Background
After more than a century from the discovery of the role of
Anopheles mosquitoes in the transmission of
Plasmodium parasites, malaria is still one of the leading causes of human morbidity and mortality. Currently, the malaria toll is especially high among young children in sub-Saharan Africa, where transmission of the most deadly malaria parasite,
Plasmodium falciparum, is mainly accomplished by two members of the
Anopheles gambiae species complex (i.e.
A. gambiae and
Anopheles arabiensis, subgenus
Cellia, Pyretophorus Series) and by
Anopheles funestus (subgenus
Cellia, Myzomyia Series) [
1]. Proper evaluation of malaria transmission intensity, of seasonal and temporal variation of vector density and of the efficacy of anti-parasite and anti-vector control measures play crucial roles in the framework of anti-malaria strategies.
Assessment of malaria transmission intensity is currently based on both parasitological and entomological measures and a key parameter is the entomological inoculation rate (EIR), which accounts for human exposure to parasite-carrying mosquitoes. However, entomological measurements are not only expensive and labor-intensive but, sometime, also difficult or impossible to apply: for example in conditions of low transmission intensity and/or low mosquito density, or for logistic restrictions. Therefore, additional and/or alternative methods to evaluate
Anopheles density and human exposure to malaria vectors would be extremely valuable allowing for epidemiological studies also in settings where classical entomological methods are of problematic use. During blood feeding, mosquitoes inject into their hosts a complex mixture of salivary components whose main role is to facilitate haematophagy by counteracting the haemostatic, inflammatory and immune responses of vertebrates [
2,
3]. These salivary components also elicit into hosts an immune response with production of anti-saliva antibodies. For example, at least 10-15 protein bands recognized by human IgG can be detected by western blot using
A. gambiae salivary gland protein extracts and sera from exposed individuals from a malaria hyperendemic area (B.A., unpublished observations). Several reports support the concept that measurement of this antibody response to saliva may represent an indicator of human exposure to
Anopheles bites and malaria risk, as well as a tool to evaluate efficacy of insecticide-treated nets (ITNs) [
4‐
8]. Moreover, the identification of
Anopheles-specific proteins, i.e. not found in other mosquitoes or blood feeding arthropods, offers the opportunity to use as markers genus-specific recombinant salivary antigens instead of saliva [
2,
9]. This enables for a significant improvement of the methodology increasing both the accuracy and the specificity by overcoming the need of obtaining large amount of saliva and potential problems of reproducibility and cross-reactivity.
Conveniently, vector's salivary antigens could be used in parallel to
Plasmodium antigens to assess, by serological determination of antibody levels, both exposure of humans to
Anopheles mosquitoes and malaria transmission intensity [
10‐
14]. This immune response to salivary antigens would represent a direct measure of intensity of mosquito biting activity on humans, both at the population and at individual level, and could provide a few additional advantages. First, it may allow to assess
Anopheles exposure in children, which is presently unworkable for ethical reasons (the method currently in use is based on human landing catches on adult volunteers). Second, it would be very helpful to evaluate the impact of anti-vector control measures on exposure of humans to
Anopheles bites. Third, it would be a tool especially needed for epidemiological assessments in areas of low malaria transmission, which are currently increasing as a consequence of the decline of the malaria burden in several areas of sub-Saharan Africa [
15]. Finally, it might be the appropriate tool to verify if, and eventually to what extent, the mosquito biting activity is heterogeneously distributed within a population. Indeed, according to the so-called heterogeneous biting model, mosquito biting may be unequally distributed, with few people receiving most of the mosquito bites (i.e. 20-30% of the population getting 70-80% of the bites). Heterogeneous biting has broad implications for malaria epidemiology and control and, as recently suggested, may provide a plausible explanation for inconsistencies related to malaria transmission dynamics and modelling [
16].
Toward the development of serological markers of exposure to malaria vectors, attention was focused on gSG6 (
g ambiae
S alivary
G land protein
6), a small protein initially identified in
A. gambiae, where it is specifically expressed in the salivary glands of adult female mosquitoes [
17]. Its specific function awaits full clarification; however, gSG6 must play some relevant role in haematophagy since its depletion by RNAi increases probing time and affects blood feeding ability [
18]. Afterwards, members of the SG6 protein family have been identified in the salivary transcriptomes of additional anopheline mosquitoes, but in no other living organisms, pointing to its genus-specificity and blood feeding role. Among the few anophelines analysed so far the SG6 protein is present in species belonging to the subgenus
Cellia (
A. gambiae species complex,
A. funestus,
Anopheles stephensi) and in
Anopheles freeborni (a member of the subgenus
Anopheles), but it is notably absent in
Anopheles darlingi, a member of the subgenus
Nyssorhynchus and vector of malaria in Central and South America [
18]. This observation suggests that SG6 family members may be widely distributed among the main African and Asian malaria vectors, but most likely absent in South American ones.
Given the anopheline-specificity and previous indications of the immunogenicity to humans of gSG6-based peptides [
19], the
A. gambiae gSG6 protein was expressed in recombinant form and the anti-gSG6 IgG response was analysed in a population from a malaria hyperendemic area of Burkina Faso. This study provided experimental evidence that gSG6 may be a good candidate as serological marker of human exposure to
A. gambiae [
20], although full validation in different epidemiological settings (i.e. low transmission conditions, macro-geographic scale) is needed. Moreover, since malaria is transmitted by multiple and often sympatric vectors, an ideal salivary marker should allow to estimate exposure to all the major vector species in the study area. The
A. gambiae gSG6 protein is highly conserved among members of the
A. gambiae species complex (99% identity with the
A. arabiensis homologue, aSG6), whereas it is more distantly related (80%, 70/87 residues) to the
A. funestus protein (fSG6). It is likely that a certain degree of cross-reactivity to the two protein exists, but the extent of the overlap of the human IgG response to gSG6 and fSG6 proteins is unknown. Some indications in this direction have been obtained using the 23 aa long gSG6-P1 peptide, which encompasses the gSG6 N-terminal region [
21]. However, this peptide is less sensitive in comparison to the whole protein (approx 5-fold) and, therefore, it would be important to experimentally validate the efficacy of using the antibody response to the gSG6 protein as marker of exposure to the three main malaria vectors in tropical Africa. The aim of this study was to evaluate cross-reactivity of human sera from exposed individuals to the gSG6 and fSG6 proteins. To this purpose the
A. funestus fSG6 was expressed in recombinant form and the IgG response to fSG6, gSG6 and to an equimolar mixture of the two proteins was compared by ELISA using sera of individuals from a malaria hyperendemic area of Burkina Faso.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CR performed the immunoassays and contributed to data analysis. RR carried out expression and purification of recombinant proteins. GF participated in the design of the study and in setting conditions for protein purification. VM participated in the design of the study and analysis of data. SB and NI contributed reagents. VP contributed to study design and to data and statistical analyses. DM conceived the study, participated to its design and analysis of data, contributed reagents. BA conceived and coordinated the study, participated to data and statistical analyses, wrote the manuscript. All authors read and approved the final manuscript.