Corresponding Author Anne Poinsignon, Unité UR 016 ‘Caractérisation et Contrôle des Populations de Vecteurs’– Institut de Recherche pour le Développement (IRD), Montpellier, France. E-mail: email@example.com
Objective The development of a biomarker of exposure based on the evaluation of the human antibody response specific to Anopheles salivary proteins seems promising in improving malaria control. The IgG response specific to the gSG6-P1 peptide has already been validated as a biomarker of An. gambiae exposure. This study represents a first attempt to validate the gSG6-P1 peptide as an epidemiological tool evaluating exposure to An. funestus bites, the second main malaria vector in sub-Saharan Africa.
Methods A multi-disciplinary survey was performed in a Senegalese village where An. funestus represents the principal anopheline species. The IgG antibody level specific to gSG6-P1 was evaluated and compared in the same children before, at the peak and after the rainy season.
Results Two-thirds of the children developed a specific IgG response to gSG6-P1 during the study period and – more interestingly – before the rainy season, when An. funestus was the only anopheline species reported. The specific IgG response increased during the An. funestus exposure season, and a positive association between the IgG level and the level of exposure to An. funestus bites was observed.
Conclusions The results suggest that the evaluation of the IgG response specific to gSG6-P1 in children could also represent a biomarker of exposure to An. funestus bites. The availability of such a biomarker evaluating the exposure to both main Plasmodium falciparum vectors in Africa could be particularly relevant as a direct criterion for the evaluation of the efficacy of vector control strategies.
Première tentative de validation du peptide salivaire gSG6-P1 comme un outil immuno-épidémiologique d’évaluation de l’exposition humaine aux piqûres de An. funestus
Objectif: Valider le peptide gSG6-P1 comme un outil épidémiologique évaluant l’exposition aux piqûres de An. funestus, le second principal vecteur de la malaria en Afrique subsaharienne.
Méthodes: Etude multidisciplinaire dans un village sénégalais oùAn. funestus est la principale espèce d’anophèle. Le taux d’anticorps IgG spécifiques à gSG6-P1 a étéévalué et comparé chez les mêmes enfants avant, durant le pic et à la fin de la saison des pluies.
Résultats: Deux tiers des enfants ont développé une réponse IgG spécifique à gSG6-P1 durant la période étudiée et - plus intéressant - avant la saison des pluies, quand An. funestus était la seule espèce d’anophèle signalée. La réponse IgG spécifique augmentait au cours de saison d’exposition à l’An. funestus et une association positive entre le taux d’IgG et le niveau d’exposition aux piqûres de An. funestus a été observée.
Conclusions: Les résultats suggèrent que l’évaluation de la réponse IgG spécifique à gSG6-P1 chez les enfants pourrait également représenter un biomarqueur de l’exposition aux piqûres de An. funestus. La disponibilité d’un tel biomarqueurs évaluant l’exposition aux deux principaux vecteurs de P. falciparum en Afrique pourrait être particulièrement pertinente en tant que critère pour l’évaluation directe de l’efficacité des stratégies de lutte antivectorielle.
Primer ensayo para validar el peptido salivar gSG6-P1 como herramienta inmuno-epidemiológica que evalúe la exposición humana frente a picaduras de An. funestus
Objetivo: Validar el péptido gSG6-P1 como herramienta epidemiológica que evalúe la exposición a picaduras de An. funestus, el segundo vector de malaria más importante en África sub-Sahariana.
Métodos: Estudio multidisciplinar, en un poblado senegalés en donde An. funestus es la principal especie anofelina. Los niveles de anticuerpos IgG específicos para gSG6-P1 se evaluaron y compararon en los mismos niños antes, durante el pico y después de la estación de lluvias.
Resultados: Dos tercios de los niños desarrollaron una IgG específica para gSG6-P1 durante el periodo de estudio y – más interesante aún – antes de la época lluviosa, cuando An. funestus era la única especie anofelina reportada. La respuesta IgG específica aumentó durante el periodo de exposición a An. funestus y se observó una asociación positiva entre el nivel de IgG y el nivel de exposición a picaduras de An. funestus.
Conclusiones: Los resultados sugieren que la evaluación de la respuesta IgG específica para gSG6-P1 en niños también podría utilizarse como un biomarcador de exposición a picaduras de An. funestus. La disponibilidad de un biomarcador que evalúa la exposición a los dos principales vectores de P. falciparum en África podría ser particularmente relevante como un criterio directo para la evaluación de la eficacia de estrategias de control vectorial.
The threat from malaria, the leading cause of children mortality in sub-Saharan Africa, is prompting research in developing new tools for efficient control. Current control measures combine anti-Plasmodium treatment supported by artemisinin-based combination therapy with anti-Anopheles interventions such as the use of bed nets (long-lasting insecticidal nets) and household insecticides. These control measures have already reduced the number of malaria cases and deaths in some areas (Unicef and Roll Back Malaria 2009), but continued effort is needed to elevate the current level of control to elimination and eradication. Such progress requires improvement of current malaria risk evaluations and the development of new prevention and case-management technologies.
The development of new tools to evaluate the level of exposure to arthropod vector bites shows promise. New initiatives such as mapping spatial limits for human malaria parasites and determining the relative geographical frequency distribution of the Anopheles vector provide information for malaria control (Hay et al. 2010). Further, epidemiological studies linked to the antigenic properties of arthropod salivary proteins have shown that quantification of the human antibody (Ab) response specific to arthropod salivary proteins is a potential biomarker for exposure to vector bites in an exposed population (Remoue et al. 2005; Billingsley et al. 2006). This research topic has been investigated for several vectors that carry human pathogens such as ticks (Schwartz et al. 1990), Aedes (Remoue et al. 2007), Glossina (Poinsignon et al. 2008b), Triatoma (Nascimento et al. 2001) and Anopheles (Remoue et al. 2006; Waitayakul et al. 2006). Such epidemiological biomarkers of exposure could be geographical indicators for the risks of disease transmission and may serve as tools for evaluating the effectiveness of a vector control programme (Schwarz et al. 2009; Drame et al. 2010). Interestingly, it has been demonstrated that the IgG response specific to the gSG6-P1 peptide is a specific biomarker for Anopheles gambiae s.l. exposure (Poinsignon et al. 2008a). The salivary peptide was based on An. gambiae s.s. SG6 protein sequence and has as yet only been validated in an epidemiological context where An. gambiae complex species were the only anopheline species (Poinsignon et al. 2008a, 2009). The specificity of this peptide to the Anopheles genus allows the specific evaluation of the human-Anopheles contact, i.e. removing possible cross-reactivity with other salivary proteins from other arthropod vectors such as Aedes, Culex and Glossina.
In sub-Saharan countries, An. funestus is the second major malaria vector. This species occupies a broad range of ecological niches, is highly anthropophilic and abounds during the dry season when An. gambiae s.l. densities are low. In most sites, An. funestus appears to maintain malaria transmission when exposure to An. gambiae s.l. subsides. Because the three major malaria vectors in Africa, An. gambiae s.s., An. arabiensis and An. funestus geographically co-inhabit across most of sub-Saharan countries (Manguin et al. 2008), an optimal exposure biomarker needs to reflect exposure to several Anopheles species. Indeed, anopheline species responsible for Plasmodium falciparum transmission can shelter in the same house and therefore blood-feed on the same individuals. The genome of An. gambiae and An. funestus is closely related (Coetzee & Fontenille 2004) and as previously reported, the salivary peptide gSG6-P1 shares 91% identity with An. funestus (Poinsignon et al. 2008a).
Malaria transmission in the northern part of Senegal is low, unstable, geographically restricted and seasonal with an average of 2–7 infective bites/person/year (Faye et al. 1998). However, the construction of two dams has decreased the salinity gradient along the Senegal River; it is thought that this has contributed to the reappearance of An. funestus in the Senegal River Basin, which had disappeared during a drought in the 1970s (Konate et al. 2001).
The aim of this study was to evaluate the use of the An. gambiae gSG6-P1 salivary biomarker in areas where malaria transmission is assumed by An. funestus. To this end, fieldwork was carried out in a northern Senegalese village where An. funestus was reported as the predominant species by classical entomological trapping (Dia et al. 2008). This study focused on the evaluation of IgG Ab response to gSG6-P1 peptide in children.
Material and methods
The study was conducted in the north of Senegal, in Gankette Balla village located along the Senegal River Basin, on the shores of the Guiers Lake. This area is a dry savannah, with rainy season from July to October, and therefore represents a typical area of the Sahelian and sub-Sahelian regions of Africa, with approximately 400 mm of rain per year. In this village, malaria transmission is seasonal from September to December.
A longitudinal study was performed in 2004 in this village and a cohort of 173 children aged 1–9 years old was followed, as previously described (Dia et al. 2008). Triplets of sera from a sub-sample (n = 87) of the same children were available from three passages (June, September and December 2004). For each child, parasitological measurements of malaria (identification of Plasmodium species and the level of intensity of infection) were performed at each passage using thick blood smears obtained by finger-prick. In the same way, capillary blood collection was completed for each child at each passage for immunological assessment.
This study followed ethical principles according to the Helsinki Declaration and was approved by the Ethical Committee of the Ministry of Health of Senegal (CNRS, June 2004). Informed consent was obtained from the parents of the children.
Adult mosquitoes were collected in June, September and December 2004 (corresponding respectively to the beginning, the peak and after the rainy season) using human-landing catches as previously described (Dia et al. 2008). Mosquitoes caught were counted and identified morphologically to Anopheles species (Gillies & De Meillon 1968). Anopheles infection rate was studied by Enzyme-Linked ImmunoSorbent Assay (ELISA) for P. falciparum circumsporozoite antigen (CSP). The number of bites per human per night (BHN) was calculated by dividing the number of mosquitoes caught by the total person-night used for the month survey.
Salivary peptide gSG6-P1
The gSG6-P1 peptide was designed using bioinformatics to maximize its Anopheles specificity and its antigenicity, as previously described (Poinsignon et al. 2008a). The gSG6-P1 peptide was synthesized, purified (>80%) by Genosys (Sigma-Genosys, Cambridge, UK). All peptides were shipped in lyophilized form and then resuspended in 0.22 μm ultra-filtered water and frozen in aliquots at −80 °C until use for immunological tests (ELISA).
Evaluation of human IgG antibody level (ELISA)
Enzyme-Linked ImmunoSorbent Assays were carried out on the individual sera to measure the level of IgG Ab reacting to the gSG6-P1 antigen as described by Poinsignon et al. (2008a). Briefly, Maxisorp plates (Nunc, Roskilde, Denmark) were coated with gSG6-P1 (20 μg/ml) in phosphate buffer saline (PBS). After washing (distilled water + Tween-0.1%), each serum was incubated in duplicate at 4 °C overnight at a 1/20 dilution (in PBS–Tween-1%). This optimal dilution was determined after several preliminary experiments. Mouse biotinylated Ab to human IgG (BD Pharmingen, San Diego, CA, USA) was incubated at a 1/1000 dilution (1 h 30 min at 37 °C) and peroxidase-conjugated streptavidin (Amersham, Les Ulis, France) was then added (1/1000; 1 h at 37 °C). Colorimetric development was carried out using ABTS (2,2′-azino-bis (3-ethylbenzthiazoline 6-sulphonic acid) diammonium; Sigma, St Louis, MO, USA) in 50 mm citrate buffer (pH 4) containing 0.003% H2O2. Optical density (OD) was measured at 405 nm. Each test sample was assessed in duplicate wells and, in parallel, in a blank well containing no gSG6-P1 antigen (ODn) to control for non-specific reactions in the plasma and the reagents. Individual results were expressed as ΔOD value calculated according to the formula ΔOD = ODx − ODn, where ODx represents the mean of individual OD in both antigen wells, as previously used (Poinsignon et al. 2008a).
All data were analysed with GraphPad Prism software® (San Diego, CA, USA). After ensuring that the results did not have a normal distribution, the Wilcoxon matched pair test was used to compare paired sera of individuals. Differences were considered statistically significant when P-values were <0.05.
Results and discussion
Because it has been previously demonstrated that the specific IgG response to gSG6-P1 salivary peptide was an immunological biomarker of the exposure level to An. gambiae bites, this study evaluated if the same peptide biomarker could be applied to detect exposure to An. funestus, another main vector of P. falciparum in Africa. The IgG response specific to the gSG6-P1, derived from the An. gambiae SG6 salivary protein, was followed in the same children before (June), at the peak (September) and after (December) the rainy season in a village where An. funestus represented the large major Anopheles species.
Entomological data in Gankette Balla, including the study period, have previously been presented and An. funestus has been reported as the main P. falciparum vector (Dia et al. 2008). In summary, human-landing catches have sampled several anopheline species in this village, with An. funestus largely predominant. An. gambiae s.l. (An. arabiensis), An. pharoensis and An. wellcomei were also collected, but in very low densities. During the study period, from June to December 2004, the average of An. funestus BHN, calculated from the surveys, was estimated to 9.8 before the rainy season (June) and to 117 and 96.3 in September and in December, respectively (Figure 1). Whereas An. funestus larvae develop in permanent or semi-permanent water collections, its dynamic was associated positively with the rainfall during the study. For the same period, the human biting rate for An. arabiensis species was reported to be very low, 0.17 BHN in September and 0.5 in December. No An. arabiensis were collected in June. Additionally, among anopheline species, only An. funestus was positive for P. falciparum CSP antigen (Dia et al. 2008).
The P. falciparum prevalence in children under 9 years old was also investigated (Figure 1). The parasite prevalence remained very low and was positively associated with the An. funestus bites rate level with an increase from the early [June, 4% (7/173)] to the peak [September, 7.4% (10/135)] of the rainy season, a level maintained until December [7% (11/156)]. Four per cent of the children presented a positive P. falciparum infection in June, before the beginning of the raining season when An. funestus was the only anopheline species in Gankette Balla. Indeed, larvae from An. gambiae complex typically breed in temporary rain-dependant pools, and as reported, no An. arabiensis mosquitoes were sampled in June. This suggests that An. funestus was the malaria vector before and in the early rainy season.
In parallel, the IgG response specific to the gSG6-P1 peptide was evaluated in the same children in June, September and in December (Figure 2). Two-thirds of the children presented an IgG response specific to gSG6-P1 during the study period, even in June when inhabitants were mainly exposed to An. funestus bites. Therefore, the development of the observed specific IgG response in the children could result in an antigenic stimulation following specific An. funestus bites.
The evolution of the IgG response to gSG6-P1 was compared between June, September and December (Figure 2). The specific IgG response was significantly higher in September (median = 0.090), corresponding to the peak of An. funestus exposure, when compared to June (median = 0.062; P = 0.02). From September to December (median = 0.080), the specific IgG response level decreased (not significantly) but remained higher than June. It appears that the IgG response level varied positively with An. funestus densities as evaluated by entomological sampling. Altogether these results suggested that the evaluation of the IgG response specific to the gSG6-P1 salivary peptide in children could also represent a pertinent biomarker of exposure to An. funestus bites.
The SG6-P1 peptide sequences between An. gambiae and An. funestus share a high level of identity. Their sequences differ only by the substitution of two amino acids, being asparagine by glutamine (position 9) and leucine by an isoleucine (position 15) between An. gambiae and An. funestus, respectively (Figure 3). The different amino acids from fSG6-P1 are closed in terms of polarity and charge to those from An. gambiae gSG6-P1. This observation, when considered in conjunction with present results, suggests that these substitutions do not alter the synthesis and the recognition of specific Ab because this epitope appears to be conserved.
An. arabiensis mosquitoes were reported to be present in very low densities in September and in December. In a previous study, the gSG6-P1 salivary peptide has been suggested as a useful sensitive epidemiological tool for identifying individuals exposed to very low An. arabiensis densities in Senegal (Poinsignon et al. 2009). Therefore, we cannot rule out that a part of the IgG response specific to gSG6-P1 observed in September and December was the result of antigenic stimulation following An. arabiensis bites. Nevertheless, the IgG response specific to gSG6-P1 observed in June can be attributed to An. funestus bites exposure regarding entomological data.
Concerning the potential applications of such an epidemiological tool of vector exposure to the malaria vector, it could be particularly relevant to have a unique indicator for evaluating the exposure to the major anopheline vector species. Such a tool could be easily applied on field conditions as a rapid diagnostic test to evaluate the exposure of human populations to African malaria vectors. In complement, it could be also a hypothesis to develop a specific biomarker of exposure to An. gambiae vs. An. funestus. Indeed, preliminary results from our group suggested some immunogenic salivary proteins could be different between both species (S. Cornelie, unpublished data). The identification of such species-specific salivary candidates as biomarkers is currently under investigation.
This study is the first attempt to validate the gSG6-P1 salivary peptide as an epidemiological tool to evaluate exposure to An. funestus bites. The availability of such a biomarker to allow the evaluation of the exposure to both main P. falciparum vectors in Africa could represent a very useful epidemiological tool for malaria control. Such a biomarker can evaluate exposure specific to Anopheles species in areas where different species of malaria vector co-inhabit.
The authors gratefully acknowledge the population of Gankette Balla for their participation in this study and the team of the ‘Pal-Fleuve program’. A Poinsignon was supported by a scholarship provided by the Research Ministry of France and a fellowship from Francois Lacoste. JB Sarr holded a fellowship from the Conseil Regional de Saint-Louis, Senegal. This research was supported by the CGGVeritas Compagny and by the Pal+ program (French Ministry of Research). The authors thank the FSD ‘Fonds Social de Développement’, Embassy of France in Senegal for their financial participation.