Elicitation of Anti-Mosquito Immune Responses in Balb/c Mice Immunized With Plasmids Containing Anopheles Gambiae Mucin 1 (Agmuc1), GM-CSF and IL-12 Genes

Abstract

Various strategies that block malaria transmission from an infected human host to a female Anopheline mosquito that involve killing of the blood fed mosquito have been studied as a potential strategy to control malaria. These include the immunization of animal hosts with either whole homogenized blood fed mosquito midguts or their extracts. In 1939, William Trager first demonstrated anti-vector immunity by immunizing guinea pigs and rabbits with extracts of the tick Dermacentor variabilis. This immunological targeting of tick midgut antigens led to the commercial development of a vaccine against Boophilus microplus ticks licenced in 1985.This study investigated the mosquitocidal effects of an immune response elicited by immunization of BALB/c mice with a gene, Anopheles gambiae mucin 1 gene (AgMUC1 gene) alone or in combination with immunostimulatory cytokine genes GM-CSF or IL-12. The AgMUC1 genes were cloned from the midgut of blood fed female Anopheles gambiae mosquitoes. Four groups of BALB/c mice, six mice per group, were immunized with AgMUC1 gene alone or in combination with plasmids containing AgMUC1, Interleukin 12 (IL-12) or Granulocyte-macrophage colony stimulating factor (GM-CSF) genes. Another group of mice was primed with AgMUC1 cDNA and boosted with recombinant mucin protein. Anti-mucin antibody secreting hybridomas were prepared from the fusion of spleen cells from AgMUC1 cDNA immunized mice and myeloma cells. Groups of female Anopheles gambiae mosquitoes were fed on the immunized mice and the anti-mucin antibody containing hybridoma supernatants and their cumulative survival to day seven post blood feeding determined by life table analysis and the Kaplan Meier log rank statistic analysis. Cryostat sections from the midguts of the blood fed mosquitoes were examined by bright light, scanning and transmission electron microscopy. Spleen cells from the immunized mice were stimulated with mucin protein. A significantly higher mortality was observed in the groups of mosquitoes fed on the groups of mice given three or four injections of AgMUC1 cDNA (p=0.00331 and 0.0173 respectively). A highly significant increase in mortality was observed in the groups of mosquitoes fed on the groups of mice immunized with AgMUC1/IL-12 cDNA (p=4.05e-10). Mean anti-mucin antibody levels varied from 1:3,000 in the AgMUC1 cDNA immunized group, 1:6,000 in the AgMUC1/IL-12 cDNA and AgMUC1/GM-CSF cDNA immunized groups to >1:100,000 in the AgMUC1 cDNA immunized / mucin protein boosted group. Cytokine production profiles from invitro stimulated spleen cells from AgMUC1 cDNA immunized mice could not be associated with a particular type of immune response. On microscopy, white blood cells were adhering onto the midgut epithelial cell lining and activated hemocytes were observed. Under the transmission electron microscope features characteristic of apoptotic and necrotic processes were observed in the epithelial cells lining the midgut. The results of this study show that the immune response elicited following AgMUC1 cDNA immunization kills blood feeding female An. gambiae mosquitoes and that the mechanisms leading to mosquito death could probably be associated with antibody dependent cellular cytotoxicity (ADCC) induced apoptosis or necrosis. In this strategy, the killing of blood feeding adult female mosquitoes has the potential to reduce mosquito density and biting intensity, an attribute required in malaria control. This strategy, combined with other anti-malaria parasite strategies aimed at killing the parasite and the responsible use of insecticides, could reduce malaria transmission in endemic areas.