Although praziquantel, is very effective for treating schistosomiasis it is generally recognised that a vaccine will be required for elimination to occur. The goals of this thesis were to: 1) Design and produce a schistosome protein microarray; 2) Probe the schistosome protein microarray with resistant and susceptible experimental schistosomiasis japonica antisera to discover novel vaccine and diagnostic antigens; 3) Investigate the endemic human antibody response to Asian schistosomiasis infection and its relation to pathology; and 4) Test the vaccine efficacy of discovered vaccine antigens.
Using hand curation and bioinformatic approaches, tegument, schistosomula and schistosome-species specific sequences were selected along with vaccine candidates, studied schistosome antigens and Schistosoma japonicum homologues of tegument-localised S. mansoni proteins. After PCR amplification, homologous cloning, plasmid purification and sequence verification, 222 plasmids containing S. japonicum and S. mansoni coding sequences were expressed in a bacterial cell-free system. The cell-free proteins, 21 controls and 46 in vivo bacterial- and yeast-expressed S. japonicum and S. mansoni recombinant proteins were contact printed onto nitrocellulose slides. Quality control screening of the protein microarrays against expression tags revealed that 90% of cell-free proteins were expressed full length. Further preliminary protein microarray testing with mouse sera demonstrated strong differential reactivity between S. japonicum and S. mansoni infection sera, and naïve sera.
Serum and local tissue antibodies from the non-permissive rat and susceptible mouse animal models were collected for the discovery of putative vaccine and diagnostic antigens. In the resistant rat host two separate time courses were completed for longitudinal serum comparisons during infection and reinfection, and for the generation of local tissue antibodies, from the immunologically important skin- and lung-draining lymph nodes, following infection and challenge reinfection. Compared with the mouse, the rat model had reduced pathology and increased protein microarray signal intensity and broader antigen recognition. In addition, local tissues antibodies, or antibody secreting cell (ASC) probes, recognised distinct antigens that contributed to the multivalent response in serum. From the set of significantly different protein microarray features found in infection serum and ASC probes were 37 antigens consisting of 13 previously trialled vaccines and therapeutic targets as well as less studied and unknown proteins. Notably, human resistance markers from the dynein light chain family, also known as the tegument allergen-like (TAL) proteins, were strongly reactive in rat serum suggesting a shared resistance mechanism between rats and humans. Importantly, nineputative vaccine and diagnostic candidates were identified for protein expression and further testing.
The antibody profile of acute, advanced, chronic and egg-negative exposed individuals was measured on the schistosome protein microarray. Over 62% of the antigens were recognised by the human samples. From these antigens a set of 20 cross-reactive and 39 serodiagnostic protein microarray features were discovered. The serodiagnostic antigens may allow discrimination of different disease groups solely on antibody recognition. Notably, membrane proteins were significantly enriched within the serodiagnostic set. In particular, numerous tetraspanin-related antigens, potentially comprising a tetraspanin web, were recognised. Intriguingly, higher pathology acute and advanced patients had a suppressed antibody response compared to the lower pathology chronic and egg-negative exposed individuals. This suggests that chronic patients and egg-negative exposed individuals may use a broad and strong antibody response, particularly against membrane proteins, to control pathology and/or infection.
One diagnostic and eight putative vaccine antigens identified in animal model experiments were PCR amplified and incorporated into expression vectors with His and MBP tags using ligation-independent cloning methods. Four antigens with high yields of soluble recombinant protein were purified and concentrated for use as vaccines and to generate antibodies. Three purified proteins (Sj200, coatomer-like, and Sj annexin 4) and a control (MBP-only) with Quil A adjuvant were used to inoculate groups of CBA mice prior to cercarial challenge. One of the vaccines, Sj200, caused significant reductions in worm length (7.5-8.1%), liver granulomas (11%), and liver (44%) and faecal (56%) egg burdens. Moreover, fluorescently labelled antibodies generated against this vaccine and a diagnostic antigen bound to the surface of live cultured schistosomules. Notably, the Sj200 antigen used in this study is a fragment of a larger protein identified in surface proteomics studies and whose S. mansoni homologue plays a key-role in parasite elimination after chemotherapy. Additional vaccine trials with different formulations and fragments are now warranted. Furthermore, the anti-pathology and anti-fecundity effects engendered by the Sj200 protein could make it useful as a veterinary vaccine in China where 75% of transmission is via bovines.
This project, one of the first to use a protein microarray in schistosome research, has revealed details about the resistant mechanisms in the long-studied experimental schistosomiasis resistance model, the rat, and has defined differences in the humoral responses in human schistosomiasis. Further, a number of novel and untested antigens were identified as putative vaccine and diagnostic candidates.