Anopheles gambiae salivary gland proteins as putative targets for blocking transmission of malaria parasites

Abstract

Anopheles gambiae is the primary vector of human malaria in sub-Saharan Africa. Invasion of Anopheles salivary glands by Plasmodium sporozoites is a necessary step in the transmission of malaria and is likely to be mediated by specific receptor-ligand interactions. We are interested in identifying putative an A. gambiae salivary gland receptor or receptors for sporozoite invasion as a possible target for blocking malaria transmission. By using monoclonal antibodies against female-specific A. gambiae salivary gland proteins, two molecules, one of 29 kDa and one of 100 kDa, were identified and characterized with respect to the age and blood-feeding process of mosquitoes. In an in vivo bioassay, the monoclonal antibody against the 100-kDa protein inhibited Plasmodium yoelii sporozoite invasion of salivary glands >/=75%. These results show that A. gambiae salivary gland proteins are accessible to monoclonal antibodies that inhibit sporozoite invasion of the salivary glands and suggest alternate targets for blocking the transmission of malaria by this most competent of malaria vectors.

Figure 1

(A) Silver-stained SDS/PAGE gel of male (M) and female (F) A. gambiae and A. stephensi salivary gland extracts. (B) Immunoprecipitation of [³⁵S]methionine-labeled A. gambiae salivary gland extracts with monoclonal antibodies 2A3 and C26. (C) Silver-stained SDS/PAGE gel of salivary glands isolated from A. gambiae females at the indicated time points (T = hours) after a blood meal. Arrows in A indicate the position of the 25-kDa, 29-kDa, 42-kDa, and 67-kDa proteins, and those in B and C indicate the position of the 29-kDa and 100-kDa proteins. Numbers on the left indicate molecular mass standards.

Figure 2

Immunoelectron and indirect immunofluorescence microscopy of female A. gambiae salivary glands. A and B show the binding of 2A3 and C26 to the distal lateral lobes of the salivary glands respectively (×15,600 magnification). C and D show the diffuse dispersion of the 29-kDa and 100-kDa proteins on the female-specific lobes of the salivary glands as revealed by immunofluorescence assay.

Figure 3

In vivo [³⁵S]methionine labeling of A. gambiae salivary proteins. Gland extracts were run on SDS/PAGE and exposed to X-ray film. A depicts salivary glands dissected at the indicated times (T = hours) after male (M) and female (F) mosquitoes (4 days after emergence) fed on [³⁵S]methionine. B shows in vivo labeling (6 h) of salivary gland proteins in A. gambiae females of the indicated adult age groups.

Figure 4

Analysis of [³⁵S]methionine-labeled salivary gland extracts and saliva from A. gambiae females. Lane 1 shows salivary gland extracts from mosquitoes that had probed. Lane 2 shows salivary gland extracts from mosquitoes before probing. Lane 3 shows saliva collected from mosquitoes.

Figure 5

In vivo binding of fed monoclonal antibodies to salivary glands (A) and in vivo blocking of sporozoite invasion of salivary glands (B). Female A. gambiae were fed monoclonal antibodies in a blood meal, and salivary gland-bound antibodies were detected by ELISA (A). The graph shows the average absorbance of three replicates with two salivary gland equivalents per well. B shows the combined results of three in vivo sporozoite blocking assays. P values as compared with the NHS control are: 2A3, 0.0604 (n = 51); C26, 0.8829 (n = 50); 6B6, 0.6865 (n = 42); NHS, (n = 71). The third experiment was performed with ammonium sulfate-precipitated mouse ascites to eliminate any inhibitory ascites factors.