In vivo and in vitro studies of immune responses induced by normal or attenuated 'Schistosoma mansoni' schistosomula

摘要

A schistosome vaccine would be of great value in controlling schistosomiasis. Exposure of experimental animals to live gamma-irradiated cercariae (GI) is highly effective and although not logistically feasible for human vaccination has provided insight into the immune mechanisms which may facilitate development of defined antigen vaccines. In mice the radiation attenuated vaccines induce IFN- γ dependent immunity following a single exposure and antibody mediated immunity following repeated exposure. In order to induce high levels of immunity the irradiated larvae need to survive for 2-3 weeks and to reach the lung stage in order to "arm" the lungs with sensitized cells, but it has been shown that larval infections with 500 larvae treated with the drug Ro11-3128 at just 2 days post-infection also results in high levels of immunity but with an apparently more focal and truncated exposure to living larvae in the skin. The focus of this thesis is to compare the immune mechanism underlying this Ro11-3128-induced vaccination (RoNI) (which induced >90% protection) with the GI (which induced 60-70% protection) and with infections which are both irradiated and drug treated which induce poor levels of immunity (RoGI) (induced only 30% protection). RoNI, GI, RoGI and a normal unattenuated infection (NI) all resulted in a Th 1 biased response (IFN-γ>IL-4) in the skin-draining lymph nodes (SLN), but RoNI and RoGI induced enhanced and more prolonged responses. RoNI was unique in causing highly elevated antigen specific IFN-γ responses in the spleen suggesting local but protracted stimulation of a strong systemic response. Studies in B6RAG 1-/- mice (no B or T cells), IFN-γ -/-, μ-MT mice (no B cells) and by in vivo depletion of IFN-γ with neutralizing monoclonal antibody during challenge showed that RoNI is mediated almost entirely by IFN-γ mediated mechanisms and antibody is not involved. Adding Ro11-3128 to cultures containing RoNI-sensitized SLN cells and living schistosomula enhanced the IFN-γ production in the presence of in vitro bone marrow (bm)-derived DCs suggesting that altered antigen presentation is induced by drug treatment. However, the superiority of Ro11-3128 in inducing immunity compared with other drugs could not be attributed simply to its ability to induce membraneous blebs as these were also produced by Ro11-3128-treated irradiated schistosomula. Furthermore, there was no evidence of the drug having a general adjuvant effect. To try to establish how the larval exposure induces the Th 1 biased response, living schistosomula were cultured with bone marrow-derived dendritic cells. This had no effect (up- or down- regulate) on surface activation maker expression (MHCII, CD86, CD40) even if the bm-DCs, were partially activated with LPS or TNF-α. There was also no cytokine production (IL-12p70, TNF-α, IL-10 or IL-6). However, schistosomula induced a differential dose-dependent reduction in cytokine production by LPS-activated bm-DCs (IL-12p70 >IL-6 and TNF-α) but IL-10 was relatively unaffected. Again this was unaffected by addition of Ro11-3128. This work indicates that it is possible to induce highly effective Th 1 mediated systemic immunity in mice by protracted but local stimulation of the skin/SLN suggesting that delivery of defined schistosome antigens by Th 1 promoting strategies such as prime- boosting with heterologous recombinant viruses delivered in the skin would be worth investigating. There was no evidence that the Th l response to larval infection is caused by direct action of larvae or larval products on dendritic cells and in fact there was evidence of an anti-inflammatory effect, which should be investigated further at the mRNA level. It is suggested that schistosomula may interact differently with other cells encountered in the skin e. g. keratinocytes in initiating the Th 1 bias.