Some evidence that seasonal malaria chemoprevention with SPAQ has an effect on blood stage antibody responses and pre-erythrocytic stage of Plasmodium falciparum infections in Niger.

Background: In endemic areas, children develop slowly and naturally develop anti-Plasmodium antibodies and become semi-immune. Seasonal Malaria Chemoprevention (SMC) with sulfadoxinepyrimethamine + amodiaquine (SPAQ) is a new strategy to reduce malaria morbidity in young children in West Africa. However, SMC may impact on the natural acquisition of anti-Plasmodium immunity. We evaluated the effect of SMC with SPAQ on malaria antibody concentration in Niger. Methods: This survey was conducted in areas targeted with SMC since 2014 (Zinder district), without SMC (Dosso district), and with one year SMC 2016 (Gaya district). To assess the relationship between SMC and P. falciparum IgG antibody responses, we compared total antibody concentrations against two P. falciparum asexual stage vaccine candidate antigens, circumsporozoite protein (CSP) and glutamate-rich protein R2 (GLURP-R2), in children aged 3-59 months across the three sites. Antibody concentrations were quantied using an enzyme-linked immunosorbent assay (ELISA) on the elution extracted from positive and negative RDT cassettes. Results: A total of 229 children aged 3-59 months were included in the analysis: 71 in Zinder, 77 in Dosso, and 81 in Gaya. In Zinder (CSP=17.5µg/ml and GLURP-R2=14.3µg/ml) median antibody concentration observed were higher than in Gaya (CSP=7.7 µg/ml and GLURP-R2=6.5 µg/ml) and Dosso (CSP=4.5 µg/ml and GLURP-R2=3.6 µg/ml) (p<0.0001). Conclusion: We


Background
Malaria caused by Plasmodium falciparum remains the major cause of morbidity and mortality in children under 5 years in Sub-Saharan Africa [1]. It is the main public health problem in Niger [2]. The national malaria control program of Niger has implemented complementary malaria control strategies based on WHO recommendations, including seasonal malaria chemoprevention (SMC) with sulfadoxinepyrimethamine + amodiaquine (SPAQ) [3], [4,5]. SMC is an administration of full therapeutic doses of these drugs to children aged 3-59 months as monthly interval during period of greatest malaria risk in endemic areas [6]. In these areas, children develop slowly and naturally anti-Plasmodium antibodies and become semi-immune [7], [8].
Sulfadoxine is an antibacterial and antimalarial of the chemical class of sulfonamides. It is a dihydropteroate synthetase (dhps) inhibitor, a key enzyme in the biosynthesis of folate. It acts by competitive inhibition of para amino benzoic acid (PABA) to block the synthesis of folic acid and Plasmodium nucleotides (purines and pyrimidines). The pyrimethamine associated with sulfadoxine is a competitive inhibitor of dihydrofolate reductase (dhfr), a key enzyme in the redox cycle for the production of tetrahydrofolate, a cofactor necessary for the biosynthesis of DNA and proteins. SP is acts on the asexual forms of the hepatic and erythrocytic stage of Plasmodium.
Amodiaquine is an antimalarial with antipyretic and anti-in ammatory properties. It is a related 4aminoquinoline related in structure and activity with chloroquine. Amodiaquine is active on the erythrocyte form of Plasmodium.
Malaria immunity is partial, short-lived, and requires exposure to infected mosquitoes bites to be maintained [7]. Monthly given SMC reduce malaria morbidity in young children in West Africa [5,[9][10][11].
However, SMC may impact on the natural acquisition of anti-Plasmodium immunity. In Senegal, Ndiaye et al., suggested that long-term SMC by SPAQ has limited impact on the development of acquired immunity [12]. In the same country, Sylla et al., showed that SMC with SPAQ can induce decreasing of IgG anti-AMA1 and anti-MSP1_ 42 [13]. In Mali, Mahamar et al., concluded that exposure to SMC/SPAQ decreasing of anti-AMA-1, MSP1_ 42 and CSP titers [14]. Other Malian study conclude that duration of exposure to SMC had no effect on antibody to MSP1_ 42 and CSP [14].
The hypothesis of this study was that SMC/SPAQ could reduce immunity to erythrocyte stage antigens but not to liver-stage malaria antigens, and RDTs materials could be used to measure IgG titers. To assess the relationship between SMC and P. falciparum antibody responses, we compared total IgG concentrations against two P. falciparum asexual stage vaccine candidate antigens, circumsporozoite protein (CSP) and glutamate-rich protein R2 (GLURP-R2), in children aged 3-59 months across the three sites. Antibody concentrations were quanti ed using an enzyme-linked immunosorbent assay (ELISA) on the elution extracted from positive and negative RDT cassettes.

Study design and sample collection
The data presented here were generated from the malaria morbidity sentinel surveillance sites within the SMC program in Zinder, Dosso and Gaya districts located in western Niger, where malaria transmission is seasonal [15,16]. Zinder and Gaya districts have implemented SMC with SPAQ respectively since 2014 and 2016; they were classi ed as mesoendemic and hyperendemic areas [15,17]. SMC was not implemented in Dosso district, which was classi ed as hyperendemic and as a control district of the study [15,17]. In 2016, SMC coverage in Zinder received once, at least 3 times and 4 times was 91%, 73% and 50%, respectively (Unpublished data). The coverage of Gaya district were 77.72%, 81.56%, 71.26%, and 69.47 respectively for round 1, 2, 3 and 4 (Unpublished data). All the 3 sites used ACTs as rst line treatment for uncomplicated malaria and received universal coverage of bed nets. The seasonality of malaria transmission in these 3 sites is the same.
To assess the impact of SMC on the titer of antibodies to two asexual P. falciparum stage antigens, 6 health facilities in Zinder, Dosso and Gaya were selected. In this health facilities, malaria RDTs (SD Bioline) of randomly selected children aged 3-59 months were collected from symptomatic cases (fever + positive or negative RDTs) for serological analysis. For all RDTs collected, the date of consultation, the age, the gender, whether a test was performed and the result (positive or negative) were reported on the cassette. Samples were collected all three month at the same time in all sites between November 2015 to December 2016. The RDTs was stored at room temperature. The analyses were performed in April 2017. The average time of the RDT before testing across the sites was : 06 month for Zinder, 05 month for Dosso and 7 month for Gaya.
The total number of children to include was 249, and calculated based on 78% circumsporozoite protein antibody prevalence in children that received SMC for 1, 2 or 3 years [14] (95% CI) with a precision of 5%.

Recombinant antigens
The malaria antigens used in this study included a recombinant antigen circumsporozoite protein (CSP) and glutamate-rich protein R2 (GLURP-R2). CSP antigen was a 44-aa NANP repeat-sequence peptide of the P. falciparum circumsporozite protein synthesised by Sygma Genosys, while GLURP-R2 (amino acids 706-1178, F32 strain) was produced at the Statens Serum Institutes of Copenhagen (Denmark) and was expressed in Escherichia coli.

Sera extraction
Sera were extracted from lter paper inside RDTs cassette collected [18]. RDTs have proximal, middle and distal parts according Cnops et al., description [19]. The distal part of RDT contains a lter paper component that absorbed the residual blood solution. The cassettes was opened by sterile tweezers and distal part of each RDT was cut with sterile scissors in two or three pieces about 2 mm and eluted (all pieces obtained) into 300 µl of Phosphate Buffered Saline (PBS) from this fragment placed in 1.5ml Eppendorf tubes. The solution was stored at 4°C overnight. The solution is equivalent to 1/100 dilution of whole blood, with about half of the concentration of antibodies in plasma or serum resulting to a dilution of 1/200 [18]. The elations CSP and GLURP-R2 total IgG antibody responses were quanti ed using ELISA [20].

Antibody measurements
The standard operating procedure developed by the African Malaria Network Trust was used to assess total IgG concentrations by Enzyme Linked Immuno Sorbent Assay (ELISA) to CSP and GLURP R2 as described previously [21]. Brie y, recombinant proteins (0.1 µg/well) diluted in Phosphate Buffered Saline (PBS) were coated on MaxiSorp Nunc plates (Thermo Fisher Scienti c, Denmark) and blocked with 3% powdered-milk + 0.1% of PBS-Tween 20. Sera samples were diluted at 1:200 for all recombinant proteins.

Statistical analysis
The Median test was used to analyze differences between IgG medians concentrations. The comparisons between IgG median concentrations in Zinder, Dosso and Gaya were performed to investigate the potential impact of SMC on Antibody responses. The comparison between IgG median concentrations were performed by Mann-Whitney test. Data were analyzed with SPSS software version 16.0. P-values ≤ 0.05 were considered were considered statistically signi cant.

Population characteristics
A total of 229 children aged 3-59 months were included in the analysis: 71 from Zinder, 77 from Dosso, and 81 from Gaya ( Figure 1). The number of samples categorized by mean age and sex was comparable between districts and differences were seen between RDT results (Table 1). Using ANOVA test no statistical difference of mean age between the sites was showed (p=0.11).
Analysis of the differences between the median concentration of anti-CSP IgG and anti-GLURP-R2 in Zinder and Gaya (D3) showed a signi cant difference (CSP: p=0.008 and GLURP-R2: p=0.017).

Anti-CSP and GLURP-R2 IgG median antibody concentrations by RDT results
The median concentrations of anti-CSP and GLURP-R2 IgG antibodies by RDTs results are in table 3. When subdividing the groups into those that were RDTs positive or negative and compared the differences between the median concentrations of antibodies responses against CSP and GLURP-R2 in each groups, no signi cant differences were observed (p=0.093 and p=0.539).

Discussion
This study assessed the impact of SMC on the antibody titers to P. falciparum antigens CSP and GLURP-R2 in children aged 3-59 months in areas that received SMC for different time period. The total IgG antibodies to liver-stage vaccine candidate antigen CSP and blood stage antigen GLURP-R2) were signi cantly higher in Zinder where SMC was implemented since 3 years, than Gaya where SMC has been implemented for one year, and Dosso, which has never received SMC. This is consistent with a previous intriguing nding that demonstrated sustained protection during one year of follow-up in children who had receive malaria intermittent preventive treatment [23]. SMC was primarily directed against bloodstage parasites, because sulfadoxine-pyrimethamine and amodiaquine association inhibits the erythrocyte stage and liver-stage, which may contribute to increase the titer of IgG against the antigen of these stages. This nding is contrasting with a recent study published SMC reduced antibody against liver-stage antigens MSP-142 and CSP in malaria journal by Mahamar et al [14]. Others studies demonstrated that sulfadoxine does not affect liver stages, pyrimethamine has some inhibitory effect on liver stages in P. yoelii models [24] but there are high levels of resistance to pyrimethamine in SMC countries [25]. In Niger a high prevalence (> 60%) of mutations N51I, C59R and S108N in Pfdhfr gene, known to be associated with resistance to pyrimethamine was observed [26]. Amodiaquine acts on blood stage parasites [24]. In vaccine trial cohorts in the Gambia [27] SP did not affect the incidence of low level P. falciparum infections detected by PCR, consistent with SP affecting blood stage and not liver stage parasites.
The concentrations of both antibodies against CSP and GLURP-R2 showed an increase with SMC implementation probably as a result of decrease of either liver-stage maturation or erythrocyte stage by SPAQ. This is contrasting with other studies [14], [13], [28], [12] which found a decrease in the titers of antibodies after SMC delivery. Previous studies established that chemoprophylaxis conferred protective immunity against reinfections when anti-malaria drugs are not present [29], [30], [31]. The inhibitory effect of SP on pre erythrocyte stage [32], [33] and AQ on erythrocyte stage was previously described [34].
Friesen J et al., showed in the redone malaria model induction of antimalarial immunity by pyrimethamine prophylaxis during exposure to sporozoites and attenuation by pyrimethamine permits hepatocyte invasion but appears to block intrahepatocytic replication [29]. The increase in antibodies concentration is believe to be linked with exposure of the immune system to an attenuate hepatic stage parasites or complete suppression of blood-stage parasites, thereby resulting in an increase of IgG antibody concentration to CSP and GLURPR2 antigen in the sites where SMC was implemented.
This is the rst used of antibody elution from RDT lter paper for the assessment of CSP and GLURPR2 antibody concentration in Niger. As demonstrated by Amrish Baidjoe et al, antibody elution from lter paper is an operationally attractive approach [18]. RDT cassette can be use to monitor molecular markers of malaria drugs resistance and study antimalarial immunity in Niger.
The comparative analysis of antibodies concentration against CSP and GLURP-R2 antigens between age groups by district, observed no signi cant difference (CSP: p= 0.6813; GLURP-R2: p= 0.0760). This difference was not statically signi cant, perhaps because of the small number of samples and the short period of following, which is a limitation of this study. There are some important limitations, we do not know have con rmation of SMC status of all children included in the study, the sample size is small, and from only three districts, and only two marker of immunity has been measured. Also we are not able to interpret the immune responses in terms of implications for the risk of malaria. But this approach might be useful for monitoring rates of acquisition of immunity in older children who have stopped receiving SMC.

Conclusion
This data suggest that SMC by SPAQ have an effect on antibody responses against pre-erythrocytic stage and blood-stage antigens. However, other factors that have a signi cant in uence on antibody titers, such as transmission intensity, may confound this. Future studies are necessary to provide a better understanding of the impact of SMC on malaria immunity in Niger. In summary, duration of SMC administration may increased the antibody concentration of P. falciparum blood stage antigen GLURP-R2 and pre-erythrocytic stage antigen CSP. RDT lter paper serum elution methodology can signi cantly reduce the workload and cost in large-scale epidemiological and immunological studies.

Declarations
Ethics approval and consent to participate All the study participants provide informed consent before their enrollment. Ethical approval was obtained from the Ethics Committee of Niger (Deliberation N°024/2015/CCNE).

Consent for publication
Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The Laboratory of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of Abomey-Calavi, Benin and the Center for Medical and Health Research (CERMES-Niger) supported the realization of this study.

Authors' contributions
MML and RA carried out the ELISA processing, the analysis and interpretation of data, and contributed to the drafting of the manuscript; ALPM and DA participated to the analysis and interpretation of data; IML participated to the conception of the study and the eld samples collection and identi cation; DC, JT and NJL coordinated the study, contributed to the analysis, interpretation of data and the drafting of the manuscript. All authors read and approved the nal manuscript.  was performed by Median test and two by two with Mann-Whitney test. The signi cance limit was p<0.05.