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Abstract

Background: Artemisinin-based Combination Therapies (ACTs) are the first line drug for the treatment of uncomplicated malaria in most malaria endemic countries. They quickly clear the parasitaemia and reduce fever. In animal models it has been found that artemisinin derivatives have an immunosuppressive effect. In the present study we assessed the effect of ACTs on malaria antigens specific antibodies production in a population living in malaria hyperendemic area.

Methods: In 2012, 102 patients aged over 6 months and adults, presenting uncomplicated malaria were recruited and allocated to receive ACTs and follow up to 2 years. Antibodies titers against three P.falciparum blood stage malaria vaccine candidates (MSP3, GLURP R0, and GLURP R2) were measured by ELISA before treatment and 28 days (D28) after treatment with ACTs.

Results: Antibody levels were always higher 28 days after the initiation of the treatment for all tested antigens but not statiscally significant. An increase in antibody responses to MSP3 and R0 from D0 to D28 was also observed in children below 5 years of age with a borderline statistical significance.

Conclusion: The ACTs were effective on parasite clearance. Rapid clearance of parasitaemia was associated with age and did not prevent antibodies production. No immuno-suppressive action was observed in our study population.

Fulltext

Introduction

Malaria is one of the diseases which causes a large number of deaths in the world, especially in Africa [1]. Pregnant women and children under five years old are those paying the heavy burden in term of mortality [1]. In 2015, 306000 deaths due to malaria in children under five years were reported [1]. In malaria endemic areas, current malaria control strategies rely on vectors control (insecticide treated materials, indoor spraying etc.) and on adequate clinical malaria management through early diagnosis and treatment of uncomplicated malaria cases. However the emergence of the resistance of the vectors to insecticide and of the parasites to most affordable drugs has compromised malaria control strategies.

Combination therapy (CT) with antimalarial drugs is the simultaneous use of two or more blood schizontocidal drugs with independent modes of action and different biochemical targets in the parasite. The last two decades, because of the resistance to malaria monotherapies drugs [2], Artemisinin-based Combination Therapies(ACTs) were introduced as first line drugs for the treatment of uncomplicated malaria in early 2000 [3]. In most endemic African countries the most commons ACT used are Artesunate-Amodiaquine and Artemether-Lumefantrine [4-6]. ACTs have been proven to be efficacious in many studies [7-13]. They were proven to efficiently clear malaria parasites and reduce fever [5,6]. However in animal model ACT and artemisinin derivatives were shown to have some immune-suppressive effect [14,15]. In mice treated with different concentrations of artemisinin derivatives, antibody production was lowered and in vitro T cells proliferation was inhibited [16]. Artemisinin derivatives suppressed T cell differentiation and exerted moderate inhibition of T cell response. They also reduced IFNγ, IL4, IL6, IL12, IL17A and IL21 production [17].

Many of artemisinin derivatives were used in animal model cancers' and auto-immune diseases. They had anti-cancer and antiinflammatory properties [15,18-22]. In this study we aimed to assess in volunteers with uncomplicated malaria living in malaria hyper-endemic and seasonal transmission area the effect of ACT on antibodies against malaria specific antigens. The selected antigens were Merozoite Surface Protein3 (MSP3) and Glutamate Rich Protein (GLURP) which were shown to elicit antibodies associated with reduced risk of clinical malaria [23-27].

Methods

Study area

This study was carried out at the Banfora and Niangoloko district trial site, amongst the most humid areas of Burkina Faso close to the border of Ivory Coast, with an annual rain fall above 900 mm per year. Both sites are located 30 km apart and 500 km from Ouagadougou, the capital city of Burkina Faso. These two sites were chosen to evaluate the malaria incidence rate as well as the efficacy and safety of Pyronaridine Artesunate (PA or Pyramax) and Dihydroartemisinin Piperaquine (DHA-PQP) when used repeatedly for consecutive clinical malaria episodes. Burkina Faso is an endemic malaria country characterized by a dry and one rainy season. The transmission of malaria is stable and seasonal. The high peak of transmission is observed during the rainy season between May and November (in Banfora and Niangoloko). The main specie of Plasmodium, which causes mortality, is Plasmodium falciparum, and the main vectors are Anopheles gambiae and Anopheles funestus [28].

Study design and population. The study volunteers were drawn from a cohort study carried out at the Banfora trial site and at Niangoloko district hospital. The cohort study was designed to compare the incidence rate of uncomplicated malaria episode in children and adults treated with repeated ACT therapy over a period of 2 years. In this 3-arms study PA and DHA-PQP were compared to either Artesunate-Amodiaquine (ASAQ) or Artemether Lumefantrine (AL) (depending on the site location). The study is designed as a comparative, randomized, multi center, open label longitudinal clinical study to assess the safety and efficacy of repeated ACT therapy over a period of 2 years in uncomplicated Plasmodium (falciparum, malaria) malaria in children and adults. Patients were followed for 2 years starting from the first enrolment with the randomized study drug. For each treatment period, patient will be followed up for 42 days primarily for safety. For each treatment arm the same ACT (PA, DHA-PQP or comparator) was given unless one of the reasons for the use of an alternative was met. Random samples from the study conducted in Burkina Faso were selected to assess the effect of ACT's treatment on antibody production in subjects with uncomplicated malaria. Prior to any study procedures, the study protocol was approved by National Regulatory Authority and Ministry of Health ethical committee for biomedical research.

Blood sampling

For the purpose of immunological response assessment, 4 milliliters of venous blood were taken at D0 and 28 from study volunteers fulfilling the study inclusion criteria and the resulting sera were stored at -40 degree C prior to antibody measurement. Finger prick blood was also collected for each study volunteer to prepare a slide with thick and thin blood films at D0 and 28.

Laboratory Methods

Malaria diagnosis

Thick and thin blood smears were collected and stained with 6% Giemsa and read by experienced microscopists for microscopic diagnosis of malaria. The number of malaria parasites of each species and stage were recorded. Each slide was read twice by two independent technicians and the final result was the average of the two eadings. A 100% of qualitative agreement for the diagnosis was required for each slide between readers, and 30% difference for quantitative diagnosis was accepted between the two readers.

A third reading was performed in case of significant discrepancy between the two readers. The number of parasites per 1 μl of blood was calculated according to the leukocyte count obtained after the full blood count for each slide collected during the malaria transmission season. A slide was declared negative if no parasite was seen after 200 HPF (High Power Field) were examined.

Antibodies measurement

The levels of antibodies (total IgG) to the 3 malaria tested antigens (MSP3,GLURP R0 and GLURP R2) were measured using enzyme-linked immunosorbent assays (ELISA), as previously described in the Standard Operating Procedure (SOP) for CNRFP. Briefly, 96-well micro-ELISA plates (NUNC F96Maxisorb, Roskilde, Denmark) were coated with each respective antigen at 0.5 μg/ ml in phosphate buffered saline (PBS), and incubated overnight at 4 degree C. After blocking at 37 degree C for one hour with blocking buffer (0.1%Tween20 +5% non-fat skimmed milk in PBS), the plates were washed with washing buffer (NaCl + 0.1% Tween20 in PBS). 50 μl/well of plasma samples diluted at 1/200 in serum dilution buffer (0.1% Tween20 + 2.5% milk in PBS) were applied to the plates and incubated for 2 hours at 37 degree C. In each plate, positive and negative control plasma pools were added, as well as two wells referred to as blank. In the blank wells only PBS-Tween20 (1%) was added. The plates were then washed, and 50 μl per well of peroxidase-conjugated goat anti-human IgG (1:80000), diluted in dilution buffer (0.1% Tween20 + 2.5% milk in PBS) (Caltag, Camarillo, CA 93012 USA) were added. After washing, the plates were developed with Diethanolamine Substrate buffer 1X + 4-Nitrophenyl phosphate disodium hexahydrate tablet substrate and reactions stopped after 30 min by adding 50 μl of 0.2 M of sulfuric acid per well. Antibody levels, measured as optical density (O.D.), were determined using a Biotek Lx808 microplate reader (Winooski, Vermont 05404-0998 USA) at 450 nm with a reference at 620 nm. The O.D. values of the testsamples were converted into Arbitrary Units (AU) by means of extrapolation from a standard curve in each plate, obtained by using 12 serial dilutions of both pools of negative and positive hyperimmune sera. Positive control plasma was obtained from positive Bukinabe adults older than 20 years living in malaria hyper-endemic and negative control plasma was from Danish individuals never exposed to malaria from the Statens Serum Institute (Copenhagen, Denmark).

Statistical analysis

Data were double entered with Epi Info and analyzed with STATA. The study population was categorized in three treatment groups to compare to their differences in parasitological and immunological variables. To assess the effect of treatment in parasite clearance, parasitaemia was evaluated for each treatment at the same time point, before treatment, and every twelve hours of treatment administration. To assess effect of treatment in antibodies production, the mean of the concentrations (AU) of total IgG to P. falciparum antigens was compared between D0 and D28 of treatment. Statistical X2 test was used to compare antibodies production. A value of P< 0.05 was considered statistically significant.

Results and Discussion

Study population characteristics

The volunteers enrolled in this study were 102, among them 46 females and 56 males (the male/female ratio was 1.21). The mean age was 5.96 years old, 47 participants were under 5 years old, 40 participants were aged between 5 to 10 years old and 15 participants were above 10 years old. Thirty-one volunteers were treated with ASAQ, 36 with DHA-PQ and 35 with Pyramax. The geometric mean of P.falciparum asexual stages was 58733 parasites/ μL. Sixty participants had parasitaemia under 50000 parasites/ μLand 42 participants had more than 50000 parasites /μL. Antibody levels before and after treatment Geometric means of antibody levels are summarized in Table 1. The level of antibody responses to GLURP- R2 was higher than that for R0 and MSP3. The antibody responses against the 3 selected antigens measured at D28 were higher than those at D0. However, no statistical significant difference was observed. The data generated in this study clearly showed that naturally exposed subjects were able to elicit antibody responses to malaria specific antigens and confirm that these antigens are immunogenic in stable and hyper-endemic area. Previous studies had similar findings [29-31]. GLURP-R2 was more immunogenic than R0 and MSP3. Comparing the antibody responses to the antigens tested, the same trend was observed. It clearly appears that ACTs do not have any immunosuppressive effect on our study

subject who showed a slight increase in antibody responses D28 following the treatment administration. These findings confirm that infected parasites can have some boosting effect on immune responses but a larger number of study participants are needed to statistically confirm the observation.

Relationship between level of antibodies and age before and after treatment

Antibodies level increases with age for all tested antigens. Young subjects, under 5 years old, produced less antibodies than the elder. The same trend was observed before (D0) and after treatment (D28). An increase in antibody responses to MSP3 and R0 from D0 to D28 was also observed in children below 5 years of age with a borderline statistical significance (P=0.05) and 0.08 for MSP3 and R0, respectively) (Table 2). A decrease in antibody responses to R2 was observed for children below 5 years of age from D0 to D28 with also a borderline statistical difference (P=0.08) (Table 2). Elder subjects produced more antibodies and this trend seems to be conserved irrespective of treatment administered.

Antibody responses and treatment arms

Antibody responses are presented in Figures 1, 2 and 3 respectively for Pyramax, DHA-PQ and ASAQ. Increasing trends were observed for antibody responses in each treatment arm after treatment except in ASAQ treatment arm where decreasing in antibody response to R2 was observed. Irrespective of their presentation formulas artemisinin derivatives do not lead to any suppressive effects in relation to antibodies production against the tested antigens.

Parasite clearance

The 3 treatments (Pyramax, ASAQ and DHA-PQ) were effective in clearing P.falciparum malaria asexual stages (Table 3). All study volunteers cleared their parasites within 60 hours, and 93.1% were parasites free before 36 hours and only 6.9% remained parasitemic between 36 and 60 hours. Medians time to parasite clearance was 27.4 hours for Pyramax and DHA-PQ and 30.8 hours for ASAQ. Pyramax appears to be more efficient on parasite clearance. Within 24 hours of treatment the percentages of patients who were parasites free were 60%, 46.9% and 60% respectively for Pyramax, ASAQ and DHA-PQ. Within 36 hours all study subjects treated with Pyramax were parasite free while 9.4% and 11.4 % receiving ASAQ and DHAPQ were still harboring asexual malaria parasites, respectively (Table 3). Having 38459 parasites/μL before treatment with ASAQ, volunteer parasitaemia decrease rapidly and, at 36 hours after treatment, density decreased to 880 asexual stages parasites/μL. Total clearance was obtained before 60 hours after treatment. In volunteers treated with DHA-PQ, parasite density decreased from 12696 before treatment to 111 parasites /μL at 36 hours after treatment, and parasites were totally cleared before 60 hours. For Pyramax treatment, parasitaemia decreased from 31920 before treatment to 182 parasites /μL at 24 hours after treatment. All parasites were cleared before 36 hours (Table 4). The advantages

of artemisinin based combination therapy (ACT) relate to the unique properties and mode of action of the artemisinin component, which include rapid substantial reduction of P. falciparum malaria parasite biomass. The three drugs were effective in parasites clearance. As DHA-PQ and ASAQ, Pyramax was also efficient in clearing. These data are comparable to those found in other countries [4-6,9]. Pyramax was clearing faster than ASAQ. The shorter parasite clearance time of Pyramax is comparable to that found in Plasmodium vivax malaria patients [32]. Pyronaridine has an estimated terminal elimination halflife of 12 to 14 days [33]. DHA-PQ also had a shorter parasite clearance time comparatively to ASAQ. Piperaquine, one of the components of this ACT, had terminal elimination half-life of around 5 weeks in comparison to 9 to 18 days for amodiaquine, the component of ASAQ [34].

Parasite clearance and age

Ageing is playing a role in parasite clearance. All study subjects above 10 years cleared their parasites within 36 hours. With ASAQ treatment patients aged less than 5 years were parasites free at 60 h following the treatment, those aged between 5-10 years old at 48 hours and those aged more than 10 years at 36 hours (Table 5a). Then with DHA-PQ treatment patients aged less than 5 years cleared all the parasites at 60 hours, and subjects aged between 5-10 years old and those aged more than 10 years parasites clearance was obtained at 36 h (Table 5b). All subjects treated with Pyramax were aparasitic before 36 hours (Table 5c). Parasites clearance is also quickly carried out by older subjects treated with ASAQ and DHA. Elder subjects achieve parasite clearance comparatively sooner than younger individuals. The higher speed in clearing malaria parasites observed in older subjects is probably due to the synergy of high antibody responses and ACTs [29,31,35-42].

Relationship between antibodies and parasite clearance

Patients with antibody titers against all tested antigens above the mean had less parasites than the subjects whose antibody titers were below the mean. Furthermore, high level of antibodies was associated with rapid parasite clearance. For MSP3and R0 total clearance is reached before 48 hours for volunteers with titers above the mean while for subjects with antibody titers less than the mean clearance is obtained before 60 hours. For R2, total clearance is independent of antibody titers but before 60 hours after treatment (Tables 6 and 7). As indicated before, high antibodies levels were associated with rapid clearance [43-46].

Conclusion

The data presented in this report indicate that the use of ACT based therapies in children infected with malaria parasites have no influence on the antibodies response against experimental malaria antigens D28 after beginning of the therapy. This in contrast with studies performed in mice where inhibition of antibody (in vivo) and T cell (in vitro) responses was observed.

This can be explained by the different protocols used for in vivo mouse studies where artemisinin derivatives were given daily for 7 days while in children three doses were prescribed. It is very likely that "in vivo" treatment and antibodies are working in synergy in clearing parasites since elder children resolve the infection in shorter time.

Author Contributions

FT participated in study design, samples collection, laboratory work, data analysis and manuscript preparation. AC participated in study design, laboratory analysis, data analysis and manuscript preparation. SMO participated in study design, laboratory analysis, data analysis and manuscript preparation. GSS participated in study design, samples collection, laboratory analysis, data analysis and manuscript preparation. MK participated in study design, laboratory analysis, data analysis and manuscript preparation. AD participated in laboratory analysis, data analysis and manuscript preparation. YT participated in study design, clinical data collection and manuscript preparation. IS participated in manuscript preparation and overall study supervision. SBS participated in study design, data analysis and manuscript preparation. IN participated in study design, data analysis and manuscript preparation and

overall study supervision.

Acknowledgement

We thank the population of the study site for their cooperation and the Ministry of Health, Burkina Faso. Especial thanks to Giampietro Corradin for his help in data analyses and manuscript review. We are grateful to the staff of CNRFP whose participation has made this study possible. This investigation received financial support from European & Developing Countries Trials Partnership (EDCTP) and Medicines for Malaria Venture (MMV)

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Tables & Figures

The white box-and-whisker plotindicates the geometric means and IC95% for antibodies concentrations at day 0, and the black box-and-whisker plot indicates the equivalent values for day 28.

Figure 1: Comparison of antibody production before (D0) and after (D28) treatment with Pyramax.

The white box-and-whisker plotindicates the geometric means and IC95% for antibodies concentrations at day 0, and the grey box-and-whisker plot indicates the equivalent values for day 28.

Figure 2: Comparison of antibody production before (D0) and after (D28) treatmentDHA-PQ.

The white box-and-whisker plotindicates the geometric means and IC95% for antibodies concentrations at day 0, and the black box-and-whisker plot indicates the equivalent values for day 28.

Figure 3: Comparison of antibody production before (Day 0) and after (Day 28) treatment with ASAQ.