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|Malaria vaccine studies on Anopheles midgut glycoproteins and a plasmodium falciparum metrozoite surface protein
|A thesis submitted to Doctor of Philosophy
|Vaccination is an alternative approach to control malaria because ofthe development of insecticide resistance in mosquito vectors and drug resistance in parasites. In this context, two different approaches viz, transmission blocking, using mosquito midgut (MG) glycoproteins, and eliciting immunity to asexual stage parasite antigens, using Plasmodium falciparum merozoite surface antigen 2 (PfMSA2) were investigated. M G glycoproteins have been shown to act as receptors for recognition of ookinetes. Effect of the antibodies raised against Concanavahn A binding proteins (ConABP) of M G of Anopheles tessellatus on mosquito physiology and infectivity were investigated. Although a reduction (14%) in fecundity and an increase (11%) in percentage mortality were noticed in mosquitoes by in vitro feeding of anti-ConABP serum the effects were not significant. Antigenic relationships between proteins of M G and peritrophic matrix (PM) of An. tessellatus larvae and aduU and M G glycoproteins of other vector mosquitoes were studied using anti-ConABP and antiW GA binding protein (WGABP) antisera. While adult and larval M G glycoproteins An. tessellatus are antigenically almost similar, glycoproteins in the PMs of the two developmental stages differed extensively. Antigenic cross-reactions are also observed between adult M G glycoproteins of different Anopheles species and, to a lesser extent, between An. tessellatus and Culex and Aedes mosquitoes. Screening an An. gambiae cDNA expression library with anti-WGABP serum identified many myosin clones and yielded a unique clone (clone E) for a protein with a transient receptor potential (TRP). Homologous search analysis showed that clone E encodes a homoiogue of TRP gamma cation ciiannel protein of Drosophila. These findings ilhistrate the potential of M G glycoproteins as targets in transmission blocking as well as drawbacks in producing specific antisera. Pf MSA2 has been identified as potential candidate molecule for developing an erythrocytic stage vaccine against malaria. PfMSA2 was inducibly expressed and displayed in lactic acid bacteria, Lactococcus lactis and Lactobacillus spp, in two different forms (1) covalently attached to the surface of live L. lactis cells through PrtP anchor system (MSA2cP) (2) non-covalently attached to L. lactis cell wail ghosts and to live Lb. salivarious and Lb. reuteh through AcmA anchor system (MSA2cA). Immunogenicity of MSA2 was investigated in three inbred strains of mice (Balb/c, C57 and C3H) and an outbred strain, ICR, by combined oronasal immunisation. Oronasal immunisation of both immunogens (MSA2cP and MSA2cA) elicited high serum IgG antibodies (highest Abs observed at 405nni was 1.99 with MSA2cA and 0.69 with MSA2cP) against MSA2 . Balb/c and C3H mice responded better to MSA2cP and MSA2cA on L lactis respectively. The IgG isotypes to both immunogens reflected Thl and Th2 influences. IgM response was significantly (p<0.001) low. IgA antibodies were observed in ICR, Balb/c and C3H strains and antisera of these" strains reacted with native MSA2 on the surface of P. falciparum merozoites. However the responses of mice to MSA2cA on Lactobacillus were weak (highest Abs observed at 405nm was 0.34). Antigen specific spleenic IFN-y secreting T cells were demonstrable by ELISPOT assay in Balb/c, C3H and C57 strains immunised with MSA2cA and in C57 mice immunised with MSA2cP. Oronasal immunisation with PfMSA2 was able to generate systemic antibodies and cellular immunity in mice and in general MSA2cA was better than MSA2cP in eliciting immune responses in mice. Also the immune responses were dependent on the strain of mice and the way of antigen presentation. Immune responses to the two immunogens were weaker and T-indcpendent in old (24-30 wks) Balb/c mice. IFN-y secreting cells were not detected in old mice. Histological studies revealed that there was a tendency for enlargement of mesenteric lymph nodes but not the spleen, increase in number of primary but not secondary follicles in mesenteric lymph nodes and spleen and presence of enlarged lymphoid aggregates in lamina propria in animals orally immunised with live L. lactis or L. lactis cell walls. However, immunisation with MSA2cP and expression inducer strain NZ9700 L. lactis together improved the immune responses in both oral and subcutaneous immunisation. Mice immunised with live L. laclis cells had significantly (p<0.05) higher levels of anti-lactococcal antibodies than mice immunised with cell walls. Levels of antibodies tended to plateau between 5x10^ and 5x10^ live L lactis per dose in ICR mice. Antilactococcal antibodies were noticeably lower against bacteria expressing MSA2 suggesting that presence of the heterologous antigen may divert the immune response from L laclis antigens. These findings demonstrate the potential use of MSA2 as a vaccine candidate and use of L. laclis as vaccine delivery system in mucosal immunisation. Also it demonstrates the need to monitor anti-lactococcal responses to avoid possible hypersensitivity reactions in the recipient, to monitor local inflammatory responses in the gut mucosa and its implications in the induction of immune responses in old people.
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