu/microgram) library. The variable regions of these antibodies were compared with the variable regions of antibodies obtained from single plasma cells. The phage libraries were panned against whole T. cruzi homogenates and recombinant T. cruzi ribosomal P proteins. Several methods, both classical and original, were used to screen for specific Fab- and scFv-bearing phages. Anti–T. cruzi and anti-P antibodies were obtained that reacted with different T. cruzi proteins and with the C-terminal ends of the ribosomal P proteins. Furthermore, the repertoire and distribution of 148 rearranged VH genes were analyzed and compared with available data of repertoires derived from healthy individuals, patients with autoimmune diseases, and patients with other infections. Preliminary results show that the VH family usage in the unselected libraries has an overrepresentation of VH5 family– and underrepresentation of VH3-23–related germline genes. Amongst the anti–T. cruzi clones, a VH4 overrepresentation was evident, contributed by VH4-59– and VH4- 39–related genes. By contrast, plasma cells from cardiac inflammatory sites showed a preference for the VH1 family. The composition of CDR3 regions from the antibodies of the phage libraries and those derived from plasma cells were different, pointing to a higher degree of affinity maturation among the latter, a finding that was confirmed by analysis of the somatic mutations. The characterization of anti-P antibodies from patients with Chagas disease allowed us to compare them with anti-P antibodies from patients with systemic lupus erythematosus (SLE). Molecular differences underscore the fact that antibodies againstT. cruzi homogenates and recombinant T. cruzi ribosomal P proteins. Several methods, both classical and original, were used to screen for specific Fab- and scFv-bearing phages. Anti–T. cruzi and anti-P antibodies were obtained that reacted with different T. cruzi proteins and with the C-terminal ends of the ribosomal P proteins. Furthermore, the repertoire and distribution of 148 rearranged VH genes were analyzed and compared with available data of repertoires derived from healthy individuals, patients with autoimmune diseases, and patients with other infections. Preliminary results show that the VH family usage in the unselected libraries has an overrepresentation of VH5 family– and underrepresentation of VH3-23–related germline genes. Amongst the anti–T. cruzi clones, a VH4 overrepresentation was evident, contributed by VH4-59– and VH4- 39–related genes. By contrast, plasma cells from cardiac inflammatory sites showed a preference for the VH1 family. The composition of CDR3 regions from the antibodies of the phage libraries and those derived from plasma cells were different, pointing to a higher degree of affinity maturation among the latter, a finding that was confirmed by analysis of the somatic mutations. The characterization of anti-P antibodies from patients with Chagas disease allowed us to compare them with anti-P antibodies from patients with systemic lupus erythematosus (SLE). Molecular differences underscore the fact that antibodies againstT. cruzi ribosomal P proteins. Several methods, both classical and original, were used to screen for specific Fab- and scFv-bearing phages. Anti–T. cruzi and anti-P antibodies were obtained that reacted with different T. cruzi proteins and with the C-terminal ends of the ribosomal P proteins. Furthermore, the repertoire and distribution of 148 rearranged VH genes were analyzed and compared with available data of repertoires derived from healthy individuals, patients with autoimmune diseases, and patients with other infections. Preliminary results show that the VH family usage in the unselected libraries has an overrepresentation of VH5 family– and underrepresentation of VH3-23–related germline genes. Amongst the anti–T. cruzi clones, a VH4 overrepresentation was evident, contributed by VH4-59– and VH4- 39–related genes. By contrast, plasma cells from cardiac inflammatory sites showed a preference for the VH1 family. The composition of CDR3 regions from the antibodies of the phage libraries and those derived from plasma cells were different, pointing to a higher degree of affinity maturation among the latter, a finding that was confirmed by analysis of the somatic mutations. The characterization of anti-P antibodies from patients with Chagas disease allowed us to compare them with anti-P antibodies from patients with systemic lupus erythematosus (SLE). Molecular differences underscore the fact that antibodies againstT. cruzi and anti-P antibodies were obtained that reacted with different T. cruzi proteins and with the C-terminal ends of the ribosomal P proteins. Furthermore, the repertoire and distribution of 148 rearranged VH genes were analyzed and compared with available data of repertoires derived from healthy individuals, patients with autoimmune diseases, and patients with other infections. Preliminary results show that the VH family usage in the unselected libraries has an overrepresentation of VH5 family– and underrepresentation of VH3-23–related germline genes. Amongst the anti–T. cruzi clones, a VH4 overrepresentation was evident, contributed by VH4-59– and VH4- 39–related genes. By contrast, plasma cells from cardiac inflammatory sites showed a preference for the VH1 family. The composition of CDR3 regions from the antibodies of the phage libraries and those derived from plasma cells were different, pointing to a higher degree of affinity maturation among the latter, a finding that was confirmed by analysis of the somatic mutations. The characterization of anti-P antibodies from patients with Chagas disease allowed us to compare them with anti-P antibodies from patients with systemic lupus erythematosus (SLE). Molecular differences underscore the fact that antibodies againstT. cruzi proteins and with the C-terminal ends of the ribosomal P proteins. Furthermore, the repertoire and distribution of 148 rearranged VH genes were analyzed and compared with available data of repertoires derived from healthy individuals, patients with autoimmune diseases, and patients with other infections. Preliminary results show that the VH family usage in the unselected libraries has an overrepresentation of VH5 family– and underrepresentation of VH3-23–related germline genes. Amongst the anti–T. cruzi clones, a VH4 overrepresentation was evident, contributed by VH4-59– and VH4- 39–related genes. By contrast, plasma cells from cardiac inflammatory sites showed a preference for the VH1 family. The composition of CDR3 regions from the antibodies of the phage libraries and those derived from plasma cells were different, pointing to a higher degree of affinity maturation among the latter, a finding that was confirmed by analysis of the somatic mutations. The characterization of anti-P antibodies from patients with Chagas disease allowed us to compare them with anti-P antibodies from patients with systemic lupus erythematosus (SLE). Molecular differences underscore the fact that antibodies againstT. cruzi clones, a VH4 overrepresentation was evident, contributed by VH4-59– and VH4- 39–related genes. By contrast, plasma cells from cardiac inflammatory sites showed a preference for the VH1 family. The composition of CDR3 regions from the antibodies of the phage libraries and those derived from plasma cells were different, pointing to a higher degree of affinity maturation among the latter, a finding that was confirmed by analysis of the somatic mutations. The characterization of anti-P antibodies from patients with Chagas disease allowed us to compare them with anti-P antibodies from patients with systemic lupus erythematosus (SLE). Molecular differences underscore the fact that antibodies against T. cruzi clearly have a different origin than true auto-antibodies.clearly have a different origin than true auto-antibodies. POSTER SESSIONOSTER SESSION Abstracts of Presentations 4949