Viral paratransgenesis in the malaria vector Anopheles gambiae

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.     

Immunogenicity of Plasmodium yoelii merozoite surface protein 4/5 produced in transgenic plants

Malaria is a major global health problem for which effective control measures are urgently needed. Considerable effort has been focused on the development of effective vaccines against the causative parasite and protective vaccine trials are now being reported. Due to the relative poverty and lack of infrastructure in malaria-endemic areas, a successful immunisation strategy will depend critically on cheap and scaleable methods of vaccine production, distribution and delivery. One promising technology is transgenic plants, both as a bioreactor for the vaccine-manufacturing process as well as a matrix for oral immunisation. In this study, we investigated the feasibility of using transgenic plants to induce protective immunity against malaria infection using Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5) in a mouse model of malaria infection. Our data show that the PyMSP4/5 protein can be produced in plants in a configuration that reacts with protective antibodies. Optimisation of codon usage for the PyMSP4/5 gene resulted in significantly increased antigen expression in plants. PyMSP4/5 protein from the codon-optimised construct accumulated to 0.25% of total soluble protein, a sixfold increase over the native gene sequence. Tobacco-made PyMSP4/5 was able to induce antigen-specific antibodies in mice following parenteral delivery, as well as boost the antibody responses induced by DNA vaccination when delivered parenterally or orally. We believe this is the first report to show that plant-made malaria antigens are immunogenic. However, the antibody levels were not high enough to protect the immunised mice against a lethal challenge with P. yoelii. Further strategies are needed to achieve a protective dose, including improvements to antigen expression levels in plants and strategies to enhance the immunogenicity of the expressed antigen.     

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.