First screening of Aedes albopictus immunogenic salivary proteins

Authors


Correspondence: Souleymane Doucoure, URMITE 198 Campus IRD-UCAD, route des Pères Maristes, BP 1386, CP 18524, Dakar, Sénégal. Tel: + 221 33,849 35 22; fax: + 221 33,832 43 07; e-mail: souleymane.doucoure@ird.fr

Abstract

Study of the human antibody (Ab) response to Aedes salivary proteins can provide new biomarkers to evaluate human exposure to vector bites. The identification of genus- and/or species-specific proteins is necessary to improve the accuracy of biomarkers. We analysed Aedes albopictus immunogenic salivary proteins by 2D immunoproteomic technology and compared the profiles according to human individual exposure to Ae. albopictus or Ae. aegypti bites. Strong antigenicity to Ae. albopictus salivary proteins was detected in all individuals whatever the nature of Aedes exposure. Amongst these antigenic proteins, 68% are involved in blood feeding, including D7 protein family, adenosine deaminase, serpin and apyrase. This study provides an insight into the repertoire of Ae. albopictus immunogenic salivary proteins for the first time.

Introduction

The last 20 years have been marked by rapid worldwide invasion of the Aedes albopictus mosquito (Benedict et al., 2007), a competent vector of at least 23 arboviruses including dengue, yellow fever and chikungunya (Gratz, 2004). One major consequence of this expansion was the recent large-scale chikungunya outbreaks in the Indian Ocean area and in Italy (Flahault, 2007; Rezza et al., 2007). This situation has prompted the need to reinforce surveillance for Ae. albopictus. The evaluation of human exposure to Ae. albopictus bites is a useful tool for monitoring vector control efficacy and assessing the risk of disease transmission. This evaluation is currently based on entomological methods, which are essentially based on identification of breeding sites in order to determine the House and Breteau entomological indices resulting from the enumeration of Aedes immature stages (Focks, 2003). However, these indices represent indirect methods for measuring the human exposure to vector bites and sometimes are inaccurate for assessing the risks of arbovirus transmission (Sulaiman et al., 1996; Sanchez et al., 2010). To overcome these limitations, individual exposure can be measured by the human landing catches technique, but this method raises ethical concerns (Focks, 2003). The current entomological methods are time-consuming and costly and require a large, qualified staff to give robust results in large-scale studies. For these reasons, much effort is being devoted to developing new simple, rapid and complementary tools to evaluate human exposure to Ae. albopictus bites. New tools should be easy to implement and sensitive enough to detect human exposure to Ae. albopictus bites both in endemic and re-emerging areas of arbovirus transmission/risks.

The study of human−mosquito interactions through vector salivary proteins has provided a new direction to develop such complementary tools. Indeed, during blood feeding, arthropods inject a mixture of salivary proteins into the host's skin to promote blood intake. The human antibody (Ab) response against these salivary components has been reported to be a useful epidemiological biomarker of exposure to arthropod bites. This approach has been applied for a wide range of hematophagous arthropods: mosquitoes (Remoue et al., 2006, 2007; Poinsignon et al., 2009, 2010), ticks (Schwartz et al., 1990; Schwarz et al., 2009a, b) and sand flies (Souza et al., 2010; Teixeira et al., 2010). However, some salivary proteins are shared between species or even genera, increasing the occurrence of cross-reactivity and leading to the overestimation of human exposure to a given arthropod bite. In addition, the composition of saliva can be influenced by several physiological factors such as age or diet (Prates et al., 2008), which restrict its use in large-scale studies.

Measuring the Ab response against specific recombinant salivary proteins instead of total saliva is an alternative method that can be used to evaluate specific human exposure to vector bites. The Ab response to specific peptides and/or recombinant proteins has already been used to evaluate exposure to Anopheles and Triatomines bites (Poinsignon et al., 2008, 2009; Schwarz et al., 2009a; Teixeira et al., 2010). For example, the salivary peptide gSG6-P1 and the recombinant salivary protein rTisP14.6 are highly effective to detect a specific Ab response in human populations with low levels of exposure to Anopheles gambiae and triatomine bugs, respectively (Poinsignon et al., 2009; Schwarz et al., 2009a). Regarding the human response to Aedes bites, early studies investigated salivary allergens involved in anaphylaxis or that caused anaphylaxis. It has been shown that strong immunoglobulin G 4 (IgG4) and immunoglobulin E (IgE) responses were associated with exposure to Aedes bites in Finland (Brummer-Korvenkontio et al., 1994; Brummer-Korvenkontio et al., 1997). Another study demonstrated that people transiently exposed to Ae. aegypti bites developed a strong IgG response to Ae. aegypti salivary antigens (Orlandi-Pradines et al., 2007). Recently, a study conducted in an urban area showed that the IgG response to Ae. aegypti saliva was positively associated with standardized entomological indicators (Doucoure et al., 2012b). In addition, weak cross-reactivity between the Ae. albopictus and Ae. aegypti IgG response has been noted, suggesting the possibility that a species-specific biomarker could be developed (Doucoure et al., 2012a).

In the current study, the main objectives were (1) to identify Ae. albopictus immunogenic salivary proteins in humans exposed to Aedes bites and (2) to provide a repertoire of antigens that are potential candidates for a specific biomarker for exposure. Proteomic tools were used to detect Ae. albopictus salivary antigens in individuals from Reunion Island, where Ae. albopictus is the only anthropophilic Aedes species. These immunogenic profiles were compared with those obtained from individuals exposed only to Ae. aegypti bites (Bolivia). Mass spectrometry was used to identify specific Ae. albopictus immunogenic salivary proteins.

Results

Aedes albopictus immunogenic salivary proteins

The antigenicity of Ae. albopictus salivary proteins was evaluated by 2D-immunoblotting. Most antigenic proteins displayed an intense signal that induced considerable overlaps between them. These antigenic proteins were distributed between pH 3 and 11 and between 12 and 70 kDa (Fig. 1, panel 1). Four sets of proteins were recognized by almost all individuals. To the acidic side of the gel, two strings of immunogenic proteins were detected at 25–40 kDa (set 1) and at 60–70 kDa (set 2), and to the basic side, two other strings at 20–40 kDa (set 3) and 60–65 kDa (set 4) were detected. In the middle of the gel, two additional sets of immunogenic proteins were observed at 25–34 kDa and 40–50 kDa. Two-dimensional blots were also carried out with sera from Bolivian individuals presenting a high response to Ae. aegypti saliva (Fig. 1, panel 2). In this case, the same major sets of immunogenic proteins were recognized. For each geographical area, strong individual variability was observed both in the intensity of the signals and in the number of spots detected. This heterogeneity within each group made it impossible to establish significant differences between the areas of exposure after SameSpots software analysis (Nonlinear Dynamic, URL http://www.nonlinear.com/products/progenesis/samespots/) (Fig. 1 and data not shown). The image analysis performed by SameSpots software revealed no significant difference between individuals and the type of exposure to Ae. albopictus or Ae. aegypti (Fig. 1 and data not shown).

Figure 1.

Profiles of Aedes albopictus immunogenic salivary proteins. Immunoblots were performed using Ae. albopictus salivary gland extracts. Individual serum samples were used to detect Ae. albopictus antigenic salivary proteins. Three individuals for Reunion Island (panel 1) and Bolivia (panel 2) are presented and were representative of the six individuals tested. The limits of isoelectric points (Pi) are indicated at the top of the figure, and molecular weight (MW) (kDa) is represented on the left side of the immunoblots.

Characterization of Ae. albopictus immuno-sialome

The second step was to characterize Ae. albopictus immunogenic salivary proteins using both Matrix-Assisted Laser Desorption/Ionisation-Time-Of-Flight (Maldi-TOF) and Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) mass spectrometry. A blue-stained gel was prepared and aligned with the immunoblots (Fig. 2). Seventy-six spots were selected for mass spectrometry analysis (Tables 1, 2); 64% (49/76) of these proteins were secreted (Tables 1, 2) and 36% were intracellular (Tables S1, S2). Amongst the secreted salivary proteins, 61% (30/49) were involved in blood feeding and mainly related to the D7 proteins family, adenosine deaminase (ADA), serpin and apyrase. The remaining 39% of salivary secreted proteins concerned mainly proteins of the 34 and 62 kDa families, with unknown function. In this set of 49 salivary secreted proteins, 46 were identified as Ae. albopictus proteins and three were identified by homology with Ae.aegypti sialome data but were not found in the Ae. albopictus transcriptome. The 2D approach shows that most of the salivary secreted proteins identified exhibited multiple isoforms. Indeed, we observed some spots on the same row (quite similar molecular weight), with the same identification (same accession number) and with different apparent isoelectric points (pIs) on the gel, so these are probably isoforms of the same protein (Fig. 2, eg spot nos 30, 31, 32, 33, 34 and 63).

Figure 2.

Profile of soluble salivary proteins of Aedes albopictus salivary gland extract. The salivary gland extracts were run on immobiline drystrips (pH 3–11) for the first dimension and then on 10.5–14% gel for the second dimension as described in the Experimental procedures. The gel was stained with Colloidal blue overnight. The number represent the Ae. albopictus antigenic salivary proteins identified by mass spectrometry after alignment of this gel with immunoblots using SameSpots software. Labelled spots were selected on this gel and identified by mass spectrometry. MW, molecular weight.

Table 1. Aedes albopictus immunogenic salivary secreted proteins identified by Maldi-ToF. Protein number corresponds to the position in 2D gel, Fig. 2
Family or sequence nameProtein numberFunctionAccession numberNominal mass (kDa)PiSequence coverage %Mascot score
  1. OS, Organism Name; PE, Protein Existence; Pi, isoelectric point; SV, Sequence Version.
  2. SwissProt or TrEMBL databases were used for identification.
Enzymes       
Putative adenosine deaminase OS = Aedes albopictus1Blood feedingQ5MIX2_AEDAL616.27853
Putative adenosine deaminase OS = Aedes albopictus4Blood feedingQ5MIX2_AEDAL616.2714113
Putative adenosine deaminase OS = Aedes albopictus PE = 2 SV = 17Blood feedingQ5MIX2_AEDAL616.271291
Putative adenosine deaminase OS = Aedes albopictus8Blood feedingQ5MIX2_AEDAL616.2723138
Salivary apyrase OS = Aedes albopictus14Blood feedingE0D877_AEDAL638.171454
Salivary apyrase OS = Aedes albopictus15Blood feedingE0D877_AEDAL638.1726121
D7protein family       
Long form D7 salivary protein D7l2 OS = Aedes albopictus3Blood feedingQ5MIW6_AEDAL378.643140
Short form D7 salivary proteinSD7-1 OS = Aedes albopictus10Blood feedingQ5MIX1_AEDAL189.13172
D7 salivary protein d7s6 OS = Aedes albopictus11Blood feedingQ5MIP1_AEDAL219.2349132
D7 salivary protein d7s6 OS = Aedes albopictus12Blood feedingQ5MIP1_AEDAL219.234697
Long form D7Bclu1 salivary protein d7l1 OS = Aedes albopictus18Blood feedingQ5MIW7_AEDAL398.8737114
Serpins       
Salivary serpin putative anticoagulant OS = Aedes albopictus13Blood feedingQ5MIW0_AEDAL476.1532154
Salivary serpin putative anticoagulant OS = Aedes albopictus16Blood feedingQ5MIW0_AEDAL476.1522106
Putative salivary serpin OS = Aedes albopictus17Blood feedingQ5MIW4_AEDAL475.542055
Putative salivary serpin OS = Aedes albopictus19Blood feedingQ5MIW4_AEDAL475.542576
34 kDa family       
Similar to Aedes aegypti 34 kDa salivary secreted protein 34k-2 OS = Aedes albopictus2UnknownQ5MIU2_AEDAL365.9538148
Similar to Aedes aegypti 34 kDa salivary secreted protein 34k-2 OS = Aedes albopictus6UnknownQ5MIU2_AEDAL365.9530115
62 kDa family       
Putative salivary secreted protein 62k-3 OS = Aedes albopictus5UnknownQ5MIU9_AEDAL656.4330205
Putative salivary secreted protein 62k-3 OS = Aedes albopictus9UnknownQ5MIU9_AEDAL656.431274
Table 2. Aedes albopictus immunogenic salivary secreted proteins identified by LC MS/MS. Protein number corresponds to the position in 2D gel, Fig. 2
Family or sequence nameProtein numberFunctionAccession numberNominal mass (kDa)PiSequence coverage%PeptidesIon score
  1. OS, Organism Name; m, methionine oxidation; Pi, isoelectric point.
  2. SwissProt or TrEMBL databases were used for identification.
Enzymes        
Probable salivary maltase OS = Aedes albopictus38Sugar feedingQ5MIZ5_AEDAL665.4416.06ImmTEGYTSLPK73
DSDGDGIGDLDGITEK60
QIQTEYGDLDAFQR50
GEFYLHQFLK44
SNWVLGNHDNPR44
NPAVVEEmKNVLR42
IDAVPYLFESGESDGR36
Putative adenosine deaminase OS = Aedes albopictus44Blood feedingQ5MIX2_AEDAL616.749.01QLALNSIEYSAmNSEEK77
QWmTNEVYDAK60
GNFGLNHGPEFK44
HIFEVLDR42
D7 protein family        
Short D7 salivary protein d7s5 OS = Aedes albopictus21Blood feedingQ5MIT1_AEDAL19826.92SDTGVLSFIK60
RPNKSDTGVLSFIK52
FmNcVFTK40
SDTGVLSFIK38
SDTGVLSFIK37
VGTFNmQDHDSQSLmDGTIK36
Short D7 salivary protein d7s5 OS = Aedes albopictus22Blood feedingQ5MIT1_AEDAL17824.36SDTGVLSFIK71
VGTFNmQDHDSQSLmDGTIK40
FmNcVFTK39
D7 salivary protein d7s6 OS = Aedes albopictus23Blood feedingQ5MIP1_AEDAL219.130.6NPDELQSIAAR68
NPDELQSIAAR67
SSScTDVFNAYK57
IMQQWETFSR50
AILFcDGK47
IMQQWETFSR46
cmLENIGFYEPAEK45
SSScTDVFNAYK44
cmLENIGFYEPAEKR44
Long form D7Bclu1 salivary protein d7l1 OS = Aedes albopictus25Blood feedingQ5MIW7_AEDAL388.666.02SSScSDVFNAYK83
AILFcDGK47
Long form D7 salivary protein D7l2 OS = Aedes albopictus26Blood feedingQ5MIW6_AEDAL368.3218.27cVLEmSGLYDAASGK64
cLVESSVANDFK45
QSYFEFcENK44
QYVVLDDAQFK43
ATSINLFHDNK41
Long form D7Bclu1 salivary protein d7l1 OS = Aedes albopictus27Blood feedingQ5MIW7_AEDAL388.6615.66SSScSDVFNAYK73
VmQQWETFNR48
NPEQLQYIAAR41
SKDYYAQITGK41
AILFcDGK40
Long form D7Bclu1 salivary protein d7l1 OS = Aedes albopictus29Blood feedingQ5MIW7_AEDAL388.6612.35NPEQLQYIAAR79
SSScSDVFNAYK59
VmQQWETFNR48
AILFcDGK38
Long form D7 salivary protein D7l2 OS = Aedes albopictus33Blood feedingQ5MIW6_AEDAL368.3213.62cVLEmSGLYDAASGK77
ATSINLFHDNK43
QPYNKPAVQAQVSEVDGK39
Long form D7Bclu1 salivary protein d7l134Blood feedingQ5MIW7_AEDAL388.669.94NPEQLQYIAAR61
SSScSDVFNAYK52
VmQQWETFNR42
Long form D7 salivary protein D7l2 OS = Aedes albopictus36Blood feedingQ5MIW6_AEDAL368.328.05cVLEmSGLYDAASGK48
QYVVLDDAQFK41
Long form D7 salivary protein D7l2 OS = Aedes albopictus37Blood feedingQ5MIW6_AEDAL368.3223.84KQSYFEFcENK70
QSYFEFcENK65
cVLEmSGLYDAASGK64
ATSINLFHDNK49
QPYNKPAVQAQVSEVDGK44
QYVVLDDAQFK41
DNILNcDEIKR41
DNILNcDEIK37
Serpins       
Putative salivary serpin OS = Aedes albopictus41Blood feedingQ5MIW4_AEDAL475.7111.85KFNDQLSIASIR85
MSEFEAFANK67
mSEFEAFANK67
VFmSMYGEmDASAK62
VFmSmYGEmDASAK62
VFmSmYGEmDASAK59
FNDQLSIASIR47
FNDQLSIASIR45
VFmSmYGEmDASAK44
VFMSmYGEmDASAK42
ETNVDEVmTQFImK40
FNDQLSIASIR40
ETNVDEVmTQFImK36
VFmSmYGEmDASAK35
VFmSmYGEmDASAK33
VFmSmYGEmDASAK30
VFmSmYGEmDASAK29
Salivary serpin putative anticoagulant OS = Aedes albopictus42Blood feedingQ5MIW0_AEDAL466.589.16APPDAAmGLADLQK71
TPFNQGVFK44
mEINmSGDGEIQGQK39
MEINmSGDGEIQGQK35
34 kDa family        
Similar to Aedes aegypti 34 kDa salivary secreted protein 34k-2 OS = Aedes albopictus28UnknownQ5MIU2_AEDAL366.2918.01DTGISEEQVDELK58
QSYEQQVNEIVK50
SVASEIATLK44
LDEmSLETNK41
YLDHLGDQPLIDK38
Putative salivary protein 34k-1 OS = Aedes albopictus30UnknownQ5MIU3_AEDAL365.3321.27MYEDmAEYIFQR52
MLNYVEQK48
cTLSEEDLDSLK45
cTLSEEDLDSLK45
TQGVSNAEVELLR43
KmYEDmAEYIFQR43
KmYEDmAEYIFQR41
TVATDMEVLK41
Similar to Aedes aegypti 34 kDa salivary secreted protein 34k-2 OS = Aedes albopictus31UnknownQ5MIU2_AEDAL366.2929.81ScTVSEEDLTTIR75
ASLDDVNLDEDLIAK60
DTGISEEQVDELK59
SVASEIATLK51
QSYEQQVNEIVK51
ScTVSEEDLTTIR40
YLDHLGDQPLIDK39
mYDDmAELIFQR33
IALKFYPK33
62 kDa family        
Putative salivary secreted protein 62k-2 OS = Aedes albopictus40Defence responseQ5MIV0_AEDAL646.234.94DLcLTYLNSNQmSNAK64
KLDEDVDTLHNK43
Putative salivary secreted protein 62k-2 OS = Aedes albopictus45Defence responseQ5MIV0_AEDAL646.2317.99MAEDYENQLK68
VAAYKELIDR67
DLcLTYLNSNQmSNAK57
YALDHLFVNQmK54
ASQAEVDHLLVmDVK54
LDEDVDTLHNK52
IQSEFNEYK47
KLDEDVDTLHNK44
KLDEDVDTLHNK44
GIEDTIIK41
RDLcLTYLNSNQmSNAK41
mAEDYENQLK40
YALDHLFVNQMK39
mAEDYENQLK39
YLPHVVVGR39
mAEDYENQLK34
Putative 62 kDa salivary secreted protein OS = Aedes albopictus46UnknownQ5MIV1_AEDAL656.397.36IQSDFNAYQTSK47
FGEYVTGR46
IAAYNSLVEK44
VTSTmSAcTSFR 43
Lectins       
Putative salivary C type lectin OS = Aedes albopictus20Blood feedingQ5MIZ0_AEDAL175.5525.49LAILDTEEK55
QQQVEEQAQKVDAFK39
NGNEHcVHLWYEPSR38
Angiopoietin        
Angiopoietin-like protein 2 (Fragment) OS = Aedes albopictus35MetabolismQ5MIV9_AEDAL266.7717.02YGGGWLVVmQR56
YGGGWLVVMQR45
YGGGWLVVmQR40
NELAYSSIcWmK39
GAVFSTYDDDEFGcSNK33
Angiopoietin like salivary protein OS = Aedes albopictus32MetabolismQ5MIV8_AEDAL325.2410.53GScYGSSLTGIWK67
GScYGSSLTGIWK49
GAGFSTYDYDDFGcSNK47
GScYGSSLTGIWK38
GAGFSTYDYDDFGcSNK32
Antigen 5 family        
Salivary secreted antigen-5 AG5-3 OS = Aedes albopictus49UnknownQ5MIV5_AEDAL298.8414.51LASGTmSSTHGmFPSAmNmPELK42
DFSHYPScSNQHPK38
LASGTmSSTHGmFPSAmNmPELK37
DFSHYPScSNQHPK32
DFSHYPScSNQHPK29
56 kDa family       
Putative 56 kDa salivary secreted protein OS = Aedes albopictus48UnknownQ5MIU5_AEDAL588.186.42LLENEVAYSAGR57
VYVQQIANYQK40
YQAATcLNEAR37
Other proteins        
Nucleoside diphosphate kinase OS = Aedes aegypti24MetabolismQ16MB4_AEDAE188.6323.81QILGATNPADSAPGTIR93
NIIHGSDAVESANK56
GDLcVQVGR42
AAEL009524-PA OS = Aedes aegypti39MetabolismA2I874_AEDAE665.457.68ImmTEGYTSLPK73
DSDGDGIGDLDGVTEK63
SFKDSDGDGIGDLDGVTEK52
NPAVVEEmKNVLR42
Myo-inositol-1 phosphate synthase OS = Aedes aegypti43MetabolismQ17H06_AEDAE616.439.87AGVLEVGLQDQVHK70
VVVLWTANTER50
TAIENILR49
AGVLEVGLQDQVHKK46
AGVLEVGLQDQVHKK44
TAIENILR41
GLNTTmAELEK40
YVPYVGDSKR37
AGVLEVGLQDQVHK37
AAEL009524-PA OS = Aedes aegypti47MetabolismA2I874_AEDAE665.454.89ImmTEGYTSLPK70
DSDGDGIGDLDGVTEK40
IMmTEGYTSLPK39

The intracellular immunogenic proteins were involved in housekeeping functions, such as energy metabolism, transcription and structural functions (Tables S1, S2). The Blastp analysis indicated that no species-specific immunogenic salivary protein could be identified (data not shown). Most of the immunogenic salivary proteins had high levels of homology with Ae. aegypti.

Discussion

This study has identified 76 Ae. albopictus salivary antigens using immunoproteomic analysis. To our knowledge, this is the first screening of Ae. albopictus mosquito immunogenic salivary proteins namely the immune-sialome. The results show that all serum samples presenting IgG Ab to Aedes saliva exhibited strong immune reactivity against Ae. albopictus salivary gland extract (SGE). The molecular weights of these antigens ranged from 12 to 70 kDa, corresponding to the range of Ae. albopictus immunogenic salivary protein described by 1D immunoblot in a previous study (Shan et al., 1995). The patterns of recognition appeared to be relatively similar to those observed with Ae. aegypti SGEs probed with sera from individuals exposed to Ae. Aegypti bites (Wasinpiyamongkol et al., 2009). The secreted proteins represented about two-thirds of the Ae. albopictus immune-sialome. These results are in accordance with proteomic data from Ae. aegypti and An. gambiae (Choumet et al., 2007; Orlandi-Pradines et al., 2007; Wasinpiyamongkol et al., 2009). Secreted salivary proteins are involved in the interactions between the mosquito and vertebrate host during blood feeding. These secreted proteins may, therefore, be expected to induce an antibody response in the human host. The antigenicity of intracellular proteins is unexpected. Several hypotheses can be advanced to explain the antigenicity of these proteins. As these proteins may also be present in the mosquito carcass (head, body), one could speculate that individuals were sensitized by their frequent contact with mosquitoes. Indeed, in several mosquitoes species, the possible antigenicity of whole body extracts (Wongkamchai et al., 2010) or one specific component of the mosquito body, such as the haemolymph (Kumari et al., 2009), has been reported. The possible glycosylation of these proteins may also explain the results observed. Indeed, it has been reported that mannosylation greatly enhanced the antigenicity of candidate vaccines (Lam et al., 2005, 2007; Doores et al., 2010). It is also possible that this immunogenicity results from human sensitization to the antigens of other arthropods and/or micro-organisms (conserved epitopes).

It has been reported previously that some salivary components are expressed only in the salivary glands or are more expressed in this tissue than in other Ae. albopictus organs (Arca et al., 2007). This is the case for the major protein families such as the D7, 34-kDa and 62-kDa families and for certain enzymes such as apyrase and ADA. However, the 30-kDa allergen and proteins from the mucin family, which are expressed specifically in female salivary glands, were not antigenic in our conditions. Our study also revealed the presence of isoforms for almost all antigens. Further studies are needed to examine fully the biological function of these isoforms and the consequences on the bloodmeal. A recent study has demonstrated that Ae. aegypti saliva may enhance dengue virus infection and suppress the human immune response (Surasombatpattana et al., 2012).

In the current study, immunoblot profiles exhibited high variability amongst individuals. This suggests variability in the human antibody response, as has been reported previously for different Aedes species (Shen et al., 1989) and An. gambiae (Cornelie et al., 2007) using the 1D immunoblot technique. Here we did not discern an antigenic profile specific to the Ae. albopictus exposure, suggesting that all of the antigens detected are shared between the two Aedes species. In contrast, a recent study using the enzyme-linked immunosorbent assay (ELISA) technique showed a weak cross-reactivity between IgG responses against Ae. albopictus and Ae. aegypti SGEs in the same two human populations as in our study (Doucoure et al., 2012a). This discrepancy could be a result of our use of denaturing 2D electrophoresis, which disrupts the protein structures, inducing loss of native and conformational epitopes. These denaturing conditions may result in the loss of species-specific epitopes, and the response detected would then involve only the shared antigens of these two closely related species. It would be useful to investigate, under native conditions, the individual immunogenic profile to Ae. albopictus secreted proteins, ie in saliva instead of SGEs. However, mosquito saliva is characterized by low protein concentrations and its collection is labour-intensive and time-consuming, thus limiting its use. The high variability of antigenicity noted here may also indicate heterogeneity in the exposure level to vector bites. Therefore, others factors should be investigated to understand better the diversity of immunogenic profiles. The occurrence of the IgG Ab may be related to several epidemiological factors, such as age and exposure history. In this study, the individuals selected belong to the same age group. It would be appropriate to investigate the pattern of immunogenic salivary proteins from childhood to adulthood, as age has been shown to affect levels of anti-Anopheles saliva Ab (Drame et al., 2010). This should give an overview of the acquisition of human Ab response against Ae. albopictus salivary proteins.

None of the salivary immunogenic proteins was found to be specific to Ae. albopictus, highlighting how difficult it is to develop a specific biomarker based only on an immunoproteomic approach. In the present study, 20 proteins, identified by homology with Ae. aegypti sequences, were genus-shared. It would be appropriate to use complementary tools to identify Ae. albopictus-specific epitopes. Previously, bioinformatics analysis has been used to identify a specific biomarker of exposure to Anopheles bites (Poinsignon et al., 2008). An in silico analysis based on the antigenic protein data provided by this study could help to develop a salivary biomarker specific to Ae. albopictus bites.

The present study provides, for the first time, the 2D profile of Ae. albopictus immunogenic salivary proteins in humans exposed to this vector. Further studies are needed to identify specific salivary biomarkers for the bite of Ae. albopictus. The present database of immunogenic salivary proteins provided in this study is a step facilitating this task.

Experimental procedures

Study individuals

Individuals were selected from two different areas, Reunion Island and Bolivia, for their specific human exposure to Ae. albopictus or Ae. aegypti, respectively. Six individuals, between 18 and 30 years of age, were selected in each area according to their IgG response against Ae. albopictus and Ae. aegypti SGEs measured by ELISA in a previous study. All individuals from Reunion Island were IgG positive to Ae. albopictus SGEs and IgG negative to Ae. aegypti SGEs and conversely, Bolivian individuals did not present an IgG response to Ae. albopictus SGEs and were IgG positive to Ae. aegypti SGEs (Doucoure et al., 2012a).Both studies followed ethical principles as stipulated in the Edinburgh revision of the Helsinki Declaration. The study in Reunion Island was approved by a French ethics committee (the Sud Ouest, Outre Mer Ethics Committee, 25 February 2009) and authorized by the French Drug Agency [Agence Française de Sécurité Sanitaire des Produits de Santé (AFSAPS), Ministry of Health; 12 January 2009]. The study in Bolivia was approved by the Bolivian Committee of Bioethics (September 2006) and the Institut de Recherche pour le Développement (IRD) ‘Comité Consultatif de Déontologie et d”Ethique’ (July 2006). Written informed consent was obtained from every subject.

Collection of Aedes salivary gland extracts

SGEs were obtained from 10-day-old uninfected females reared in an insectary. Ae. albopictus was bred from larvae collected in the field on Reunion Island (Direction Regionale des Affaires Sanitaires et Sociales, Saint Denis, Reunion Island). Briefly, 2 days after a bloodmeal, the mosquitoes were sedated with CO2 and their legs and wings were removed and the salivary glands were dissected out and transferred into a tube containing 30 μl isoelectric focusing (IEF) buffer {7 M urea, 2 M thiourea, 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.8% Immobilized pH Gradient (IPG) buffer, 0,2% tergitol, 100 mM dithiothreitol (DTT)}. The dissected glands were pooled in 30 or 60 pairs per batch and frozen at −80 °C before protein extraction. A simple technique consisting of three successive freeze−thaw cycles was used to extract the soluble fraction of proteins from the salivary gland homogenate. The soluble proteins of SGE were then collected after centrifugation for 20 min at 30 000 g at +4 °C. Protein extracts were then desalted using a 2-D Clean-Up Kit (GE Healthcare, Germany). The concentration of proteins was evaluated by the Bradford method (OZ Biosciences, Marseille, France) after pooling the different batches to generate a homogenous SGE. The proteins extract were then resuspended in IEF buffer for proteomic analysis and stored frozen.

Two-dimensional electrophoresis and immunoblotting

IEF was carried out using Immobiline DryStrips [11 cm, pH 3–11 non-linear (NL), GE Healthcare]. The strips were rehydrated for 16 h at room temperature with 8 μg Ae. albopictus or Ae. aegypti SGE made up to 180 μl by adding IEF buffer containing 2% DeStreak reagent (GE Healthcare). Strips were run at 20 °C at 50 μA per strip; 60 V (step) for 1 h, 500 V [gradient (grad)] for 1 h, 1000 V (grad) for 1 h, 6000 V (grad) for 2 h and then 6000 V steps up to 30 000 Volt/hours.

After IEF, the strips were reduced for 10 min with reduction buffer at pH 6.8 (65 mM DTT, 50 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS) HCl pH 6.8, 6 M urea, glycerol 30%, sodium dodecyl sulphate 2%) and then alkylation was performed with alkylation buffer at pH 8.8 (81 mM iodoacetamide, 50 mM TRIS HCl pH 8.8, 6 M urea, glycerol 30%, sodium dodecyl sulphate 2%) for 15 min. The strips were then equilibrated in pH 6.8 buffer for 5 min and 2D electrophoresis was performed on 10.5–14% TRIS-HCl gels (Bio-Rad, Marmes de la coquette, France). The gels were run at 30 V for 20 min and then 200 V for 55 min.

For immunoblotting, the proteins were transferred onto a polyvinydilfluoride (PVDF) membrane (Bio-Rad). The transfer was run at 30 V for 16 h at 4 °C in TRIS 25 mM, glycine 192 mM and 15% ethanol buffer. The PVDF membrane was then washed in TRIS buffer saline (TBS) and blocked in TBS 0.05% Tween 20, 5% dry milk for 1 h 30 min. After washing three times with TBS 0.1% Tween, the membrane was incubated overnight at 4 °C with serum diluted at 1/100 in TBS 0.05%, Tween 20, 2.5% dry milk. An alkaline phosphatase mouse antihuman IgG was used as a secondary antibody at a dilution of 1/5000 (Sigma, St Louis, MO, USA) for 2 h 30 min at room temperature. The membrane was revealed using Lumi-Phos Western Blot (WB) Chemiluminescent substrate (Thermo Fischer Scientific, Waltham, USA), and exposed to CL-Xposure film (Thermo Fischer Scientific) for 1 min.

Protein identification

Two-dimensional electrophoresis was coupled to mass spectrometry analysis for protein identification. Thirty micrograms of Ae. albopictus SGE were used in Immobiline DryStrips (11 cm, pH 3–11 NL, GE Healthcare) as described above. After the second dimension, the gel was stained with PageBlue protein staining solution (Fermentas, Saint-Remy les Chevreuse, France) overnight. After washing, the gels were scanned and images were superposed and aligned with immunoblot images using SameSpots v. 4.1 (NonLinear dynamics) software. The matching proteins were then excised manually under a laminar flow hood and enzymatic in-gel digestion was performed automatically (Tecan freedom evo proteomics, Tecan Group Ltd., Männedorf, Switzerland) according to a modified protocol described by Shevchenko et al. (1996). Briefly, protein spots were digested using 150 ng trypsin, extraction was performed using five sonication cycles lasting 2 min each and peptides were concentrated for 1 h at 50 °C. Then, 0.5 μl sample peptide and 0.5 μl alpha-cyano-4-hydroxy-trans-cinnamic were deposited on a 384-well MALDI anchorship target using the dry-droplet procedure (Karas & Hillenkamp, 1988) and air dried at room temperature. MALDI-TOF MS analysis was performed using an UltraFlex MALDI TOF-TOF mass spectrometer (Brucker Daltonics, Bremen, Germany) in the reflectron mode with a 26 kV accelerating voltage and a 50 ns delayed extraction. The AutoXecute module of Flexcontrol v. 3.0 (Bruker Daltonics; laser power ranged from 40 to 50%, 600 shots) was used to acquire mass spectra. Spectra were analysed using FlexAnalysis v. 3.0 software (Bruker Daltonics) and calibrated internally with the autoproteolysis peptides of trypsin [mass-to-charge ratio (m/z): 842.51; 1045.56; 2211.10]. Peptides were selected in the mass range of 900–3000 Da.

Peptide mass fingerprint identification of proteins was performed by searching against the Insecta entries (653 632 sequences) of either the SwissProt or TrEMBL databases (http://www.uniprot.org/) using the Mascot v. 2.3 algorithm (http://www.matrixscience.com). Mascot scores higher than 71 were considered as significant (P < 0.05) for the SwissProt and TrEMBL databases.

Protein samples that could not be identified by Maldi-TOF MS analysis were subjected to nano LC electrospray ionization MS/MS analysis with a linear trap quadrupole (LTQ) Orbitrap XL mass spectrometer (Thermo Fisher, San Jose, CA, USA). Samples were dehydrated in a vacuum centrifuge, solubilized in 2 μl 0.1% formic acid-2% and analysed online by nano-flow high-performance liquid chromatography (HPLC)-nanoelectrospray ionization using a LTQ Orbitrap XL mass spectrometer coupled with an Ultimate 3000 HPLC (Dionex, Amsterdam, the Netherlands). Desalting and preconcentration of samples were performed online on a Pepmap precolumn (0.3 × 10 mm; Dionex). A gradient consisting of 0–40% A in 30 min, 80% B in 10 min (A = 0.1% formic acid, 2% acetonitrile in water; B = 0.1% formic acid in acetonitrile) at 300 nl/min was used to elute peptides from the capillary (0.075 × 150 mm) reverse-phase column (Pepmap, Dionex). LC-MS/MS experiments comprised cycles of two events: an MS1 scan with Orbitrap mass analysis at 60 000 resolution followed by collision-induced dissociation (CID) of the 20 most abundant precursors. Fragment ions generated by CID were detected at the linear trap. Normalized collision energy of 35 eV and activation time of 30 ms were used for CID. All spectra were recorded under the positive ion mode using Xcalibur 2.0.7 software (Thermo Fisher Scientific). Spectra were acquired with the instrument operating in the information-dependent acquisition mode throughout the HPLC gradient. The mass scanning range was m/z 400–2000 and standard mass spectrometric conditions for all experiments were: spray voltage, 2.4 kV; no sheath and auxiliary gas flow; heated capillary temperature, 200 °C and capillary voltage, 40 V. For all full scan measurements with the Orbitrap detector, a lock-mass ion from ambient air (m/z 445.120024) was used as an internal calibrant as described previously (Olsen et al., 2005).

Regarding protein identification, all MS/MS spectra were searched against Insecta entries (690 637 sequences) of either the SwissProt or TrEMBL databases (v. 2011_08 http://www.uniprot.org/) by using the software ProteomeDiscoverer v. 1.2 (Thermo Fisher Scientific) and the Mascot v. 2.3 algorithm (MA; Matrix Science Inc. http://www.matrixscience.com/) with trypsin enzyme specificity and one missed trypsin cleavage. Carbamidomethyl was set as fixed cysteine modification and oxidation was set as variable methionine modification for searches. A peptide mass tolerance of 5 ppm and fragment mass tolerance of 0.5 Da were allowed for identification.

Management and validation of mass spectrometry data was carried out using ProteomeDiscoverer v. 1.2. Peptides with scores greater than the identity score (P < 0.01) were considered significant. Only proteins identified with two or more peptides were considered further.

After protein identification, the National Center for Biotechnology Information Blast T Non Redundant bank (http://blast.ncbi.nlm.nih.gov/Blast) was run to check the protein species specificity. To determine if the proteins are secreted or not, the SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP/) was used to predict the presence and location of signal peptide cleavage.

Acknowledgements

The authors gratefully acknowledge the populations of La Reunion and Santa Cruz de la Sierra for their participation in this study. The authors thank Vincent Robert for reviewing the draft of the manuscript.

Financial disclosure

This work was supported by a GIS CRVOI (Centre de Recherche et Veille en Océan Indien –Projet N° PRAO/AIRD/CRVOI/08/01) funded project and by IRD. Souleymane Doucoure was supported by a PhD fellowship provided by the Infectiopole Sud Foundation (Marseille, France) and Sirilakassana Patramool by a PhD fellowship from IRD. The funding partners had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Conflict of interest statement

The authors declared no conflict of interest concerning the work in this paper.

Authors' contributions

Conceived and designed the experiments: S. D., S. C., S. P., F. M., D. M., F. R.

Conducted the experiments: S. D., S. C., S. P., E. D., M. S.

Contributed reagents/materials/analysis tools: S. D., S. C., J. S. D., E. D., M. S., A. H. R., J. P. H., D. M., F. F., P. G., F. R.

Wrote the paper: S. D., S. C., F. R.

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