# Human antibody responses to the Anopheles salivary gSG6-P1 peptide: a novel tool for evaluating the efficacy of ITNs in malaria vector control.

Papa Makhtar Drame, Anne Poinsignon, Patrick Besnard, Sylvie Cornélie, Jacques Le Mire, Jean-Claude Toto, Vincent Foumane, Maria Adelaide Dos-Santos, Mbacké Sembène, Filomeno Fortes, et al.

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Papa Makhtar Drame, Anne Poinsignon, Patrick Besnard, Sylvie Cornélie, Jacques Le Mire, et al. Human antibody responses to the Anopheles salivary gSG6-P1 peptide: a novel tool for evaluating the efficacy of ITNs in malaria vector control. PLoS ONE, 2010, 5 (12), pp.e15596. 10.1371/journal.pone.0015596. hal-03021404

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# Human Antibody Responses to the *Anopheles* Salivary gSG6-P1 Peptide: A Novel Tool for Evaluating the Efficacy of ITNs in Malaria Vector Control

Papa Makhtar Drame¹*, Anne Poinsignon², Patrick Besnard³, Sylvie Cornelie², Jacques Le Mire³, Jean-Claude Toto⁴, Vincent Foumane⁴, Maria Adelaide Dos-Santos³, Mbacké Sembène⁵, Filomeno Fortes⁶, Francois Simondon⁷, Pierre Carnevale², Franck Remoue¹

1 UR016 « Contrôle et Caractérisation des Populations de Vecteurs », Institut de Recherche pour le Développement, Cotonou, Benin, 2 UR016 « Contrôle et Caractérisation des Populations de Vecteurs », Institut de Recherche pour le Développement, Montpellier, France, 3 Malaria Control Program-SONAMET Clinic, Lobito, Angola, 4 Laboratoire de Recherche pour le Paludisme, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, Yaoundé, Cameroun, 5 Department of Animal Biology, Cheikh Anta DIOP University, Dakar, Senegal, 6 Malaria Control Program, Ministry of Public Health, Luanda, Angola, 7 UMR145 « VIH/SIDA et maladies associées », Institut de Recherche pour le Développement, Montpellier, France

## Abstract

To optimize malaria control, WHO has prioritised the need for new indicators to evaluate the efficacy of malaria vector control strategies. The gSG6-P1 peptide from gSG6 protein of *Anopheles gambiae* salivary glands was previously designed as a specific salivary sequence of malaria vector species. It was shown that the quantification of human antibody (Ab) responses to *Anopheles* salivary proteins in general and especially to the gSG6-P1 peptide was a pertinent biomarker of human exposure to *Anopheles*. The present objective was to validate this indicator in the evaluation of the efficacy of Insecticide Treated Nets (ITNs). A longitudinal evaluation, including parasitological, entomological and immunological assessments, was conducted on children and adults from a malaria-endemic area before and after the introduction of ITNs. Significant decrease of anti-gSG6-P1 IgG response was observed just after the efficient ITNs use. Interestingly, specific IgG Ab level was especially pertinent to evaluate a short-time period of ITNs efficacy and at individual level. However, specific IgG rose back up within four months as correct ITN use waned. IgG responses to one salivary peptide could constitute a reliable biomarker for the evaluation of ITN efficacy, at short- and long-term use, and provide a valuable tool in malaria vector control based on a real measurement of human-vector contact.

**Citation:** Drame PM, Poinsignon A, Besnard P, Cornelie S, Le Mire J, et al. (2010) Human Antibody Responses to the *Anopheles* Salivary gSG6-P1 Peptide: A Novel Tool for Evaluating the Efficacy of ITNs in Malaria Vector Control. PLoS ONE 5(12): e15596. doi:10.1371/journal.pone.0015596

**Editor:** Etienne Joly, Université de Toulouse, France

**Received:** August 17, 2010; **Accepted:** November 15, 2010; **Published:** December 14, 2010

**Copyright:** © 2010 Drame et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Funding:** Papa-Makhtar Drame was supported by a fellowship provided by the IRD (Département Soutien et Formation). Anne Poinsignon was supported by fellowships provided by the Research Ministry of France then by François Lacoste (Fonds Inkermann). This work was supported by ACERGY-SONAMET Company (Malaria Control Program), the French Ministry of Defense (Délégation Générale pour l'Armement) and the IRD (Département Expertise et Valorisation). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing Interests:** Funding received from ACERGY-SONAMET (a commercial company), as from the other funders, is not accompanied by any other relevant declarations relating to employment, consultancy, patents, products in development or marketed products and does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

* E-mail: papa-makhtar.drame@ird.fr

## Introduction

Vector-borne diseases constitute major public health problems in developing countries and currently present high risks of re-emergence in the developed world. Developing tools for disease control is a priority, especially new anti-vector strategies to prevent transmission. Among these diseases, malaria represents the greatest worldwide problem, causing at least 400 million acute cases every year with around 1 million deaths [1]. Most of these deaths occur in children from Sub-Saharan Africa and are due to *Plasmodium falciparum* species. In these areas, the *Anopheles gambiae* complex is the major vector [2].

Preventive methods are used against both parasite (chemoprophylaxis) and vector (insecticide-based control). Among anti-vector strategies, Insecticide Treated Nets (ITNs) are currently the most efficient strategy for reducing human exposure to the vector, *Plasmodium* transmission and malaria morbidity [3,4,5]. When

correctly used, even moderate coverage of populations (35–65%) can afford substantial community benefit as well as personal protection [6,7,8,9]. Moreover, implementation of Long-Lasting Insecticidal Nets (LLINs) represents an achievable means of rapidly improving ITN coverage [10]. The evaluation of ITN efficacy is currently based on entomological methods (entomological inoculation rate, *Anopheles* abundance and aggressivity) and, in humans, on parasitological and clinical assessments [4,11,12]. The reference WHO method for phase 3 evaluation of ITNs efficacy is based on the measurement of *P. falciparum* density in human populations [12]. However, these methods present limitations when it comes to large-scale field studies, especially when transmission rates and exposure levels are low (dry season, high altitude, urban settings or after vector control). Moreover, evaluating *Plasmodium* density in human individuals is labour-intensive by active follow-up of populations. Entomological methods are mainly applicable at the population/area level and

<figure><img src="image_2.png" /></figure>
do not give a measure of the heterogeneity of individual exposure
in a given area. Human-landing catch measurements (adult
volunteers) are currently the reference method for evaluating
individual human exposure but it raises ethical questions and it
may not be relevant to children [13]. In addition, as exposure
levels drop with ITNs use, all these monitoring methods become
less effective for evaluation by National Malaria Control Programs
[14].

In order to improve vector control, much effort is being devoted
to developing new indicators to evaluate, at the individual level,
the efficacy of control strategies. One promising approach is based
on the idea that exposure to arthropod vector bites can be assessed
by directly measuring real human-vector contact. Indeed, the
human antibody (Ab) response to arthropod salivary proteins
could give a measure of exposure to vector bites [15,16]. At the
time of biting, the female mosquito injects saliva containing
bioactive molecules which facilitate the blood meal and some of
these are antigenic [17,18,19]. Human Ab responses to the saliva
of various vectors, e.g. *Triatoma* (Chagas' disease) [20], *Ixodes* ticks
(Borrelia) [21,22], phlebotomes (Leishmania) [23,24] and *Glossina*
(African trypanosomiasis) [25] have been reported as reliable
immunological markers for vector exposure. For mosquitoes, anti-
saliva Ab responses has been related to exposure to *Culex*, *Aedes*
[26,27,28,29], *An. gambiae* [30], *An. dirus* [31] and *An. darlingi* [32].
Recently, it has been shown that the Ab response to whole *An.
gambiae* saliva could be a useful biomarker for evaluating ITN
efficacy in phase 3 studies [33]. Even if this concept may appear to
be valid, whole vector saliva could not be used, as pertinent
indicator, because of i) potential cross-reactivity with salivary
epitopes of other hematophagous arthropods; ii) lack of reproduc-
ibility between saliva batches and iii) the adequate production
needed for large-scale studies. For use as a biomarker for *Anopheles*
exposure, the specific [34,35] and antigenic [36] *Anopheles* SG6
salivary protein has been identified as an encouraging candidate
[37]. The gSG6 protein, first identified in *An. gambiae* [38], was
further reported as being highly conserved among *Anopheles* species
[39,40]. To optimize this biomarker candidate, peptide design has
recently been applied using bioinformatics approach to generate
five *Anopheles* specific peptides (gSG6-P1 to gSG6-P5). Among
them, only the gSG6-P1 peptide was validated as a specific
biomarker of exposure to malaria vectors. Indeed, the level of
human IgG to gSG6-P1 peptide evaluated the level of exposure to
*An. gambiae* bites in human populations from a rural area in
Senegal [37]. IgG response to this peptide has been also confirmed
as biomarker for evaluating very low-level exposure to *An. gambiae*
[41] as well as *An. funestus* (the second major malaria vector in
Africa) [42]. In addition, the gSG6-P1 peptide can be easily
synthesized in large quantity and offers an efficient solution to the
lack of reproducibility observed with whole salivary extracts [37].

The present study addresses a potentially important application
of such biomarker as a tool to evaluate the efficacy of ITN-based
strategies. Human IgG responses to the gSG6-P1 peptide were
evaluated before and after the introduction of ITNs in individuals
living in a malaria-endemic area. The results focused on the
biomarker's potential for evaluating short-term ITN efficacy.

**Materials and Methods**

Ethics Statement

This study was conducted in accordance with the Edinburgh
revision of the Helsinki Declaration, and was approved by the
National Malaria Control Program of the Ministry of Health of
Angola (October 17th 2008), the only one Ethical authority in
2008 for approving studies on malaria research in Angola. Written

informed consent (signed by the head of each household) was
obtained for all individuals enrolled in the study, by the
SONAMET Malaria Control Program (MCP) which supervise/
control malaria infection of all workers for SONAMET and their
family. This consent procedure was regularly approved by
SONAMET workers, benefiting from several malaria studies/
survey by MCP, and was approved by the involved Ethical
authority in Angola.

Study population

This study was conducted in Lobito, a coastal city of Western
Angola, from March 2005 to January 2007. The site is in the
tropical savannah with a rainy season from October to May, with
approximately 600-700 millimetres of rain per year. The duration
of the malaria transmission season varies between 7 and 12
months with a peak between March and May. The major malaria
vector is here the *An. gambiae* s.l. complex [43,44].

The population has been previously described [33]. Briefly, all
workers of the SONAMET company lived in 250 households.
Residents of these households were followed in the SONAMET
in-patient clinic. In 2004, the presence of malaria parasite was
diagnosed in sixty (60) households (positive, at least, in one
member of household) by SONAMET Malaria Control Program
(MCP). Twenty-one (21) of these 60 households were then
randomly selected for the present study. These families lived in
the Bella Vista district where exposure to *An. gambiae* s.l bites was
relatively uniform. All residents of the 21 selected households,
corresponding to two hundred and fifty (250) individuals (children
and adults), were included for longitudinal follow-up, with
evaluations every 6 weeks from March 2005 to January 2006
(before ITNs), and from April 2006 to January 2007 (after ITNs).
The families were given LLIN treated with deltamethrin
(Permanet®) in February 2006 (according to the number of rooms
and beds per household). At each visit, samples were collected
from each individual: thick blood smears for parasitological
measurements and dried blood spots (on filter paper) for
immunological analysis. Parasite density (parasitaemia) was
calculated as the number of *P. falciparum* parasites per microliter
of blood; mean parasitaemia values (x+1) were calculated [33].
Immunological tests were performed in a sub-sample (n = 105,
children and adults) of the whole study population (n = 230).
Individuals who missed more than 2 of the 14 visits (travel, illness,
change of household,...) were excluded from the immunological
assessments as described previously [33]. Filter papers were kept at
+4°C in Silicagel before testing.

Entomological analysis and survey of ITN use

Mosquitoes were collected every six weeks during the study
period at 6 reference households, representative of the studied
area. *An. gambiae* density was evaluated using capture by CDC
miniature light traps, deployed from 19:00 hours to 07:00 hours
for two consecutive nights. PCR was used to confirm species to
yield an estimate of the number of *An. gambiae* /trap/night.

After the introduction of ITNs, their use by individuals and their
quality were inspected, the night before each blood sampling, by
the MCP team. Information were then collected for all studied
individuals by questionnaires, covering: i) the number of installed
ITNs, ii) the number of exchanged ITNs and iii) the number of
damaged ITNs (hole, torn, etc.), as previously described [33].

Salivary gSG6-P1 peptide

The gSG6-P1 peptide was designed as previously described
[37], synthesized and purified (>95%) by Genepep SA (St-
Clément de Rivière, France). All peptide batches were shipped in

<figure><img src="image_3.png" /></figure>
lyophilized form and then suspended in 0.22 µm ultra-filtered water and frozen at -80°C until their use.

## Evaluation of human IgG antibody levels (ELISA)

Standardized dried blood spots (1 cm diameter) were eluted by incubation in 300 µl of phosphate buffered saline (PBS-Tween-0.1%) at 4°C for 24 hours [33]. ELISAs were carried out on eluates to assay IgG to the gSG6-P1 antigen. Maxisorp plates (Nunc, Roskilde, Denmark) were coated with gSG6-P1 (20 µg/mL) in PBS. Each eluate was then incubated (duplicate) at 4°C overnight at a 1/20 dilution (PBS-Tween-1%). This optimal dilution had been determined in preliminary experiments. Mouse biotinylated Ab against human IgG (BD Pharmingen, San Diego, CA, USA) was incubated at a 1/2000 dilution and peroxidase-conjugated streptavidin (Amersham, Les Ulis, France) was then added (1/2000) Colorimetric development was carried out using ABTS (2,2'-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) diammonium; Sigma, St Louis, MO) in 50 mM citrate buffer (pH 4) containing 0.003% H₂O₂. Optical Density (OD) was measured at 405 nm. In parallel, each test sample was assessed in a blank well containing no gSG6-P1 antigen (ODn) to measure non-specific reactions. Individual results were expressed as the ΔOD value: ΔOD = ODx-ODn, where ODx represents the mean of individual OD in both antigen wells. Specific anti-gSG6-P1 IgG levels were also assayed in non-*Anopheles* exposed individuals (n = 14 - neg; North of France) in order to quantify the non-specific background Ab level and to calculate the specific immune response threshold (TR): TR = mean (ΔOD<sub>neg</sub>) + 3SD = 0.204, i.e. an exposed individual was classified as a responder if its ΔOD > 0.204.

## Statistical analysis

All data were analyzed with GraphPad Prism4 software® (San Diego, CA, USA). After checking the non-Gaussian distribution, the non-parametric Mann-Whitney U-test was used to compare Ab levels between two independent groups, the Wilcoxon matched pairs test was used for comparison between two paired groups, the non-parametric Kruskal-Wallis test for comparisons between more than two groups and the Chi-2 test to compare two proportions. All differences were considered significant at P<0.05.

## Results

### Anti-gSG6-P1 IgG responses before and after ITN use

The evolution of the percentage of “immune responders” (Fig. 1A) and the levels of anti-gSG6-P1 IgG Abs (Fig. 1B) were evaluated before (March 2005 to January 2006) and after (April 2006 to January 2007) the installation of ITNs (February 2006).

Before the introduction of ITNs, the percentage of anti-gSG6-P1 IgG responders appeared relatively constant according to months (64 to 81%) (Fig. 1A). Within two months of the ITNs introduction (April, 2006), the percentage of immune responders had decreased significantly (P<0.0001) down to 5% from 64% in January 2006. In parallel, the density of *An. gambiae* (classical entomological methods) and the intensity of *P. falciparum* infection (mean parasitaemia) are shown for the same period (Fig. 1A). Whereas peak of parasitaemia was observed in May 2005, only low-level infection was observed after the introduction of ITNs (2006). In particular, parasitaemia was lower in April 2006 compared to May 2005, which suggests that ITNs were effective, as previously described [33]. In addition, the densities of *An. gambiae* peaked in May 2005 with a small rise from October 2005. After the introduction of ITNs, a similar peak was observed (April to July 2006), indicating that entomological exposure was comparable in 2006 to that in 2005 in the studied area.

A similar trend was observed in the percentage of immune responders (Fig. 1A) and specific IgG Ab levels (Fig. 1B). In spite of inter-individual heterogeneity, the anti-gSG6-P1 IgG level was strong and relatively stable before the introduction of ITNs (Fig. 1B) although small but significant seasonal variations were seen in median specific IgG levels (P<0.0001). The major peak of Ab level was detected in May 2005, as observed for both entomological *An. gambiae* exposure and *P. falciparum* density (Fig. 1A). Immediately after the introduction of ITNs, a marked decrease in anti-gSG6-P1 IgG was observed between April and October 2006. Specific IgG levels were markedly lower in 2006 than in 2005 in June-July (P<0.001), August (P<0.001) and October (P<0.001). Interestingly, the greatest drop in Ab levels was observed in all individuals just two months after ITN introduction in April 2006, the month corresponding to the peak anti-IgG level before ITNs (P<0.001 for April 2006 compared to March-April 2005 and P<0.0001 for April 2006 compared to May 2005). The still positive Ab responses to gSG6-P1 (Fig. 1B) just after ITN installation (April and June-July 2006) were almost exclusively observed in older children and adults: the 5 responders in April 2006 and 15/16 responders in June-July 2006 were from 7-14 and >14 years age groups.

The percentage of specific immune responders (Fig. 1A) and the level of anti-gSG6-P1 IgG Ab (Fig. 1B) increased significantly in October 2006 compared to August 2006 (P<0.001) to reach a high level in December 2006 and January 2007, similar to that observed before ITN introduction in 2005. Interestingly, the major results of the ITN survey (loss rate and correct/damaged-usage rate) evaluated by two horizontal surveys (April and June/July 2006) indicated that: i) after just 4 months (June/July), only 63% of the ITNs were installed, and ii) only 53% were being used correctly (in use and undamaged), as previously described in detail [33].

### Individual IgG responses to gSG6-P1 and short-term ITN efficacy

Previous analyses have been performed at the population level. To investigate changes in anti-gSG6-P1 IgG levels as a biomarker at the individual level and immediately after ITN installation, IgG level was individually evaluated just before (January 2006) and just after (April 2006) the introduction of ITNs (Figure 2). For almost all individuals, specific IgG levels decreased significantly in April 2006 compared to January 2006 (P<0.0001 – Fig. 2A). Other patterns - an increase or no change - were observed in only 5% of individuals. In addition, a new indicator of the January/April difference in IgG level was defined (ΔOD<sub>ITNs</sub> = ΔOD<sub>April</sub> - ΔOD<sub>January</sub>) to assess individual trends (positive, negative or unchanged) between both months (Fig. 2B). After applying a threshold for positive IgG response (ΔOD>0.204), 60% of individuals showed a decrease (ΔOD<sub>ITNs</sub> <-0.204) whereas only 1% showed an increase (ΔOD<sub>ITNs</sub> >0.204) and 39% (39/100) showed no significant change (-0.204<ΔOD<sub>ITNs</sub> <+0.204).

### Changes in anti-gSG6-P1 IgG responses according to age

The changes in anti-gSG6-P1 IgG level was analysed (Figure 3) according to age of individuals in 3 age groups: 0–6 (n = 49), 7–14 (n = 34) and >14 years (n = 25). Similar trends in all age groups were observed between 2005 and 2006 (Fig. 3A), in particular a substantial drop in April 2006 after the introduction of ITNs. Nevertheless, there are significant age-related differences in some months, e.g. during the peaks before ITN introduction (May and October 2005) and in June-July 2006, where anti-gSG6-P1 IgG levels were significantly higher in older-children and adults (>14

<figure><img src="image_4.png" /></figure>
A

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /></figure>

**Figure 1.** IgG Ab responses to gSG6-P1 before and after ITN use. The percentage (%) of anti-gSG6-P1 IgG immune responders (thick-dotted line) in the "immunological" sub-population (n = 105), before (2005) and after (2006 and January 2007) the installation of ITNs (A). These results are presented together with the intensity of *P. falciparum* infection (mean parasitaemia – fine-dotted line) measured in the same population and the

<figure><img src="image_7.png" /></figure>
mean of number of *An. gambiae* (solid line) in the studied area (A). Entomological data (number of *An. gambiae*) were not available in December 2006 and January 2007 (the two last months of the study). This arrow indicates the installation of Insecticide Treated Nets (ITNs) in February 2006. Individual anti-gSG6-P1 IgG levels (ΔOD) are presented before (2005) and after (2006) the installation of ITNs (B). Bars indicate the median value for each studied month. Statistically significant differences between months are indicated.
doi:10.1371/journal.pone.0015596.g001

years) than in younger children. Before ITN installation (2005), seasonal variations were most marked in the >14 years age group.

The relationship between short-term ITN efficacy and anti-
gSG6-P1 IgG levels was analysed according to age group (Figure 3
B, C and D). A marked decrease was observed between January
and April 2006 in all age groups. In particular, IgG level decreased
clearly in all young children from 0–6 years (Fig. 3B) whereas some
individuals of 7–14 years (Fig. 3C) and >14 years (Fig. 3D)
presented increased or unchanged Ab responses.

Discussion

This study focused on the application of a new biomarker based
on evaluation of the human Ab response to an *Anopheles* salivary
peptide, as an indicator for evaluating the efficacy of malaria
vector control. In this study, a step-by-step approach was adopted
to validate the anti-gSG6-P1 IgG response as a biomarker for ITN
efficacy from the whole population level to the individual level
with a view to identifying a potential tool for evaluation
immediately after the introduction of ITNs.

Firstly, the present study and previous data [33] indicated that
the correct use of deltamethrin ITNs was followed by a
considerable decrease of *P. falciparum* parasitaemia, the current
WHO criterion for vector control efficacy [13]. It indicated that
ITNs installation was short-term effective in the studied popula-
tion. In the whole population, the percentage of immune
responders and the level of anti-gSG6-P1 IgG Ab in most
individuals decreased considerably just after ITNs introduction
(April-October 2006). The drop was particularly marked in April-
August 2006, corresponding to the peak of *An. gambiae* exposure.
Interestingly, the entomological data indicated that this season-
dependent peak was similar before (2005) and after (2006) ITN
use. This suggests that ITN installation had no impact on *An.
gambiae* density, probably because of the low percentage of the
overall population covered in the studied area [45]. It indicated
also that the drop of anti-gSG6-P1 IgG response was associated

with correct ITN use and not due to low *Anopheles* density. As
described [37,41], Ab responses to gSG6-P1-peptide could
accurately reflect human-vector contact. A marked drop after
ITN installation was observed in all age groups studied (<=7 years,
7-14 years, and >14 years) suggesting that this biomarker is
relevant for ITN evaluation in all age groups. Taken together,
these results confirm that - as previously shown with whole *An.
gambiae* saliva [33] - the estimation of human Ab responses to
*Anopheles* salivary proteins could provide a reliable biomarker for
evaluating the efficacy of malaria vector control. It validates the
general approach to use Ab to salivary antigens for evaluating the
quantitative human exposure to mosquito bites.

The specific gSG6-P1 peptide is more reliable than whole saliva which may show cross-reactivity with salivary epitopes from other arthropods and which could skew and/or overestimated described effects [37]. Interestingly, compared to IgG responses to whole saliva [33], the percentage of responders and the levels of anti-gSG6P1 IgG showed less variability before ITN installation (2005) and especially remained high in January 2006, one month before ITN installation. The use of gSG6-P1 peptide increases the sensitivity and the specificity of such type of biomarker. It strengthens such effective peptide biomarker for the evaluation of ITN efficacy at a large scale. The smallness of seasonal variations could preclude the need for demanding longitudinal evaluation as needed for parasitaemia indicator (full year before introduction and especially during the peak of transmission). One measurement of anti-gSG6-P1 Ab just before ITN installation could provide a representative picture of the specific Ab response, i. e. exposure level to *Anopheles*, prior to ITN introduction.

The gSG6 peptides were designed on the basis of the *An. gambiae* s.s. sequence, the only *Anopheles* genome completely available [46]. In the studied area, the *An. gambiae* s.l. complex, especially *An. gambiae* s. s., is the main vector of *P. falciparum* as previously described [44]. However other *Anopheles* species, such as *An. arabiensis* and *An. melas*, were locally caught in the study area and had been reported to participate to malaria transmission as

<figure><img src="image_8.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_9.png" /></figure>

**Figure 2.** IgG response to gSG6-P1 as biomarker for short-term ITN efficacy. Changes in individual IgG levels (ΔOD) are presented between "just before" (January 2006) and "just after" (April 2006) ITN introduction (n = 105; children and adults) (A). The arrow indicates the installation of Insecticide Treated Nets (ITNs) in February 2006. Individual IgG level changes from January (before) to April are presented (B) by individual ΔOD<sub>ITNs</sub>, value (ΔOD<sub>ITNs</sub> = ΔOD<sub>April06</sub> - ΔOD<sub>January06</sub>). The threshold of specific IgG responders (TR = 0.204) is indicated (dotted line). Significant positive (ΔOD>0.204) or negative (ΔOD<−0.204) changes are therefore individually presented.
doi:10.1371/journal.pone.0015596.g002

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 3.** Changes in anti-gSG6-P1 IgG levels before and after the introduction of ITNs according to age group. Median anti-gSG6-P1 IgG levels in 2005–2006 are presented according to three age groups (A): 0–6 years-old (solid line; n = 49), 7–14 years-old (thick-dotted line; n = 34) and >14 years-old (fine-dotted line; n = 25). Individual, short-term changes in specific IgG levels from January (just before) to April (just after ITN installation), are presented according to age group (0–6 years = B; 7–14 years = C and >14 years = D). The arrow indicates the installation of Insecticide Treated Nets (ITNs) in February 2006. Statistically significant differences between age groups are indicated for respective months (*: P<0.05; **: P<0.0001).</figcaption></figure>

doi:10.1371/journal.pone.0015596.g003

secondary vectors [44]. The exposure of individuals to these "secondary" *Anopheles* species have been probably evaluated using gSG6-P1 peptide which presents a very stable domain of the gSG6 *Anopheles* salivary protein [37] and monoacids sequences highly conserved among *Anopheles* species [40]. For example, it has been shown that gSG6-P1 peptide shares 82% and 91% identity with *An. stephensi* and *An. funestus* [37] and that IgG response to this peptide was also biomarker of *An. funestus* exposure [42]. All these data tends to support the idea that gSG6-P1 can be used to evaluate the exposure to major *Anopheles* species known to be vector of malaria.

After an analysis at the whole population level, an ideal biomarker has to be used i) at the individual level and ii) to evaluate short-term ITN efficacy. The individual results showed

that few individuals presented a positive anti-gSG6-P1 IgG response just after ITN installation (from April to August 2006) and that most of these were older children or adults (>14 years). It cannot be ruled out that these older individuals may stay outside the house until late in the evening when *Anopheles* starts biting again so they therefore derive less protection from ITNs than young children who go to sleep earlier in the evening. Individual specific IgG response between January (before) and April 2006 (2 months after ITNs) showed a considerable decrease in all age groups. This rapid decrease after correct ITN usage appears to be a special property of the anti-saliva Ab response which is short-lived in the absence of ongoing antigenic stimulation, at all ages [30,36,47]. The response does not seem to build up but wanes rapidly, when exposure failed. This property represents a major

<figure><img src="image_13.png" /></figure>
strength for use such salivary biomarker for evaluating the human
exposure to mosquito bites, especially for efficacy of vector control.
In addition, using a response threshold (ΔOD = 0.204) combined
with ΔOD<sub>ITNs</sub> - the difference between April (after ITNs) and
January 2006 (before) - makes possible the use of this operational
biomarker large-scale [40]. If the ΔOD<sub>ITNs</sub> value is between -
0.204 and +0.204, no clear difference can be defined. In contrast,
if the individual ΔOD<sub>ITNs</sub> value < -0.204, it could be concluded
with a high level of confidence that this individual is benefiting
from ITN installation. The ΔOD<sub>ITNs</sub> parameter could therefore
provide a measure of ITN efficacy at the individual level. An
individual biomarker would also be relevant at the large-scale,
operational level in the field, e.g. in National Malaria Control
Programs (NMCP), since it is pertinent for the evaluation of short-
term ITN efficacy, as demonstrated in the study (April, 2 months
after ITN installation). Short-term and individual changes in Ab
responses to gSG6-P1 provide a superior biomarker to *P. falciparum*
parasitaemia, especially in a context of low malaria transmission
and by evaluation of ITN efficacy, only in small sub-sample
(around N = 100) from populations receiving ITN (parasitaemia
needing a larger population of the order of N = 1000). We have
previously shown the high sensitivity and specificity of the gSG6-
P1 Ab response make it ideal for the evaluation of low-level
exposure to *An. gambiae* [41] which is relevant in areas where
exposure/transmission is being curtailed by NMCP efforts. In
addition, its evaluation can be performed from blood spots on filter
paper, which is easy and operational to be integrated in NMCP
strategies.

The ITN survey, as previously reported in the studied area [33],
indicated that only 53% of ITNs were being correctly used and
were undamaged after just 4–5 months of use (June/July 2006), as
also observed in several other endemic areas [14]. Because of the
high sensitivity of gSG6-P1, we were able to observe an increase in

specific IgG levels from October 2006 (8 months after ITN
installation). The strong decrease of anti-gSG6-P1 IgG response
was therefore transient after ITN installation and the biomarker
reappeared as correct ITN usage waned and individuals were once
re-exposed to *An. gambiae*. These results point up the relevance of
this salivary biomarker for the evaluation of short-term efficacy as
well as longer-term monitoring.

The results suggest that this salivary biomarker for *Anopheles*
exposure could constitute an efficient, reliable and new tool for
evaluating the efficacy of malaria vector control, at both
population and individual levels. Further studies are however
needed to confirm this in other areas and for different vector
control strategies. Finally, this first approach could be similarly
applied to vector-control strategies for other mosquito-borne
diseases such as emergent arbovirus diseases.

Acknowledgments

The authors gratefully acknowledge the population of Bella Vista area (Lobito, Angola) for their participation in this study; all the technical personal of the MCP team, especially Maria Chilombo (SONAMET Lobito); and Cheikh Sow, Amady Ndiaye and Souleymane Doucoure from the IRD-EPIVECT group, for their technical support. We gratefully acknowledge Dr Jean-François Foucher ACERGY Company) for his helpful contribution, Dr Karine Puget (Genepep SA) for helpful advices on peptide synthesize and Dr Anthony Molloy for the English review of the manuscript.

**Author Contributions**

Conceived and designed the experiments: PMD AP PB FS PC FR.
Performed the experiments: PMD AP JCT VF MD. Analyzed the data:
PMD PB VF PC FR. Contributed reagents/materials/analysis tools: PMD
AP PB SC JM JCT VF MD MS PC FR. Wrote the paper: PMD AP SC
MS FS FF PC FR. Logistical help in the field: FF PB JCT.

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