Wide cross-reactivity between Anopheles gambiae and Anopheles funestus SG6 salivary proteins supports exploitation of gSG6 as a marker of human exposure to major malaria vectors in tropical Africa

<p>Abstract</p> <p>Background</p> <p>The <it>Anopheles gambiae </it>gSG6 is an anopheline-specific salivary protein which helps female mosquitoes to efficiently feed on blood. Besides its role in haematophagy, gSG6 is immunogenic and elicits in exposed individuals an IgG response, which may be used as indicator of exposure to the main African malaria vector <it>A. gambiae</it>. However, malaria transmission in tropical Africa is sustained by three main vectors (<it>A. gambiae</it>, <it>Anopheles arabiensis </it>and <it>Anopheles funestus</it>) and a general marker, reflecting exposure to at least these three species, would be especially valuable. The SG6 protein is highly conserved within the <it>A. gambiae </it>species complex whereas the <it>A. funestus </it>homologue, fSG6, is more divergent (80% identity with gSG6). The aim of this study was to evaluate cross-reactivity of human sera to gSG6 and fSG6.</p> <p>Methods</p> <p>The <it>A. funestus </it>SG6 protein was expressed/purified and the humoral response to gSG6, fSG6 and a combination of the two antigens was compared in a population from a malaria hyperendemic area of Burkina Faso where both vectors were present, although with a large <it>A. gambiae </it>prevalence (>75%). Sera collected at the beginning and at the end of the high transmission/rainy season, as well as during the following low transmission/dry season, were analysed.</p> <p>Results</p> <p>According to previous observations, both anti-SG6 IgG level and prevalence decreased during the low transmission/dry season and showed a typical age-dependent pattern. No significant difference in the response to the two antigens was found, although their combined use yielded in most cases higher IgG level.</p> <p>Conclusions</p> <p>Comparative analysis of gSG6 and fSG6 immunogenicity to humans suggests the occurrence of a wide cross-reactivity, even though the two proteins carry species-specific epitopes. This study supports the use of gSG6 as reliable indicator of exposure to the three main African malaria vectors, a marker which may be useful to monitor malaria transmission and evaluate vector control measures, especially in conditions of low malaria transmission and/or reduced vector density. The <it>Anopheles stephensi </it>SG6 protein also shares 80% identity with gSG6, suggesting the attractive possibility that the <it>A. gambiae </it>protein may also be useful to assess human exposure to several Asian malaria vectors.</p

Humoral Response to the Anopheles gambiae Salivary Protein gSG6: A Serological Indicator of Exposure to Afrotropical Malaria Vectors

Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens

Seasonal variation of the IgG response to the <i>P. falciparum</i> CSP in Mossi and Fulani.

<p>Scatter plot of OD values representing the IgG response to the <i>P. falciparum</i> CSP among gSG6 IgG responders of the Mossi and Fulani ethnic groups in the three different surveys. C, unexposed controls (n = 28). Bars, number of Mossi and Fulani analyzed, p values and pairwise comparisons as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096130#pone-0096130-g001" target="_blank">Figure 1</a>. Note that three data points are outside the axis limits.</p

Seasonal variation of the gSG6-specific IgG1 and IgG4 antibody titers in the sympatric ethnic groups Mossi and Fulani.

<p>Scatter plots reporting IgG1 (left panel) and IgG4 (right panel) antibody titers among gSG6 IgG responders of the Mossi and Fulani ethnic groups in the three different surveys. C, unexposed controls. Bars indicate median values. Number of individuals analyzed (n) and average age in years ±95% CI were as follows. August: Mossi n = 60 (22.0±4.5), Fulani n = 63 (16.1±3.9); October: Mossi n = 56 (13.0±3.4), Fulani n = 30 (21.2±7.0); March: Mossi n = 32 (15.3±5.1), Fulani n = 29 (11.6±4.2); Controls n = 44 (33.1±7.1). P values determined according to the Kruskal-Wallis test. Pairwise comparisons refer to the Mann-Whitney U test (*, 0.01</p

Humoral response to the gSG6 and CSP proteins according to age.

<p>Scatter plot reporting the antibody responses to gSG6 (IgG1, black; IgG4, red) and to CSP (IgG, green) as function of age among gSG6 IgG responders of the Mossi (n = 148) and Fulani (n = 122) ethnic groups from the three different surveys. Anti-gSG6 IgG1 and IgG4 are expressed as titers (ng/ml, left Y axis); IgG response to CSP is expressed as OD₄₀₅ (right Y axis). The best-fit lines are shown (Mossi, solid lines; Fulani dashed lines). Spearman correlation coefficients: (i) anti-CSP IgG (Mossi, r = 0.53, p<0.0001; Fulani, r = 0.41, p<0.0001); (ii) anti-gSG6 IgG1 (Mossi, r = −0.25, p = 0.0019; Fulani, r = −0.27, p = 0.0024); (iii) anti-gSG6 IgG4 (Mossi, r = −0.12, p = 0.1359; Fulani, r = −0.29, p = 0.0013). Note that twenty-six data points are outside the axis limits.</p

IgG4/IgG1 ratio by age group in Mossi and Fulani.

<p>Scatter plot reporting the IgG4/IgG1 ratio by age group in Mossi (n = 120) and Fulani (n = 95). Data from the three different surveys were pooled together and individuals with no detectable IgG1 or IgG4 (or both) were excluded from the analysis. The five different age groups (years) are indicated at the bottom. The number of individuals for each age group is given in parenthesis. Bars indicate median values. P values determined according to the Kruskal-Wallis test. Pairwise comparisons refer to the Mann-Whitney U test (*, 0.01</p

Monthly follow up data

<p>Children were visited at home 1) to collect blood samples for malaria serology and 2) to the detect the first malaria infection.</p