Reduction of Mosquito Survival in Mice Vaccinated with Anopheles stephensi Glucose Transporter

Abstract

Despite the fact that recent efforts to control/eradicate malaria have contributed to a significant decrease in the number of cases and deaths, the disease remains a global health challenge. Vaccines based on mosquito salivary gland antigens are a potential approach for reducing vector populations and malaria parasites. The Anopheles AGAP007752 gene encodes for a glucose transporter that is upregulated during Plasmodium infection, and its knockdown decreases the number of sporozoites in mosquito salivary glands. These results together with the fact that glucose is a vital source of energy suggested that a glucose transporter is a candidate protective antigen for the control of mosquito infestations and Plasmodium infection. To address this hypothesis, herein we investigate the effect of mice vaccination with an immunogenic peptide from mosquito glucose transporter on Anopheles stephensi fitness and Plasmodium berghei infection. We showed that vaccination with a peptide of glucose transporter reduced mosquito survival by 5% when compared to controls. However, the reduction in Plasmodium infection was not significant in mosquitoes fed on vaccinated mice. The effect of the peptide vaccination on mosquito survival is important to reduce infestation by malaria vectors. These results support further research on developing glucose transporter-based vaccines to reduce mosquito fitness.

Figure 1

Molecular Phylogenetic Analysis by Maximum Likelihood Method. Using the Maximum Likelihood method based on the Le_Gascuel_2008 model [20], the tree with the highest log likelihood (−3359.935) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.65)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 5 amino acid sequences with respective VectorBase accession number: AGAP007752: Anopheles gambiae, ASTE006385: A. stephensi, ASIS016443: A. sinensis, ADAC010569: A. darlingi, and CG15406: Drosophila melanogaster (outgroup). All positions containing gaps and missing data were eliminated and bootstrap values for internal branches are shown. Evolutionary analyses were conducted in MEGA6 [21].

Figure 2

Fluorescence Microscopy of A. stephensi-Infected Salivary Glands Triple-Stained. Salivary glands were incubated with PBS, preimmune and anti-GTp immune serum. Green fluorescence corresponds to GFP-expressing P. berghei, blue to the nuclei stained with DAPI and magenta for Cy5-Alexa Fluor 647 to localize specific anti-GTp antibodies. The fluorescence intensity of SG Z-stack and merged images was compared and then analyzed in the same conditions. A specific and localized recognition was evident when SGs were incubated with the anti-GTp serum. Z-stack maximum intensity projections and a merge image of the three channels are given. Green: GFP-expressing P. berghei. Blue: DAPI-counterstaining of the nuclei. Magenta: Cy5-Alexa Fluor 647. Bar: 25 µm.

Figure 3

Schematic Overview of Vaccination Trial. From twelve five-week-old female Balb/c mice, six were primed with PBS and the others with GTp-formulated vaccine. Every fortnight, mice were boosted five times intraperitoneally. Four days after the last immunization, mice were infected with P. berghei parasitized red blood cells by intraperitoneal inoculation or left uninfected as control. Blood samples were collected to determinate antibody titers during immunization and after Plasmodium infection. For five days the infection was monitored and when the parasitaemia reached 10–20% and 4–6 exflagellations/field were observed, mice were anesthetized and used to feed and infect female mosquitoes (N = 300/mice). Mosquito survival and oviposition were assessed until the 18th day after blood meal (PBM).

Figure 4

Mice Immunization with GTp after Challenge or Not with P. berghei. Comparison between GTp-immunized and control mice with and without Plasmodium infection. Antibody titers were determined by ELISA assays and Plasmodium inoculation is represented with a red arrow. Negative control is represented in a red dashed line. Statistical analyses were performed using Mann–Whitney test (P < 0.05). MONT NINF: control mice without Plasmodium infection; MONT INF: control mice with Plasmodium infection; GTp NINF: GTp-immunized mice without Plasmodium infection; GTp INF: GTp-immunized mice with Plasmodium infection.

Figure 5

The Effect of Anti-GTp Antibodies on A. stephensi Mosquitos and Malaria Parasites. (a) Oviposition. Total number of eggs laid after each experiment (N = 3) were counted and compared between GTp-immunized and nonimmunized groups. (b) Mosquito survival after blood meal with anti-GTp immune serum and preimmune serum. (c). Plasmodium mRNA levels at 18th day after blood meal in SGs tissues. Statistical analyses of (a) and (b) results were performed using Mann–Whitney test and for (c) using CFX Manager Software (^∗P < 0.05). MONT INF: control mice with Plasmodium infection; GTp INF: GTp-immunized mice with Plasmodium infection.