An insight into immunogenic salivary proteins of Anopheles gambiae in African children

Morbidity and mortality of malaria are closely linked to exposure of the human host to the Anopheles vector. Among the 60 Anopheles species which transmit malaria worldwide, Anopheles gambiae is the most competent and the primary vector of Plasmodium falciparum in sub-Saharan Africa [1]. Human infection by P. falciparum can be measured by several diagnostic tests, such as thick smears or rapid diagnostic tests (RDT, Dipstick) [2]. The evaluation of exposure to risk of malaria is currently based on entomological methods (traps, household/indoor spraying, etc.), but such methods are mainly applicable to the population level and do not enable evaluation of the heterogeneity of individual exposure. Trapping methods using adults volunteers can estimate individual exposure, but may be limited due to ethical constraints and limitations for extrapolation to the incidence in children [3].

During the bite, the female mosquito injects saliva containing bioactive molecules such as vasodilatators, anti-clotting and anti-hemostatic proteins, which permit a successful blood meal [4]. During the last decade, research on mosquito salivary extracts has benefited from advances in genomic and proteomic tools generating salivary transcriptomes for several mosquito species, including An. gambiae [5‐8]. In particular, interactions between salivary proteins and the host immune system have aroused great interest [9, 10]. Indeed, some of these salivary components are recognized by the human immune system and induce specific responses [11]. Interestingly, a connection between the level of specific saliva antibodies (Ab) and exposure to vector borne-diseases has been reported for Lyme disease, Chagas disease, leishmaniasis [12‐14], and recently, human African trypanosomiasis [15]. Surprisingly, few data have described an immunomodulatory role for salivary proteins from Anopheles species and little is known about the human host response to Anopheles saliva. The Ab response to Anopheles stephensi saliva has been detected in allergic individuals [16]. Owashi and colleagues identified an inflammatory protein from An. stephensi saliva, which is recognized by sera from individuals exposed to malaria [17]. A recent report indicated that the IgG response to whole saliva from An. gambiae was found in children living in a malaria endemic area and was correlated with the intensity of exposure to An. gambiae bites [18]. That study led to the hypothesis that this specific Ab response could represent a marker of exposure to Anopheles bite. Thus, the objective of the present work was to describe immunogenic proteins of An. gambiae saliva so as to elucidate whether detection of immunogenic components depends upon the malaria transmission season.

This immunogenic band did not appear to be strongly expressed in saliva, as it did not match abundant proteins clearly visible on silver-stained gel. Along this line, sparse reports mentioned high molecular masses protein in Anopheles salivary glands; one is an anti-inflammatory factor described in An. stephensi salivary extracts [17], and the other a member from the SGS family which may play a role in malaria parasite invasion of salivary glands [22]. A putative assignation to the 72 kDa immunogenic band would be the apyrase, an inhibitor of platelet aggregation, or the maltase involved in sugar meal [23]. But the methodology used in the present study (1-D electrophoresis) appears limited to clearly associate one immunogenic band to one protein. However, less than 80 sequences have been defined as secreted products from An. gambiae saliva [7, 8]. Discriminating proteomic tools could achieve the characterisation of these salivary antigens.

Surprisingly, only half of the children developed IgG Abs against Anopheles saliva. Previous studies in the field of allergy to mosquito bites have reported considerable variations of Abs response associated with mosquito species and population densities [24, 25, 16]. Here, no significant variation was observed according to villages: 40% of the children from the high exposed village were responders versus 60% in the low exposed village. However, a spatial heterogeneity in malaria transmission is also observed elsewhere depending on the repartition of individuals at the village level [26]. Some of the children, because of the distance from major breeding sites, could have sparse contacts with Anopheles mosquito that may be insufficient to elicit anti-saliva Abs [18]. In addition, 20% of the population studied slept under a bednet also limiting the Anopheles bites [20]. Finally, the majority of the studies investigating human response to mosquito salivary proteins were performed in adults (over fifteen years old) [27, 28] who have a longer history of exposure to mosquito bites and an immune response which could be slightly different than the one observed in young children [13]. The results presented here show variability between individuals in the immune response to salivary antigens. For example, two individuals presented a decrease in the 175 kDa band but an increase in the 72 kDa band intensity in December compared to September. Further studies integrating strong data on the evaluation of the intensity of Anopheles exposure are needed to determine whether one immunogenic band could reflect a "pattern" of specific exposure. Several epidemiological parameters, such as genetic background or the possibility of co-infections could also involved in the variations in immunogenicity observed between children [29‐31].

The results presented here show a seasonal decrease in the intensity of immunogenic bands suggesting that the Ab response to Anopheles salivary antigens was transient, as previously observed in the same area [18]. As a matter of fact, it has been reported elsewhere that Ab against tick salivary antigens declines after a period of non-exposure [32] and clear seasonal difference in the anti-mosquito saliva Ab response has been described in Tanzania [11]. This transient character of the anti-saliva Ab response could be useful for the evaluation of Anopheles exposure, especially in areas with low intensity of exposure. Interestingly, the seasonal influence was significant for the 175 kDa band suggesting that this band could represent a good candidate for a marker of intensity of exposure to Anopheles bites. The results also showed a positive association with the intensity of immunogenicity for the 175 kDa band and the malaria morbidity in children over the same period. Indeed, the children developing malaria morbidity showed the higher intensity for this band in September which decreased during the malaria season. A previous study reported that the children presenting high IgG Abs in September are those will suffer from malaria attack within the next three months [18]. However, further studies including more subjects are necessary to confirm the association between anti-saliva Abs and malaria morbidity. This study described for the first time the presence of immunogenic bands against Anopheles saliva in children living in malaria endemic area. Few studies have described the anti-saliva Abs response to mosquito bite in children [13] whereas they are the main target for malaria parasite and other diseases transmitted by mosquito bites. Interestingly, the children who respond to Anopheles bites have common immunogenic bands suggesting that major common epitopes could be found in exposed populations. Theses results represent the first step toward the identification of immunogenic protein as a marker of exposure. Studies in other areas of malaria transmission and within a larger population were necessary to reinforce this hypothesis. But the methodology presented here, associating immunoblots and measurements of band intensity, appears very convenient for initial screening of molecules with an immunological and epidemiological interest. It provides qualitative and semi-quantitative data on the immunogenic components detected. Furthermore, such an approach could be applied to other vector-borne diseases which represent public health priorities in the developing world.

The last ten years have witnessed a renewed interest in host/vector studies for controlling vector-borne diseases. Major efforts have been made in sequencing the genomes of most "killing" vectors [33] and the saliva proteome of the Anopheles vectors [7, 23, 34]. The physiological function of the salivary proteins has been confirmed for only a few of them. This work suggests the importance of the human/vector relationship to develop an integrated strategy for the evaluation of exposure to vector bites and thus the risk of malaria. These results suggest that salivary markers of exposure to mosquito bite may ultimately be identified.