A recent study performed in the laboratory shown that 35% of soldiers who travelled for a few month period in tropical Africa, presented a serological response against pre-erythrocytic antigens. Among them, only 2% had experienced a clinical malaria attack . Others have reported that travellers from malaria-free countries can develop antibody response against pre-erythrocytic [20, 21] and blood stages P. falciparum antigens without progressing to a symptomatic illness. Thus, the characterization of erythrocytic stage antigens specifically detected by healthy adults shortly exposed to malaria and under chemoprophylaxis could be informative to estimate individual exposure to malaria transmission, and to clarify the mechanisms involved in the development of immune responses after malaria infection. In this aim, a comparison and an analysis of serological responses between NEI, BEI and HEI against membrane protein extracts from uninfected and P. falciparum infected RBC were performed. Although the use of a prophylactic treatment limiting the abundance of the blood malaria parasite could affect the antibody response development, it was observed that 5% of soldiers shown an IgG reactivity directed against the surface of iRBC. Among them, only a minor part (21%) presented a history of clinical malaria. Thus, although exposure to malaria transmission was brief and probably low because of the use of chemoprophylaxis and anti-vector devices, some BEI could develop an antibody response against blood stage antigens without history of clinical malaria attacks.
Analysis of IgG immune response indicated an association between the exposition level and the number of bands revealed by 1-D immunoblotting against iRBC membrane protein extracts. A thorough qualitative analysis of the immune profiles shown that eight protein bands from iRBC membrane protein extracts were significantly more frequently recognized by BEI sera than NEI sera. To identify unambiguously these antigenic bands, an original 2-D immunoproteomic approach using a fluorescence-based method was performed. Almeras et al previously demonstrated that it was possible to align, with a good confidence, antigenic bands detected by 1 D immunoblot with the corresponding spots onto 2 D immunoblot . Here, this study shown that a perfect match is now conceivable between antigenic spots from the blot and their corresponding spots from the preparative gel, as recently described by Donoghue et al . This method allowed us to identify 13 protein spots corresponding to six out of the eight antigenic bands.
Surprisingly, spots corresponding to bands "I", "II" and "VII" were identified as H. sapiens proteins. Auto-antibodies (aabs) against RBC membrane proteins have been already described in malaria patient's sera [34, 35]. Little is known about the mechanisms that underlie the aabs production during parasite infection. Aabs production could arise from cross reactivity between host and parasite antigens. This molecular mimicry phenomenon could reduce the tolerance to self antigens and develop an auto-immunity during parasite infection . However, here, all discriminatory antigenic proteins were exclusively recognized on iRBC membrane protein extracts (Additional file 3). The aabs emergence against host proteins on iRBC membrane protein extracts could be attributed to others phenomenon such as protein post-translational modifications. Effectively, it was demonstrated that protein phosphorylations of host and parasite occur during P. falciparum infections [37, 38]. Moreover, ankyrin (gi|178646; band "I"), which was identified as a host discriminatory antigenic protein, was reported to be targeted by falcipain-2, a P. falciparum protease . This protein cleavage could produce neo-antigens which are then recognized only in infected conditions. Nevertheless, aabs directed against these structural membrane proteins were also observed in other Plasmodium species infections  and could occur in other diseases involving RBC abnormalities like thalassaemia or autoimmune haemolitic anaemia [40, 41]. Despite a significant recognition of these host proteins by BEI sera, these discriminatory proteins did not seem to be relevant markers of malaria exposure. Another explanation of the identification host proteins could be attributed to that the spotted area could contain a mixture of both parasitic and human proteins, and the amount of parasite proteins is under-represented compared to human proteins, and so parasitic antigenic proteins may be missed by MS.
Four P. falciparum proteins (exported protein 2 (Exp-2) PF14_0678, band "VI"; early transcribed membrane protein (Etramp5) PFE1590w, band "VI"; Heat shock 70 kDa protein (Pf-Hsp70-1) PF08_0054, band "III"; elongation factor 1α (EF1 α) PF13_0304, band "IV") corresponding to three discriminatory antigenic bands were also identified. Exp-2 and Etramp5 which are iRBC membrane associated proteins, have been described to participate to protein transport between parasite and iRBC [42–44]. An immune response against these proteins was previously observed using sera of travellers and individuals from endemic areas [17, 44]. The absence of signal detection by sera from never-exposed individuals suggests that Etramp5 could be an interesting marker of P. falciparum exposure.
The Pf-Hsp70 was detected at several parasite stages into the human host, in sporozoites , in liver stage [46, 47], in all parasite blood stages [48, 49] or in association with the iRBC plasma membrane , and is also possibly present on the merozoite surface . Here, a specific antibody response against Pf-Hsp70 was revealed by BEI sera. This protein has been described as a major target in the acquisition of immunity in naturally infected humans living in areas of endemic malaria [17, 49, 51]. Moreover, Pf-Hsp70 is able to elicit protection of Saimiri sciureus monkey against the asexual blood stage of P. falciparum . Collectively, these data suggest that Pf-Hsp70 seems to be an immunodominant antigens following parasite infection, and could be considered as a biomarker of malaria exposure.
The EF1 α is an abundant protein constituting 1-2% of the total protein in eukaryotic cells , and is an essential component of the translational machinery. This protein is involved in other processes as protein degradation, signal transduction and in the regulation of cytoskeletal rearrangements [54, 55]. EF1 α protein was unambiguously recognized by BEI sera reflecting an exposition to the immune system. It is the first time that EF1 α protein was described as antigenic in individuals exposed to malaria.
The present study failed to identify several well described P. falciparum antigenic proteins from the iBRCs plasma membrane. The major part of these P. falciparum antigens are large hydrophobic proteins (> 150 kDa) which are generally under represented and can be difficultly detected on 2-D electrophoresis , such as PfEMP1 , Pf332 [58, 59], the cytoadherence linked asexual protein 9 (Clag 9) [60, 61] or the erythrocyte-binding antigen 175 (EBA-175) . Furthermore, it was reported that serological immune responses against several P. falciparum antigens is conformation dependent [63, 64]. Thus, the reducing and denaturing conditions used for the immunoblot analysis in the present study, did not allow to take into consideration conformational epitopes. Conversely, antibody reactivities against linear epitopes derived from PfEMP1 or other P. falciparum proteins have been already observed under reducing conditions [65, 66]. The non-detection of these large proteins on 1-D and 2-D immunoblotting could be then attributed to incomplete protein transfer onto nitrocellulose membrane as previously described . The use of only one parasite strain with limiting variant antigen repertoire, for BEI sera selection, could also induce a bias for identifying more antigenic membrane parasite proteins. Moreover, Fried et al. reported that continuous in vitro culture of laboratory isolates could conduct to the loss or truncation of some parasite proteins , which could participate to this underestimation of antigenic parasite protein detection. Additionally, a lot of known antigens from the asexual blood stage are proteins from the merozoite surface (eg. MSP1, MSP2 or RESA) . In the sample condition preparations used here, iRBC membrane extracts were exempted of merozoites. Moreover, although some merozoite proteins are described to associate to the iRBC membrane during merozoite invasion (eg RSP2) [70, 71], these proteins are largely under-representated on the iRBC membrane fraction. Other iRBC membrane proteins such as RIFIN (30-45 kDa)  and STEVOR (30-40 kDa) , were reported to be antigenic in individuals which have experienced several malaria infections [74, 75]. A short exposure to malaria might not be sufficient to induce an antibody response against these proteins in BEI. However, complementary methods, such as the screening of P. falciparum expression library with BEI sera, could be envisaged for the characterization of others antigenic parasite proteins, as described previously .
The detection of a specific immune response against iRBC membrane extract could be unexpected using sera from individuals which have a mandatory chemoprophylaxis during their journey. Doxycycline was reported to be partially efficient on liver stages of P. falciparum parasites , and to alter asexual parasite blood stage at the end of the second erythrocytic cycle [78, 79]. Thus, a possible discontinuity in chemotherapy observance would have increased the risk to develop a specific IgG response against blood stage antigens.