Infection with Schistosoma japonicum remains prevalent in China and the Philippines despite initially successful Government and World Bank sponsored multidisciplinary control programs. Immunity can be induced in mammals by multiple vaccinations of irradiated cercariae. Logistical, practical and quality-control issues prevent large scale animal or human applications. Human resistance to reinfection has been described and is age-dependant. Defined molecule vaccines either as proteins or DNA constructs have had some demonstrated efficacy in animals - but humans trials are yet to commence. Treatment or retreatement with Praziquantel (PZQ) does not induce protection in humans. PZQ resistance in S. mansoni is emerging in North Africa. It is currently the only anti-schistosomal drug available on the world market.
Artemether (ART) is a well described antimalarial with emerging efficacy against juvenile schistosomes. 7-day old larvae are particularly susceptible. Both ART-affected worms and worms developing from irradiated cercariae die at similar times. The basic hypothesis in this thesis is that ART-treatment of prepatent schistosomiasis japonica may expose molecules previously hidden from the host immune repertoire that are able to generate a subsequent protective immune response. In this thesis, female CBA mice are treated with a single dose of ART by gavage feeding at defined time points following a baseline percutaneous infection of either Chinese or Philippine strain S. japonicum cercariae. Half are randomly subject to homologous strain rechallenge.
Experimental work commenced using a field strain of S. japonicum from Anhui Province (PR China). ART induced an expected reduction in juvenile larvae and also a significant reduction in egg production. Homologous strain rechallenge resulted in reproducible (50-75%) and statistically significant reduction in challenge worm burden following reinfection if those mice were treated with ART at two weeks p.i.. One round of irradiated cercariae vaccination failed to induce protection. Additionally, antifecundity and anti-pathology effects were maintained following rechallenge. Western Blot analysis for IgG and IgE responses to adult worm antigens (SWAP) and ELISAs for IgG, IgM and IgE failed to identify a specific immune correlate with this protection. Splenic lymphocyte proliferation assays reproducibly demonstrate a loss of schistosome-induced mitogen resistance with ART treatment. This does not however associate with protection. A reduction in IL-4 response to SWAP is also seen in ART-treated mice. In the Chinese mainland strain used, ART-treatment of prepatent infection at the appropriate time point induces resistance to reinfection -probably by an immune mechanism. There is also an additional anti-disease effect observed that is maintained following reinfection.
Despite 96% genomic DNA homology, the Philippine strain of S. japonicum differs from its Chinese counterpart in several important characteristics. Importantly, irradiated cercariae vaccination with this strain has never induced protection in any animal model. Results from three replicates of the protection trials were mixed. The initial trial demonstrated 50-65% reduction in challenge worm burdens in all three ART-treated groups. However, two subsequent replicates could not reproduce this or similar results. Similar worm maturational effects to the Chinese strain were observed and the anti-fecundity and anti-disease effects were similar. Lymphocyte proliferation assay results were similar to the Chinese strain. No real effect on IFN-γ or IL-4 was observed. No humoral immune mechanism was identified. While ART has clear antifecundity and antipathology effects on the Philippine strain that persist after reinfection, no reduction in challenge worm burden was convincingly demonstrated.
A highly significant polynomial correlation between IgG response to egg antigens (SEA) and liver disease burden was demonstrated for groups of mice in the Chinese strain trials and in individual micein the Philippine strain trials.
The antipathology effect of ART treatment was explored - with emphasis on the origin of the granuloma-associated fibrosis. Granuloma diameter is significantly less in Philippine strain infection compared to Chinese strain at both 5 and eight weeks p.i,. In a 14- week time course study of untreated Philippine strain infection, statistically significant down regulation in granuloma diameter was observed by 10 weeks, however overall disease burden had plateaued. Immunohistology demonstrates the presence of perisinusoidal cells containing cytoplasmic vacuoles that have strong cytoplasmic staining for a-smooth muscle actin in the periphery of egg granulomas. These cells are in the same region as the type 1 collagen gene is expressed and gene product is localised. This data is supportive of activated hepatic stellate cells being the effector cell for granuloma-associated fibrosis. Quantitative histological analysis displays a significant reduction of SMA-α and collagen expression in response to ART treatment - of similar proportions to disease burden. This is confirmed by real time PCR - which also confirmed the intense Th2 nature of the granulomatous inflammation and the peak and subsequent decline of SMA-α and type 1 collagen RNA expression. Data are displayed implicating activated hepatic stellate cells being the effector cell for fibrosis in experimental and human schistosomiasis.
Overall, this research project has demonstrated that appropriately-timed ART-attenuation of S. japonicum (Anhui strain) infection reliably induces resistance to homologous rechallenge in early patency. This protection was superior to one round of irradiated cercariae vaccination. An antipathology effect is also observer, which is maintained following reinfection. Protection was not observed after reinfection of mice with ART-treated Philippine strain S. japonicum infection. The antipathology effect was preserved and is accompanied by a reduction in granuloma-associated fibrosis. ART-treatment facilitates normalization or adult worm-induced mitogen insensitivity. Histological and molecular evidence supports the role of activated hepatic stellate cells as the effector cell of granuloma-associated fibrosis.