Expression of recombinant Anopheles gambiae salivary proteins: implications for malaria control and epidemiology.

The saliva of blood-feeding arthropods is a complex mixture of bio-active compounds whose role is to facilitate blood feeding by affecting host hemostasis, inflammation and immunity. As a “side-effect” salivary components also interfere with other aspects of host-vector-pathogen interactions: indeed, several pieces of evidence reveal an unexpected role of saliva of blood-feeding arthropods in transmission of disease agents and host immune response. As far as salivary secretions of Anopheles gambiae are concerned, two aspects of relevance for malaria studies emerged recently. Firstly, human antibody response to salivary proteins may represent an indicator of exposure to bites of anopheline mosquitoes and, therefore, a potential marker of malaria risk (Remoue F et al, 2006, Trans R Soc Trop Med Hyg, 100: 363-70). Secondly, salivary proteins may be useful vaccine components as suggested by the protective effect on Plasmodium infection of mice pre-immunization through exposure to bites of uninfected mosquitoes (Donovan MJ et al, 2007, Infect Immun, 75: 2523-30; Fonseca L et al, 2007, Malaria J, 6: 77). We have analyzed the An. gambiae salivary transcriptome assembling a catalogue including 71 bona fide secreted proteins (Arcà B et al, 2005, J Exp Biol, 208: 3971-86): these are components of adult female saliva and are injected into the host during the blood meal. Almost one third of these proteins are anopheline-specific, i.e. they are not found in the saliva of Aedes and/or Culex mosquitoes, as indicated by comparative transcriptome analysis (Ribeiro JMC et al, 2004, Insect Biochem Mol Biol, 34: 543-63; Lombardo F et al, 2006, Parassitologia, 48: 573-80; Ribeiro JMC et al, 2007, BMC Genomics, 8:6). These proteins, if immunogenic, could represent ideal serological markers of exposure to Anopheles mosquitoes’ bites. Indeed, they would allow to overcome the complication of obtaining large amounts of mosquito saliva as well as the potential problem of cross-reactivity to salivary antigens from other mosquitoes or other blood-sucking arthropods. AIM: With this in mind, and in the effort of gaining insights into the role of mosquito salivary proteins in parasite-vector-host interactions, we selected a group of An. gambiae salivary proteins for expression in recombinant form. METHODS: Proteins that are mainly, or exclusively, expressed in the saliva of anopheline mosquitoes, and whose functions are still under investigation were initially chosen. The cDNAs encoding gSG1, gSG5, gSG6, gSG7, gSG8, gVAG, cE5/anophelin and hyp37.7 were cloned in suitable expression vectors. So far five proteins have been succesfully expressed either as secretory products in the yeast Pichia pastoris (gSG6, gSG7, and gVAG) or as intracellular proteins in Escherichia coli (gSG5, gSG6, gSG8, and gVAG). Expression in E. coli yielded insoluble products and protocols for protein-specific recovery from inclusion bodies are presently being developed. Among the proteins expressed in P. pastoris, gSG6 was purified to homogeneity and subjected to different functional assays including knock-down by RNA interference. RESULTS: Despite the fact that gSG6 must play some important role in blood feeding, so far its specific function has remained elusive. On the other hand, as suggested by ELISA assays, gSG6 appears to be immunogenic and able to elicit a specific IgG response in humans. Preliminary analysis using sera from exposed individuals of the Mossi and Fulani ethnic groups from Burkina Faso suggests that the anti-gSG6 IgG response increases with the level of exposure and it is short-lived. Moreover, as expected on the basis of previous results (Modiano et al, 1996, Proc Natl Acad Sci USA), a higher response was found in the Fulani ethnic group as compared to Mossi, although no significant difference was observed between Mossi and a control group. CONCLUSION: In conclusion, these results provide a proof-of-principle that individual recombinant An. gambiae salivary proteins may be developed as serological markers of exposure to Anopheles mosquitoes for malaria epidemiological studies.