Mosquitoes are the most menacing worldwide arthropod disease vectors. They transmit a broad range of viral, protozoan and metazoan pathogens responsible of the most devastating human and animal diseases . Among the main frequent mosquito-borne diseases, malaria represents the most widespread and serious infection in terms of heavy burden on health and economic development throughout the world. Despite substantial efforts and increasing international funding to eliminate it, malaria is still a major public health problem with nearly a million of deaths per year, especially in children younger than 5 years old (86%) . Approximately two thirds of the world's population live in areas at risk for malaria [3, 4]. Understanding mechanisms that govern its transmission remains therefore a major scientific challenge, but also an essential step in the design and the evaluation of effective control programs [5, 6].
Entomological, parasitological and clinical assessments are routinely used to evaluate the exposure of human populations to
Malaria is a parasitic disease caused by protozoan agents of the genus
However, whole saliva could be inadequate as a biomarker tool, because it is a cocktail of various molecular components with different nature and biological functions. Some of these elements are ubiquitous and may potentially cause cross-reactivities with common salivary epitopes of other haematophagous arthropods . In addition, a lack of reproducibility between collected whole
The present chapter contributes therefore to a better understanding of the human-mosquito immunological relationship. It resumes most of the studies highlighting the roles of mosquito saliva on the human physiology and immunology, approaches, techniques, and methods used to develop and validate specific candidate-biomarkers of exposure to
2. Human host-mosquito relationship: Roles of mosquito saliva
Arthropods represent the vast majority of described metazoan life forms throughout the world, with species’ richness estimated between 5 to 10 million . The blood feeding habit has arisen and evolved independently in more than 14,000 species from 400 genera in the arthropod taxonomy . In mosquitoes, only the adult female is hematophagous, whereas both male and female take sugar meals . During the probing and the feeding stages, like all blood-sucking arthropods, female
2.1. Pharmacological properties of mosquito saliva
The first-line of the human host non-specific defense to the insect bite is the haemostatic reaction. It provides an immediate response to the vascular injury caused by the intrusion of the mosquito mouthparts in host vessels, thus preventing the extensive loss of host blood [32, 34]. The haemostatic reaction consists of three not physiologically distinct mechanisms: i) the blood coagulation that leads to the production of fibrin clots, ii) the thrombus formation and wound healing mediated by platelet aggregation, and iii) the vasoconstriction that leads to restricted influx of blood to the injured site. Each mechanism is activated by several pathways, in response to different exogenous and endogenous stimuli. Platelet aggregation is the first step in the haemostatic cascade and follows the interaction between blood platelets and the exposed extracellular matrix. This latter contains a large number of adhesive macromolecules such as collagen which is abundant underneath endothelial cells (not found in blood). This interaction results to the activation of platelets by mainly collagen and adenosine diphosphate (ADP, released by damaged cells and by activated platelets), the primary agonists of platelet aggregation. Platelets can be also activated by other agonists such as thrombin (produced by the coagulation cascade) and thromboxane A2 (TXA2, produced by activated platelets) . Activated platelets release endogeneous secretions such as serotonin and TXA2, two potent vasoconstrictors. In parallel, the blood coagulation mechanism is getting underway. The main task of the coagulation cascade is to produce fibrin that supports aggregated platelets in a thrombus formation. The coagulation process consists of an enzymatic cascade with two ways of activation, the exogenous and the endogenous, where several amplification points and regulatory mechanisms are known.
However, mosquitoes can successfully engorge on their hosts within a half-minute because antihemostatic components of their saliva facilitate location of blood vessels and the blood sampling . These salivary secretions, named sialogenins (from the Greek
2.1.1. Inhibition of platelet aggregation
Compared to other blood-sucking arthropods like ticks and sand flies, only a limited number of
2.1.2. Inhibition of blood coagulation cascade
Arthropod anticoagulants mostly target factor X-active (fXa), which plays a central role at the nexus of the intrinsic and extrinsic pathways, as well as an ultimate role of thrombin in driving production of fibrin from fibrinogen. However,
2.1.3. Vasodilator effect on host blood vessels
In human, various types of endogenous vasoconstrictors (serotonin, TXA2, noradrenalin…) are released few seconds after tissue injury in order to stop the blood flow locally at the bite site. Diverse types of vasodilators have been characterized in the saliva of hematophagous arthropods.
2.2. Immunological effects of mosquito saliva
The tissue injury causes an immediate onset of acute inflammation and innate immunity, which promote tissue repair, prevent colonization of the damaged tissues by opportunistic pathogens and initiates adaptive immunity, which is more specific . These responses mobilize multiple elements such as phagocytes and antigen-presenting cells, cytokine-producing cells, T and B lymphocytes (TL and BL) and complement (classical and alternative pathways). It may result to the development of strong cell and humoral immune reactions, thereby altering physiologically the environment at the bite site and leading to the rejection of the blood-sucker . The saliva of
2.2.1. Inhibition of host inflammatory reaction
The host inflammatory reaction following tissue injury consists of the triple response of Lewis: redness, heat and pain, triggering the awareness of the host to the blood sucker action . If redness and heat are ones of the direct consequences of the dilatation of blood vessels, pain is induced by an increased vascular permeability under the effect of ADP, serotonin and histamine released by platelets and mast cells, following activation of the fXII by tissue-exposed collagen . The fXIIa converts prekallikrein to kallikrein, which hydrolyzes blood kininogen to produce the vasodilator peptide, bradykinin. This latter induces TNF-α (Tumor Necrosis Factor alpha) release by neutrophils , which in turn stimulates the release of IL (interleukin)-1β and IL-6 from various cell types. These cytokines contribute to the phenomenon of hyperalgesia (increased sensitivity to pain) that accompanies inflammation. Host inflammatory reaction to bites has been described as mast cells-dependent in individuals bitten by
2.2.2. Modulation of host immune response
A role for arthropod saliva in modifying the outcome of transmission and infection is not a novel idea introduced in the context of mosquitoes and malaria parasites. The increased pathogen infectivity in association with ticks, sand flies, and mosquitoes saliva has been described previously . If ticks that take a long time to engorge must additionally necessitate in their saliva anti-inflammatory and immunosuppressive factors, rapidly feeding dipterans, in particular mosquitoes and sand flies, clearly have evolved salivary factors that directly modulate host immune defenses . One possible explanation is that these molecules have evolved because they have long-term beneficial effects for the populations rather than to the individual at the time of feeding . Although the molecular mechanisms by which mosquito saliva induces alteration of the host immune response are unclear [59, 60], data evidently demonstrate that effects depend on the global regulation of the Th1/Th2 cytokines’ balance, as it has been described in sand flies
Therefore, future studies are needed for an overall understanding of mosquito saliva effect, especially
2.2.3. Human host-Anopheles vector immune relationship and applications
The study of immunological properties of salivary proteins of
The development of parasite transmission-blocking vaccines, by stimulating the immune response against the vector is an attractive alternative way for malaria control. Several studies targeted the effect of Abs specific to the mosquito midgut antigens have shown promising results [71-73]. The study of the immune response induced by vector saliva at the biting site and its potential effect on the transmission and the development of pathogens suggests the possibility to control parasite transmission by vaccinating the host with immunogenic salivary compounds [54, 74]. In a mouse model, it has been shown that two salivary proteins (29 and 100 kDa) of the female
In the field of allergic reactions to salivary proteins of mosquitoes, the first studies were mainly conducted in Canada and Finland. They concerned
The study of immunological relationship between human-vector by quantifying specific Ab responses to salivary proteins may also allow the identification and characterization of biological markers for epidemiological assessment of the exposure of individuals and populations to the
3. Development of biomarkers of human exposure to
Anophelesbites and indicators of malaria vector control effectiveness
3.1. Validation of concept with whole
To improve the fight against malaria and regarding numerous limitations described with current entomological and parasitological tools, the World Health Organization (WHO) has emphasized the need of new indicators and methods to evaluate, at individual and population levels, the exposure level to
The epidemiological importance of human exposure to the saliva of vectors has been firstly described in Lyme disease [80, 81], leishmaniasis  and Chagas disease . During the last decade, studies have provided data on human exposure to anopheline saliva and its interaction with malaria transmission. In particular, Remoue
Importantly, IgG response to
Anti-mosquito saliva Ab appeared transitional. Soldier travelers transiently exposed to
Taken together, these studies indicated that the estimation of human IgG Ab responses specific to
3.2. Methods for the identification of specific
The isolation of salivary components has been a challenge for many years. Many functional active salivary proteins have been isolated following classical biochemical and molecular biology approaches . Protocols mainly consisted of the isolation of salivary components from hundreds of salivary gland pairs, obtaining amino-terminal or internal peptide sequence of the purified component, screening of a salivary gland library with the information obtained, and isolation of the cDNA or gene of interest (Fig. 6).
During the last decade, technical advances in molecular biology have allowed the sequencing of the genome, including transcripts of salivary glands , of most disease vectors, comprising
3.3. Salivary proteins (sialome) of
The increasing power of large-scale genomic, transcriptomic and proteomic analyses allowed the accumulation of a considerable amount of information on the salivary secretions of blood-sucking arthropods . As far as mosquitoes are concerned, the analysis of salivary transcriptomes of a number of
The analysis of the adult
3.3.1. Ubiquitous salivary proteins
AG5 family proteins are found in the salivary glands of many blood-sucking insects and ticks [102, 103]. In
Enzymes such as maltase, apyrase, 5′ nucleotidase, and adenosine deaminase, are also secreted during the bite of many blood-sucking arthropods, including
3.3.2. Salivary proteins found exclusively in Diptera
Other Diptera-specific protein families or peptides have also been described in the sialome of blood-feeding mosquitoes . However their function is still unknown, even if some were known to play a role in antimicrobial property of mosquito saliva.
3.3.3. Protein families found exclusively in mosquitoes
The 30-kDa antigen family found exclusively in the SGs of adult female mosquitoes has been found in both culicine and anopheline mosquitoes [95, 100, 101, 106-108]. Only one gene enriched in SGs of adult females is known in
Various types of
3.3.4. Protein families found exclusively in Anophelines
The 8.2-kDa family is represented in several
The 6.2-kDa family was first described in a sialotranscriptome of
The SG-2 family proteins were identified from
3.4. Specific salivary biomarker of exposure to
Anophelesbites: The gSG6-P1 peptide candidate
The SG6 salivary protein has been reported to be immunogenic in travelers exposed for short periods to
3.4.1. Identification and sequence of gSG6-P1 peptide
Several algorithms were employed for prediction of potential immunogenic sites of
Similarities were also searched using the Blast family programs, including both the genome/EST libraries of other vector arthropods available in Vectorbase and of pathogens/organisms in non-redundant GenBank CDS databases. No relevant identity was found with proteins of other blood-sucking arthropods. Indeed, the longest perfect match was 6 AAs between a putative protein from
3.4.2. Antigenicity of gSG6 peptides
IgG Ab responses to the five gSG6 peptides were evaluated by ELISA in a randomly selected subsample of children (n<30) living in a rural area of Senegal. All peptides were immunogenic, but the intensity of the IgG level was clearly peptide-dependent; weak immunogenicity was observed for gSG6-P3, gSG6-P4 and gSG6-P5, whereas gSG6-P1 and gSG6-P2 appeared highly immunogenic (Fig. 10).
3.4.3. Validation as a biomarker of exposure in several epidemiological settings
The specific IgG level to the two most antigenic gSG6 peptides (gSG6-P1 et gSG6-P2) was then evaluated according to the level of exposure (estimated by entomological data) in a larger sample (n=241) of children living in a malaria seasonal area . A positive trend was found for both peptides, but only significant for gSG6-P1 (Figure 11). Altogether, these results indicated that only the IgG response to gSG6-P1 is suitable to be a pertinent biomarker of exposure to
22.214.171.124. Biomarker of Anopheles vector bites
As previously suggested, anti-gSG6-P1 IgG response was described as a biomarker of
One study aimed to evaluate the risk of malaria transmission in children and adults living in urban area of Senegal (Dakar region) by using the gSG6-P1 peptide biomarker. Results showed considerable individual variations in anti-gSG6-P1 IgG levels between and within districts, in spite of a context of a global low
In a population from a malaria hyperendemic area of Burkina Faso, the use of gSG6 recombinant protein as reliable indicator of exposure to the 3 main African malaria vectors (
AAs from fSG6-P1 are close in terms of polarity and charge to those from
All mentioned studies were conducted on subjects older than 1 year. However, to be more relevant in epidemiological surveys and studies on malaria, such biomarker tool must pertinently be applicable to all human age-classes, including newborns and young infants (<1 year old) who can be also bitten by
Indeed, the presence of anti-gSG6-P1 IgG and IgM in the blood of respectively 93.28 and 41.79% of 3-months old infants (the majority of infants) and their gradual increasing levels until 12 months (Fig. 13), whatever the
126.96.36.199. Factors of variation of antibody response to gSG6-P1 and their consequences
Specific gSG6-P1 Ab responses can be influenced by several determinant factors in their variations between individuals, districts, villages, regions... Therefore, identifying effects of human intrinsic (gender, age…) and extrinsic (period of sampling, use of vector control measure…) factors will be useful to the application of the gSG6-P1 biomarker in epidemiological studies or monitoring, evaluation and surveillance of risk of malaria programmes.
Studies have globally reported an increasing anti-gSG6-P1 Ab level according to individual age. In a moderate transmission semi-urban area in Angola, the lowest and highest specific IgG levels have been described in young children (0-7 years old) and in teenagers/ adults (>14 years old) respectively . In a low malaria transmission urban area (Dakar region) in Senegal, specific IgG levels were significantly higher in adults (>18 years old) compared to 6-10 years old children and in this latter group compared to those aged from 2 to 5 years  . In Tori Bossito, moderate-high rural transmission area of Benin, both anti-gSG6-P1 IgG and IgM levels were low at 3 months of age and gradually increased until 12 months after birth (Drame
Some studies have reported higher levels of anti-gSG6-P1 in female individuals (children and women) compared to males (children and men) [27, 30] (; Drame
The season of individual sampling may be also a factor of confusion in the use gSG6-P1 biomarker in epidemiological studies on malaria risk assessment or control. Indeed, significant seasonally variations in anti-gSG6-P1 IgG or/and IgM levels have been reported in studies conducted in newborns, children or/and adults from endemic malaria areas in Senegal [27-29, 124], Angola  and Benin (Drame
One direct application of a salivary biomarker of exposure could serve in the elaboration of maps representing the risk of exposure to
188.8.131.52. Indicator of malaria vector control effectiveness
A longitudinal study associating parasitological, entomological and immunological assessments of the efficacy of ITN-based strategies using the gSG6-P1 biomarker has been conducted in a malaria-endemic area in Angola. Human IgG responses to gSG6-P1 peptide were evaluated in 105 individuals (adults and children) before and after the introduction of ITNs and compared to entomo-parasitological data. A significant decrease of anti-gSG6-P1 IgG response was observed just after the effective use of ITNs (Fig. 14). The drop in gSG6-P1 IgG levels was associated with a considerable decrease of
The response does not seem to build up but wanes rapidly, when exposure failed. This property represents a major strength when using such salivary biomarker of exposure for evaluating the efficacy of vector control. In addition, using a response threshold (ΔOD=0.204) combined with ΔODITNs - the difference between April (after ITNs) and January 2006 (before) - makes possible the use of this operational biomarker at individual level (Fig. 15). The threshold response (TR) represents the non-specific background IgG response (the cut-off of immune response) and was calculated in non-
A recent cross-sectional study conducted from October to December 2008 on 2,774 residents (children and adults) of 45 districts of urban Dakar (Senegal) has validated IgG responses to gSG6-P1 as an epidemiological indicator evaluating the effectiveness of a range of VCMs. Indeed, in this area, IgG levels to gSG6-P1 as well as the use of diverse malaria VCMs (ITNs, mosquito coils, spray bombs, ventilation and/or incense) highly varied between districts . This difference of use suggests some socio-economical and cultural discrepancies between householders as described in large cities of Ivory Coast  and Tanzania . At the district level, specific IgG levels significantly decreased with VCM use in children as well as in adults. Among used VCM, ITNs, the 1st chosen preventive method (43.35% rate of use), by reducing drastically the human-
Taken together, these results suggest that the assessment of human IgG responses to
In parallel to an entomological and parasitological evaluation, IgG responses to gSG6-P1 were also used to assess, in a randomized controlled trial in 28 villages in southern Benin, four malaria vector control interventions: Long-Lasting Insecticide-treated Net (LLIN) targeted coverage to pregnant women and children younger than 6 years (TLLIN, reference group), LLIN universal coverage of all sleeping units (ULLIN), TLLIN plus full coverage of carbamate-indoor residual spraying (IRS) applied every 8 months (TLLIN+IRS), and ULLIN plus full coverage of carbamate-treated plastic sheeting (CTPS) lined up to the upper part of the household walls (ULLIN+CTPS). Results from this study have shown that specific IgG levels were similar in the 4 groups before intervention and only significantly lower in the ULLIN group compared to the others after intervention. In contrast to immunological data, clinical incidence density of malaria, the prevalence and parasite density of asymptomatic infections, and the density and aggressiveness of
3.4.4. Importance to develop a specific biomarker of infecting Anopheles bites
Recent data have shown that the use of the gSG6-P1 biomarker for the assessment of the differential risk of the disease transmission may have some limitations in high exposure areas (Drame
The principle of biomarker of infective bites is based on the use of immunogenic salivary protein like marker of transmission. The expression of some salivary proteins could be induced or regulated when the salivary glands are infected. Therefore, if one of such protein presents also immunogenic properties, we can probably use the specific immune response to this protein like a marker of transmission in human. Such a biomarker will be also particularly relevant in the context of re-emergence after malaria transmission reduction or in area of low exposure. This tool will allow focusing the intervention (vector control strategies and drugs distribution) on the most exposed and the most susceptible population.
In the present chapter, we have described the development of a biomarker (the
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