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. 2007 May;75(5):2523-30.
doi: 10.1128/IAI.01928-06. Epub 2007 Mar 5.

Uninfected mosquito bites confer protection against infection with malaria parasites

Michael J Donovan  1 , Andrew S Messmore, Deborah A Scrafford, David L Sacks, Shaden Kamhawi, Mary Ann McDowell
Affiliations

Uninfected mosquito bites confer protection against infection with malaria parasites

Michael J Donovan et al. Infect Immun. 2007 May.

Abstract

Despite decades of research and multiple initiatives, malaria continues to be one of the world's most debilitating infectious diseases. New insights for malaria control and vaccine development will be essential to thwart the staggering worldwide impact of this disease (A. Bjorkman and A. Bhattarai, Acta Trop. 94:163-169, 2005); ultimately successful vaccine strategies will undoubtedly be multifactorial, incorporating multiple antigens and targeting diverse aspects of the malaria parasites' biology (M. F. Good et al., Immunol. Rev. 201:254-267, 2004). Using a murine model of malaria infection, we show here that exposure to bites from uninfected mosquitoes prior to Plasmodium yoelii infection influences the local and systemic immune responses and limits parasite development within the host. In hosts preexposed to bites from uninfected mosquitoes, reduced parasite burdens in the livers were detected early, and during the blood-stage of the life cycle, these burdens remained lower than those in hosts that received mosquito bites only at the time of infection. Repeated exposure to bites from uninfected mosquitoes skewed the immune response towards a T-helper 1 (Th1) phenotype as indicated by increased levels of interleukin-12, gamma interferon, and inducible nitric oxide synthase. These data suggest that the addition of mosquito salivary components to antimalaria vaccines may be a viable strategy for creating a Th1-biased environment known to be effective against malaria infection. Furthermore, this strategy may be important for the development of vaccines to combat other mosquito-transmitted pathogens.

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Figures

FIG. 1.
FIG. 1.
Lower parasite burdens in mice presensitized with A. stephensi saliva. Mice in naïve and presensitized (Pres) groups were exposed to the bites of P. yoelii-infected A. stephensi mosquitoes. (A) The parasite burdens in the livers were detected by real-time reverse transcriptase PCR, and levels of infection were normalized to levels of mouse HPRT mRNA and expressed as relative parasite burdens (40 − ΔΔct). (B) Naïve and presensitized RNA isolated from the livers was also analyzed by comparison to a standard curve generated using RNA harvested from known numbers of salivary gland sporozoites. Ct, cycle threshold value. (C) Blood-stage infection was monitored each day for 7 days via blood smears from both naïve and presensitized mice. These data are representative of results from three independent experiments (n, 4 to 5 mice per experiment).*, P < 0.05; **, P < 0.10; Student's t test.
FIG. 2.
FIG. 2.
Presensitization skews the response towards that of the Th1 phenotype. Cytokine levels in naïve and presensitized (Pres) mice and naïve (Naïve Bit) and presensitized (Pres Bit) mice that received one final exposure to A. stephensi mosquito bites 24 h prior to analysis were quantified. Local (ear, panels A to C) and systemic (liver, panels D to F, and spleen, panels G to I) tissue IFN-γ (A, D, and G) and IL-4 (B, E, and H) mRNA levels were quantified by real-time reverse transcriptase PCR. (C, F, and I) Levels of IFN-γ and IL-4 expression were used to create a cytokine ratio (IFN-γ/IL-4). Data are representative of results from four independent experiments (n, 5 mice per experiment). Error bars represent standard errors of the means. *, P < 0.05; **, P < 0.001; Student's t test.
FIG. 3.
FIG. 3.
Lower levels of hepatic parasitemia are IFN-γ dependent. BALB/c WT (closed symbols) and IFN-γ KO (open symbols) animals in both naïve (squares) and presensitized (Pres; triangles) groups were exposed to the bites of P. yoelii-infected A. stephensi mosquitoes. Parasite burdens (A) and iNOS mRNA levels (B) were quantified by real-time reverse transcriptase PCR 40 h postinfection. Levels of infection were normalized to mouse HPRT mRNA levels and expressed as relative parasite burdens (40 − ΔΔct). Data are representative of results from two independent experiments (n, 5 mice per experiment). Error bars represent standard errors of the means. *, P < 0.05.
FIG. 4.
FIG. 4.
Local induction of iNOS occurs 5 h after exposure to infected A. stephensi mosquitoes. BALB/c animals in both naïve and presensitized (Pres) groups were exposed to the bites of P. yoelii-infected A. stephensi mosquitoes, and iNOS mRNA levels were quantified by real-time reverse transcriptase PCR 5 h postinfection. Error bars represent standard errors of the means. Data are representative of results from two independent experiments (n, 5 mice per experiment). *, P < 0.05; Student's t test.
FIG. 5.
FIG. 5.
IL-12p40 is induced by presensitization. BALB/c animals in both naïve and presensitized (Pres) groups were exposed to the bites of P. yoelii-infected A. stephensi mosquitoes, and IL-12p40 mRNA levels in livers (A) and spleens (B) were quantified 40 h postinfection by real-time reverse transcriptase PCR. Error bars represent standard errors of the means. Data are representative of results from two independent experiments (n, 4 mice per experiment). *, P < 0.05; Student's t test.
FIG. 6.
FIG. 6.
The protective phenomenon associated with presensitization to mosquito saliva is evident at 20 h postinfection and is localized to the liver. Presensitized (Pres) and naïve animals were subjected to the bites of P. yoelii 17XNL-infected A. stephensi mosquitoes. Animals were sacrificed at 5, 10, 20, and 30 h postinfection, and 18S rRNA levels in ears (A) and livers (B) were quantified. Squares, naïve mice; triangles, presensitized mice. (C) Presensitized and naïve animals were infected with 1,000 P. yoelii 17XNL sporozoites intravenously through the tail vein immediately following a fourth presensitization to uninfected bites and euthanized 40 h postinfection; subsequently, parasite 18S rRNA levels were quantified by real-time reverse transcriptase PCR. Levels of infection were normalized to mouse HPRT mRNA levels and expressed as relative parasite burdens (40 − ΔΔct). Data are representative of results from two independent experiments (n = 4). Error bars represent the standard errors of the means. *, P < 0.05; Student's t test.
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