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. 2021 Oct 19;11(11):465. doi: 10.1007/s13205-021-03022-0

Chemoprophylaxis under sporozoites-lumefantrine (CPS-LMF) immunization induce protective immune responses against Plasmodium yoelii sporozoites infection in mice

Arif Jamal Siddiqui 1,3,✉,#, Jyoti Bhardwaj 2,3,#, Walid Sabri Hamadou 1, Manish Goyal 3, Syed Amir Ashraf 4, Sadaf Jahan 5, Arshad Jamal 1, Pankaj Sharma 3,6, Manojkumar Sachidanandan 7, Riadh Badraoui 1, Mohd Adnan 1
PMCID: PMC8526652  PMID: 34745816

Abstract

Malaria represents one of the major life-threatening diseases that poses a huge socio-economic impact, worldwide. Chemoprophylaxis vaccination using a relatively low number of wild-type infectious sporozoites represents an attractive and effective vaccine strategy against malaria. However, the role of immune responses to pre-erythrocytic versus blood-stage parasites in protection against different antimalarial drugs remains unclear. Here, in the present study, we explored the immune responses against the repetitive inoculation of live Plasmodium yoelii (P. yoelii) sporozoites in an experimental Swiss mouse model under antimalarial drug lumefantrine chemoprophylaxis (CPS-LMF). We monitored the liver stage parasitic load, pro/anti-inflammatory cytokines expression, and erythrocytic stage patency, following repetitive cycles of sporozoites inoculations. It was found that repetitive sporozoites inoculation under CPS-LMF results in delayed blood-stage infection during the fourth sporozoites challenge, while sterile protection was produced in mice following the fifth cycle of sporozoites challenge. Intriguingly, we observed a significant up-regulation of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-12) and iNOS response and down-regulation of anti-inflammatory cytokines (IL-4, IL-10 and TGF-β) in the liver HMNC (hepatic mononuclear cells) and spleen cells after 4th and 5th cycle of sporozoites challenge in the CPS-LMF mice. Meanwhile, we also noticed that the liver stage parasites load under CPS-LMF immunization has gradually reduced after 2nd, 3rd, 4th and 5th sporozoites challenge. Overall, our study suggests that chemoprophylaxis vaccination under LMF drug cover develops strong immune responses and confer superior long-lasting protection against P. yoelii sporozoites. Furthermore, this vaccination strategy can be used to study the protective and stage-specific immunity against new protective antigens.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-03022-0.

Keywords: CPS-immunization, Plasmodium yoelii, Lumefantrine, Cytokines response, mRNA expression, Chemoprophylaxis

Introduction

Despite the global efforts, malaria is still represented as one of the major life-threatening diseases. The advent of drug-resistant parasite, insecticide-resistant vector, and lack of effective vaccine emerges as a major threat to global malaria eradication program (Siddiqui et al. 2020a). In the year 2019, approximately 229 million new cases and 500,000 deaths of malaria have been reported (WHO 2020; Possemiers et al. 2021). Thus, there is an urgent need for effective and reliable intervention strategies against malaria to prevent infection and disease transmission.

The clinically silent liver stage (pre-erythrocytic) of the malaria parasite is a promising target for chemotherapeutic and immune-prophylactic based intervention strategies (Vaughan et al. 2008; Molina-Franky et al. 2020). It has been observed that immunization with radiation attenuated sporozoites lessens the impact of antigenic variation and prompts a robust cellular immune response, i.e., protective immunity (Rénia and Goh 2016; Marques-da-Silva et al. 2020). However, this requires a consistent and precise inactivation of sporozoites (inconsistent or incomplete attenuatuation can result in ineffective immunity or infection), appropriate delivery/administration, cold-chain transport/storage and relatively large doses of sporozoites to achieve a high level of efficacy and therefore not ideal for mass vaccination (Marques-da-Silva et al. 2020; Doll and Harty 2014). A feasible alternative for this approach is the use of viable sporozoites in combination with drug treatment to curb the infection (targets liver-stage and/or blood-stage parasites), i.e., chemoprophylaxis vaccination with sporozoites (CVac) (Bijker et al. 2015; Bhardwaj et al. 2016). Antimalarial drugs that target specifically blood-stage parasites were successfully used with sporozoites immunization (either through the bites of infected mosquitoes or direct injection of purified sporozoites) to stimulate the immune responses in mice model of malaria, control human malaria infection and in malaria-naïve individuals (Hill 2011; Duffy et al. 2012; Bijker et al. 2013).

Majority of drugs used in chemoprophylaxis vaccination are those that specifically kill blood-stage parasites and therefore induces sterile immunity by targeting the sporozoites, liver, or blood-stage antigens with much smaller parasite inoculums (Healy et al. 2019; Richie et al. 2015). Some common antimalarial drugs examined so far for CVac-induced immunization are chloroquine (CQ), mefloquine (MQ), artesunate, arteether, azithromycin (Az,) piperaquine (PPQ), isopentane, primaquine (PQ) and pyrimethamine (PYR) (Bijker et al. 2014, 2015; Bhardwaj et al. 2016; Pichyangkul et al. 2017; Friesen et al. 2011; Healy et al. 2020). However, chemoprophylaxis vaccination under antimalarial drug cover need to revalidate several critical issues associated with drug stability, multiple-dose regimen, bioavailability, clinical pharmacology, drug resistance, adverse effects, and safety issues (Fernando et al. 2011; Chakraborty and Parvez 2020).

LMF is a synthetic amino alcohol fluorene derivative similar to antimalarial mefloquine and halofantrine (Quiliano et al. 2016; Ezzet et al. 2000a; Scholar 2007). LMF was found superior to chloroquine and exhibited activity against chloroquine-resistant strains of P. falciparum and P. vivax (Basco et al. 1998b; Ma et al. 2014). LMF is a safe (no adverse side effect), stable, highly effective antimalarial drug used in uncomplicated chloroquine-resistant malaria (Scholar 2007). At present, the drug LMF is widely used in combination with artemisinin and its derivatives as the first-line artemisinin-based combination therapy (ACT) (Pousibet-Puerto et al. 2016; Eastman and Fidock 2009).

Here, in this study, we have examined the feasibility and efficacy of antimalarial drug LMF with concurrent sporozoites administration (CPS-LMF) in the murine P. yoelii, N67 chloroquine (CQ) resistant Swiss Albino mice infection model. We reported that CPS-LMF immunization induces protective sterile immunity in mice with an efficacy comparable to previous findings using chloroquine chemoprophylaxis. We also emphasized the finding that CPS-LMF immunization resulted in an expansion of pro-inflammatory cytokines and down-regulation of anti-inflammatory cytokines. In summary, the CPS-LMF immunization approach is safe and efficacious against the CQ resistant rodent malaria model and can be an important advancement in addressing the bottlenecks of chemoprophylaxis immunization.

Material and methods

Ethics statement

Mice were sacrificed using deep ether anesthesia and all possible efforts were made to reduce the animal suffering in this study. Ethical approval (No: IAEC/2012/67-N) to perform experiments on animals was obtained from CSIR-Central Drug Research Institute’s ‘Institutional Animal Care and Use Committee’ recognized by ‘Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)’, Government of India.

In vivo experimental model

All the animal experiments were performed as per the Institutional Animal Ethics Committee (IAEC) protocol. 6–8-week-old laboratory-bred female Swiss Albino mice (21–23 g body weight) were used for the whole experimental period. P. yoelii N67 rodent malaria parasite strain (CQ resistant) was used in this study. Parasites and mice were maintained at the institute’s laboratory/animal facility as detailed (Siddiqui et al. 2012, 2015; Soni et al. 2015). In brief, the parasite was routinely maintained by the alternating passage of blood-stage parasite (1 × 106 infected RBC) in Swiss Albino mice. P. yoelii infected Swiss mice were further adapted to nourish Anopheles stephensi (A. stephensi) mosquitoes by infectious blood meal for collection of sporozoites (Bhardwaj et al. 2016; Azad et al. 2017). Sporozoites were collected on day 12 post-infection from the mosquito’s salivary gland. Before collection, the infection rate was routinely checked in mosquitoes by microscopic examination of the mosquito’s midgut for the availability of infectious oocysts. On day 12 post-infectious blood meal, control uninfected and/or infected mosquitoes were dissected and salivary glands were ground with a probe in RPMI1640 medium followed by centrifugation at 1000 rpm for 1 min at 4 °C. After centrifugation, the supernatant was collected and transferred in a new tube and checked under a phase-contrast microscope for the presence of sporozoites. Hemocytometer was used for counting the sporozoites number and 1 × 104 sporozoites or salivary gland debris from uninfected mosquitoes (as control) was injected in each mouse intravenously. The parasitemia was observed under the microscope from Giemsa stained thin blood smears.

LMF drug preparation and curative dose determination

LMF is water-insoluble. Hence, the drug was first dissolved in Tween-80 (1–3 drops) to make a clear suspension and further diluted in an applicable amount of sterile water. The LMF was administrated orally via syringe gavage in 0.5 ml volume per mice as milligram/kilogram/day. The curative blood schizonticidal dosage of LMF drug was determined by inoculating the groups of mice (n = 10, each group) intraperitoneally with 1 × 106 P. yoelii infected red blood cells (iRBCs) followed by administration of various doses of LMF drug (5, 10, 20 and 40 mg/kg/day). The drug dosing was continued from the day of P. yoelii infection to the 3 following days (0 to + 3). The untreated control group mice (n = 10) received the same vehicle used for making the LMF drug. Following post iRBC inoculation, parasitemia was monitored daily in both control and LMF drug-treated groups throughout the experimental observation period (28 days). The group of mice which did not develop the parasitemia (erythrocytic stage infection) up to 26–28 days was considered as cured.

LMF drug treatment and immunization procedure

For the CPS-LMF immunization, a group of female Swiss mice was used. A total of 100 mice (n = 100) were injected with freshly isolated 1 × 104 P. yoelii sporozoites via intravenous route, while 15 mice (n = 15) were injected with salivary gland debris from uninfected mosquitoes (control) as shown illustratively (Fig. 1). After the 48 h post sporozoites infection, the curative dose (40 mg/kg/day) of LMF drug was administrated in sporozoites infected mice group (n = 85), while 15 mice from the same group (n = 15) were left untreated as primary inoculum. LMF dosing was continued for four days at the curative dose of 40 mg/kg/day. The LMF-untreated control mice (n = 15) were used to check the blood stage infection (percentage parasitemia, n = 5), liver load (n = 5) and cytokines expression (n = 5) by RT-PCR after primary sporozoites infection. Blood stage infection from control and CPS-LMF-treated mice was checked during the entire experimental course and continued until 21st-day post P. yoelii sporozoite infection.

Fig. 1.

Fig. 1

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Flow chart. Diagrammatic representation of the experimental protocol for the sporozoite inoculation/challenge, drug treatment and allotment of mice at different time points

Following the primary inoculation, LMF treated mice (n = 70) were again infected with the same number of P. yoelii sporozoites (i.e. 1 × 104) (1st challenge). However, some mice (n = 15) were again kept as untreated control group (without LMF) to monitor the parasite burden in the blood (n = 5), liver (n = 5) and the cytokines expression analysis (n = 5) as 1st challange. The remaining mice (n = 70) were further treated with LMF drug 48 h post sporozoites infection as described above. The infection-treatment cycle was repeated till the complete absence of blood-stage patency in the sequentially withdrawn LMF-untreated control mice (n = 10 for 2nd and 4th Challenge and n = 15 for 3rd and 5th challenge). The criteria of sterile protection were defined as the complete absence of erythrocytic blood stage infection till the 28th day of the observation period (after post sporozoite infection). Once the sterile protection was achieved, some of the remaining mice of CPS-LMF immunized group (n = 10) were finally challenged with P. yoelii 1 × 104 sporozoites (n = 5) and 1 × 106 infected RBCs (n = 5). Additionally, a group of mice (n = 10) was also added as naïve control which were also challenged with the same quantity of either P. yoelii sporozoites (1 × 104) or iRBC (1 × 106). Following sporozoites and iRBC challenge, the mice were monitored to check the blood stage infection by Giemsa staining (percentage parasitemia).

Quantification of pre-erythrocytic stage parasite load by Real-Time PCR

The pre-erythrocytic liver stage parasite load was determined by the Real-Time PCR (RT-PCR) assay, as described previously (Bhardwaj et al. 2016; Siddiqui et al. 2012, 2020b). In brief, the liver was individually dissected after 40 h post sporozoites challenge from both LMF untreated, sporozoites infected control (n = 5) and CPS-LMF (n = 5) groups of mice and homogenized in Trizol reagent (Sigma®, St Louis, Missouri, USA). Total RNA from the individual mice liver was isolated and quantified using NanoDrop®. Subsequently, the total RNA was converted to cDNA, and stored at −20 °C for further use. The pre-erythrocytic stage parasite load was monitored by considering the P. yoelii 18S rRNA (copy number) in untreated control and CPS-LMF immunized mice liver samples as described previously (Siddiqui et al. 2015, 2020b). Primers used for 18S rRNA (copy number) analysis are listed in Supplementary Table 1. The percent parasitemia (erythrocytic stage parasite infection) in the untreated control (n = 5) and CPS-LMF (n = 5) groups were measured from Giemsa stained thin blood smears every day using the standard method (Giemsa staining of thin blood smears).

Cytokines expression in HMNC liver and spleen

The pro and anti-inflammatory cytokine responses in the liver HMNCs and spleen of control LMF untreated and CPS-LMF groups were monitored by mRNA-based cytokines expression using the RT-PCR. The HMNCs were isolated from the liver via perfusion as described previously (Siddiqui et al. 2015, 2020b). In brief, mice were anesthetized by deep ether anesthesia and an incision was made on the stomach. Following incision, the needle of the syringe was injected into the specific portal vein. Afterward, inferior vena cava was cut to permit the drainage of blood and the liver perfusion was performed with 1X PBS buffer (pH 7.2). After the perfusion, the liver samples were meshed on a cell-specific strainer to make single HMNCs suspension. The cell suspension was centrifuged at 80×g for 2 min at 25 °C and supernatant was collected and again centrifuged at 460×g for 7 min at 25 °C. Subsequently, the supernatant was discarded and the HMNCs pellet was dissolved in 6 ml of 40% Percoll (Sigma®, St Louis, Missouri, USA), mixed well and gently put over the 3 ml of 70% Percoll. The Percoll gradient tube was centrifuged at 800×g for 25 min at 25 °C to make the clear separation of HMNCs. After centrifugation, the middle layer containing HMNC was carefully taken out and washed with double volume of 1X PBS buffer by centrifugation at 800×g for 6 min at 25 °C.

To analyse the cytokine response, spleen from control untreated (n = 5) and CPS-LMF (n = 5) treated mice were carefully removed and crushed on a 70 µm specific cell strainer to create the single-cell suspension. The single-cell suspension was then centrifuged at 450×g for 6 min and the pellet was washed two times using 1X PBS buffer by centrifugation at 450×g for 6 min at 4 °C. Total RNA was isolated from liver HMNC and spleen using the RNA isolation kit (Cat no.74106, Qiagen® Hilden, Germany), and then converted to cDNA using Thermofisher® cDNA synthesis kit. The cDNA was subsequently used as the template for RT-PCR analysis of various pro and anti-inflammatory cytokines. The reaction conditions for RT-PCR analyses were: primary denaturation; 95 °C for 5 min, followed by amplification of the target cDNA for 45 cycles at 95 °C for 15 s (denaturation), 60 °C for 25 s (annealing) and 72 °C for 30 s (extension). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as a housekeeping gene. Primers used for mRNA expression analysis of pro and anti-inflammatory cytokines are listed in supplementary table 1. For primer designing the Gene Runner® software version 3.05 was used.

Residual effect of LMF drug

To check the residual effect of the LMF drug, we administrated the curative dose (40 mg/kg/day) of LMF to an additional group of Swiss Albino mice (n = 5). All these mice were administrated the curative dose (40 mg/kg/day) of LMF and dosing was continued for 4 days. After the LMF administration, mice were rested for 28 days and again cautiously administrated LMF (sequential drug treatment for five times similar to that of CPS-LMF group except P. yoelii sporozoites infection). After the 5th LMF drug treatment cycle, LMF-treated mice (only drug-treated group n = 5) and naïve control (n = 5) mice were used for P. yoelii sporozoites (1 × 104) challenge studies. After the sporozoites infection, mice were checked daily for blood stage patency (percentage parasitemia).

Statistical analysis

All data are presented as mean ± SD values. Statistical analysis was carried out using GraphPad Prism 5.0 Software. Statistical significance between different groups, i.e., between control and CPS-LMF immunized groups was determined using Student’s t test or one-way ANOVA test with Tukey’s multiple comparison method with significance set at the levels of p ≤ 0.05 (*), p < 0.01 (**) and p < 0.001 (***) which corresponds to significant, highly significant and most significant values, respectively.

Results

Curative dose determination of LMF

The curative dose of LMF (i.e., the dose at which blood-stage infection is completely inhibited) was determined by direct intraperitoneal inoculation of CQ-resistant P. yoelii-infected RBCs (1 × 106) in Swiss Albino mice. Following P. yoelii infected RBCs inoculation; the mice were either left untreated (control) or treated with different doses of LMF for 4 days (0 to + 3). Blood-stage patency (till 28 days post-infection) was considered as the endpoint for the curative dose determination (Fig. 2 and Supplementary Table 2). In this study, we found that daily treatment of mice with LMF at 20 and 40 mg/kg/day for 4 days provides a complete protection against P. yoelii infection, since none of the mice developed parasitemia during the observation period (Fig. 2 and Supplementary Table 2). However, the lower doses of LMF, i.e., 5 and 10 mg/kg/day were unable to inhibit the blood-stage infection in mice and developed the patent parasitemia similar to control LMF untreated mice (Fig. 2 and Supplementary Table 2).

Fig. 2.

Fig. 2

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Determination of blood schizonticidal curative doses of LMF. Kaplan–Meier graph representing the time to malaria infection following Plasmodium yoelii iRBCs infection. To determine the curative doses of LMF, groups of mice (n = 10, each group) was infected with P. yoelii iRBCs (1 × 106) intraperitoneally and treated with different doses of LMF (5, 10, 20, and 40 mg/kg) on the day of P. yoelii infection and for three subsequent days (0 to + 3). Parasitemia was checked regularly using microscopic observation of thin blood smears. The graph shows the day of blood-stage patency, where the y-axis shows the percentage of mice protected from infection at each time point, and the x-axis corresponds to the day of parasitemia onset after challenge

Repetitive inoculation of P. yoelii sporozoites under LMF prophylaxis and protective immunity

Next, we checked whether repetitive infectious P. yoelii sporozoites inoculation under LMF prophylaxis (CPS-LMF) can drive protective immunity/protection in mice from the subsequent sporozoites challenge, even in the absence of LMF drug or not. For this purpose, we first inoculated a group of mice with a single intravenous dose of 1 × 104 live infectious sporozoites (termed as primary inoculation). After 48 h post sporozoites inoculation, we left mice either LMF untreated or treated with LMF drug at a dose of 40 mg/kg/day (curative dose as determined) in a four-dose regimen. The LMF-treated mice were rested for 21 days and were re-inoculated (1st challenge) with 1 × 104 P. yoelii sporozoites and subsequently either treated with LMF drug cover again or left untreated and this infection challenge cycle was continued multiple times. After each sporozoite infection cycle, some mice were withdrawn to analyze blood stage (n = 5) patency, liver parasite burden (n = 5), and cytokines expression (n = 5). Blood stage patency was used as an endpoint criteria for complete protection and mice were monitored continuously (control LMF untreated and LMF treated sequentially withdrawn).

We observed that after the primary sporozoite inoculation, LMF-untreated mice developed a blood-stage infection at day 4 post sporozoites inoculation, which further reached to ~ 35% parasitemia at day 12 post sporozoites infection (Fig. 3). Similarly, LMF treated mice that were rested for 21 days and re-challenged again (1st and 2nd challenge) also did not show any significant change in time and level of blood-stage patency, when compared to control LMF untreated mice (Fig. 3). Conversely, after 3rd and 4th sporozoites challenge, CPS-LMF group mice showed a slight delay in the time of blood stage patency and percentage parasitemia, when compared with control (Fig. 3). Intriguingly, after the 5th sporozoites challenge, we observed complete sterile protection in mice withdrawn from CPS-LMF group (without LMF cover), as none of the mice developed a blood-stage infection during the entire surveillance period of 21 days. As opposed to this, control mice developed patent blood-stage infection at day 4 (Fig. 3). In short, these results suggested that repetitive sequential live infectious sporozoites inoculation under LMF drug cover advance sterile protection in mice.

Fig. 3.

Fig. 3

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Effect of sporozoites immunization under LMF prophylaxis (CPS-LMF) on blood-stage patency. Swiss Albino mice were inoculated with 1 × 104 P. yoelii sporozoites via intravenous route, i.e., primary inoculation (n = 100). Following infection, mice were either remains untreated (n = 15) or treated (n = 85) orally with a curative dose of LMF, i.e., 40 mg/kg/day, 48 h post sporozoites inoculation for 4 days. Mice that had been infected with P. yoelii sporozoites under LMF cover were rested for 21 days and challenged again with 1 × 104 P. yoelii sporozoites with (n = 75) or without (n = 15) LMF treatment, i.e., 1st challenge. The LMF-treated mice were further rested and challenged again with infectious sporozoites for multiple cycles in the presence or absence of LMF cover until protective immunity achieved, i.e. 2nd challenge, 3rd challenge, 4th challenge, and 5th challenge. Each time a new group of mice was also inoculated with 1 × 104 P. yoelii sporozoites shown as control (n = 10–15). Blood stage parasitemia was monitored by Giemsa stained thin blood smear in control LMF-untreated mice from each infection cycle (after sporozoites inoculation). y-axis shows Mean ± SD (n = 5) % parasitemia of LMF untreated withdrawn mice (n = 5) from each sequential CPS-LMF cycle (primary inoculation and subsequent challenge) and the x-axis corresponds to days post sporozoites infection. Error bars represent Standard Deviation. Statistical significances between different groups were determined using student’s t test (indicates ***p < 0.001, **p < 0.01 and ns p > 0.05)

Liver stage parasite burden during the CPS-LMF immunization

Next, we assessed the impact of CPS-LMF immunization on liver stage parasite burden through RT-PCR assay. For that purpose, liver samples were used to extract the total RNA from control LMF untreated and withdrawn mice after each sequential sporozoites challenge group. In our experiments, LMF treatment was initiated only 48 h post sporozoites inoculation, while liver samples were collected 40 h post sporozoites infection. Therefore, we compared the liver stage parasite load between control sporozoites-infected group (LMF untreated, primary inoculation) and mice withdrew after 1st to 5th sporozoites challenge groups (Fig. 4). The parasite liver load was shown in terms of 18S rRNA copy number as calculated from the standard curve described previously (Siddiqui et al. 2015, 2020b). Our results showed a comparable liver stage parasite load in control sporozoites-infected (LMF untreated) and LMF-treated 1st sporozoites challenge group, thus suggesting that LMF treatment was unable to inhibit the parasite liver stage development after 1st sporozoites challenge (Fig. 4).

Fig. 4.

Fig. 4

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Effect of CPS-LMF prophylaxis on pre-erythrocytic liver stage parasite burden. To evaluate the effect of CPS-LMF on liver-stage parasite load, liver samples from control sporozoites-infected (LMF untreated, n = 5) and sporozoites infected, LMF drug-treated mice (n = 5) from each sequential CPS-LMF cycle (challenge 1st to 5th) were dissected 40 h post sporozoites infection. The parasite burden was assessed through Real-time PCR by calculating the relative expression levels of the Py18S rRNA gene normalized to the mouse GAPDH gene expression. Individual values for each mouse (n = 5) and median are plotted. The y-axis shows Mean ± SD (n = 5) values of Py18S rRNA copy numbers. Statistical significances between different groups were determined using student’s t test (indicates ***p < 0.001, **p < 0.01 and ns p > 0.05)

Moreover, we observed a gradual reduction in liver-stage parasite load in mice withdrawn after subsequent challenge groups (i.e., 2nd to 4th challenge). The 18S rRNA copy number in mice withdrawn after 2nd, 3rd and 4th sporozoites challenge (704,403.76 ± 78,302, 260,622.03 ± 114,533 and 8373 ± 2281) was steadily decreased when compared with the control sporozoites infected group (without LMF treatment, 1,011,484.87 ± 66,902, 974,941.57 ± 40,378 and 1,398,144.45 ± 212,969) (Fig. 4). Interestingly, after 5th sporozoites challenge, we observed a complete loss of pre-erythrocytic liver stage parasite load, when compared with the control group (Fig. 4). These results were in accordance with blood-stage parasitemia, where we also observed complete sterile protection after the 5th sporozoites challenge in the absence of LMF drug cover (Fig. 3). Thus, our results showed that repetitive sporozoites inoculation under CPS-LMF drug cover induces protective immunity in mice against subsequent sporozoites challenge.

Pro-inflammatory and anti-inflammatory cytokines responses during CPS-LMF immunization

Since repetitive sporozoites inoculation under LMF cover developed the protection against further sporozoites challenge, we then assessed the role of immune responses during this immunization process. For that purpose, we measured mRNA-based cytokines expression level at 40 h post sporozoites infection in liver HMNC and spleen from control uninfected, control sporozoites infected LMF untreated (primary inoculation) and sporozoites infected LMF-treated mice (CPS-LMF immunized mice) withdrawn after 1st, 3rd, and 5th sporozoites challenges (Figs. 5 and 6). For pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) response, we measured the mRNA expression of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-12 (IL-12) and iNOS, while for anti-inflammatory cytokines we measured mRNA expression of interleukin-4 (IL-4), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-β). Our results showed that the mRNA expression of pro-inflammatory cytokines in liver HMNCs (IFN-γ, TNF-α, IL-12, and iNOS) was down-regulated at 40 h post sporozoites inoculation (primary inoculation, without LMF treatment), when compared with control uninfected mice (4.5, 4.3, 11.1 and 9.1 relative fold change, respectively) (Fig. 5, Supplementary Table 3). However, in the subsequent sporozoites challenges, the mRNA expression of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-12) and iNOS response gradually up-regulated with almost > 10 and > 20–95 fold up-regulation after 3rd (14.7, 9.07, 9.37 and 10.17 relative fold change, respectively) and 5th challenge (95.0, 46.28, 57.14 and 54.45 relative fold change respectively) in comparison with the untreated control group (Fig. 5, Supplementary Table 3). On the other hand in mice spleen, mRNA expression of pro-inflammatory cytokines such as IFN-γ, and TNF-α did not show any marked changes, while IL-12 and iNOS response were down-regulated at 40 h post sporozoites inoculation (primary inoculation, without LMF treatment) as compared with control uninfected mice (1.4 and 2.4 relative fold change) (Fig. 5, Supplementary Table 3). Similar to liver HMNCs, the mRNA expression of IFN-γ, TNF-α, IL-12, and iNOS was radically amplified after sporozoites challenges with ~  > 10 and > 20–40 fold after the 3rd (9.2, 7.7, 8.0 and 6.1 relative fold change, respectively) and 5th sporozoites challenge (48.8, 35.6, 50.8 and 37.2 relative fold change, respectively) (Fig. 5, Supplementary Table 3).

Fig. 5.

Fig. 5

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Relative mRNA expression of pro-inflammatory cytokines in HMNCs and spleen cells. Liver HMNCs and spleen cells were isolated from control uninfected and P. yoelii sporozoites infected mice (n = 5) at different time points during infection and challenge cycles, i.e., 40 h post-infection (primary inoculation), after 1st, 3rd, and 5th sporozoites challenges. Total RNA was isolated, converted into cDNA, and used as a template to measure mRNA expression of pro-inflammatory cytokines using RT-PCR as described in the material and method section. Each bar represents the Mean ± SD (n = 5) fold changes of respective cytokines in HMNCs and splenocytes. Statistical significances between different groups were determined using one-way ANOVA test with Tukey’s multiple comparison method (indicates ***p < 0.001, **p < 0.01, *p < 0.05 and ns p > 0.05)

Fig. 6.

Fig. 6

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Relative mRNA expression of anti-inflammatory cytokines in HMNCs and spleen cells. Liver HMNCs and spleen cells were isolated from control uninfected and P. yoelii sporozoites infected mice (n = 5) at different time point during infection and challenge cycles, i.e., 40 h post-infection (primary inoculation), after 1st, 3rd and 5th sporozoites challenges. Total RNA was isolated, converted into cDNA, and used as a template to measure mRNA expression of anti-inflammatory cytokines using RT-PCR as described in the material and method section. Each bar represents the Mean ± SD (n = 5) fold changes of respective cytokines (IL-4, IL-10, and TGF-β) in HMNCs and splenocytes. Statistical significances between different groups were determined using one-way ANOVA test with Tukey’s multiple comparison method (indicates ***p < 0.001, **p < 0.01, *p < 0.05, and ns p > 0.05)

However, anti-inflammatory cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β) were first upregulated to > four fold (after 40 h post sporozoites inoculation, i.e., primary inoculation) and then gradually down-regulated during the CPS-LMF immunization in sequential sporozoites challenge group compared to control uninfected in both liver HMNCs and spleen (Fig. 6, Supplementary Table 3). It has also been observed that after the 5th CPS-LMF immunization, the mRNA expression of anti-inflammatory cytokines in liver HMNCs declined below the level of control uninfected group (2.9, 4.5 and 9.1 relative fold change, respectively) (Fig. 6, Supplementary Table 3). The anti-inflammatory cytokines in the spleen of CPS-LMF immunized group were also down-regulated in a similar manner as compared with control uninfected group (Fig. 6, Supplementary Table 3). Thus, collectively our results showed that the down-regulation of anti-inflammatory cytokines and upregulation of pro-inflammatory cytokines were associated with the sterile protection in CPS-LMF immunized mice.

Determination of residual effect of LMF drug

To check, whether the protection in mice after 5th sporozoites challenge (CPS-LMF immunized group) was due to the residual effect of LMF or not, we incorporated an additional group of mice as prophylactic drug control in our study. These mice have been cautiously administrated with the sequential drug treatment (each time point after 28 days interval) without P. yoelii sporozoites infection. The prophylactic control group of mice was then finally challenged with P. yoelii sporozoites (1 × 104) after 28 days of last LMF drug administered. The results of this study showed that the prophylactic control (with LMF only) and naïve control (without LMF) mice both developed blood-stage patency on the same day with a comparable level of parasitemia. Thus, completely ruled out the possibility of the residual effect of the LMF drug in the protection against sporozoites challenge (Supplementary Fig. 1).

Sporozoites and iRBCs challenge studies in post-immunized mice

Next, to verify the nature of CPS-LMF induced protection, we again challenged the protected mice (after 5th sporozoites challenge) with the same quantity of P. yoelii sporozoites (1 × 104) and iRBC (1 × 106) (Fig. 7). Following the sporozoites challenge, we found that none of the CPS-LMF immunized mice developed blood-stage patency as compared to the naive control group which developed a blood-stage infection at day 4 (Fig. 7A). On the other hand, when CPS-LMF immunized mice were challenged with iRBC blood-stage infection (parasitemia) was noticed. However, the immunized group of mice showed delayed in blood-stage infection (4 days), when compared to naïve control group (Fig. 7B). Thus, our results showed that sporozoites immunization of mice through CPS-LMF approach protect against the subsequent sporozoites challenge, while a partial protection was achieved against P. yoelii iRBC, and mice developed the blood-stage infection similar to naïve control.

Fig. 7.

Fig. 7

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Post immunization challenge studies in CPS-LMF immunized mice. Mice protected following CPS-LMF immunization were challenged (1 month after last sporozoites inoculation) with 1 × 104 P. yoelii sporozoites or 1 × 106 iRBCs. Graphs show the Mean ± SD (n = 5) % parasitemia following A sporozoites and B iRBCs challenge in CPS-LMF immunized and naïve control groups of mice. Statistical significances between different groups were determined using student’s t test (indicates ***p < 0.001 and **p < 0.01)

Discussion and conclusion

The clinically silent liver stage of malaria parasite is widely used to induce protective immunity by targeting parasite before, during or shortly after liver stage growth (Bijker et al. 2015; Goh et al. 2019). However, this immunization approach needs sufficient, precise and specific attenuation and large scale production of sporozoites (Itsara et al. 2018). In recent years, infection-treatment vaccination with antimalarial drug cover targeting blood stages (CPS-immunization) has gained global attention and a considerable progress was seen with several antimalarial drugs in human studies, and mouse malaria models (Bhardwaj et al. 2016; Bijker et al. 2013, 2014). CPS immunization emerges as one of the most potent whole sporozoites vaccination (WSV) approach that elicits protective immune responses by fewer immunizations (dose of parasites) than radiation-attenuated sporozoites or genetically attenuated parasites (Nahrendorf et al. 2015; Itsara et al. 2018; Doll and Harty 2014). One possible explanation for this phenomenon is accessibility of additional antigenic targets due to complete liver-stage development and aborted blood-stage infection. However, emergence of drug resistance in malaria parasite, poor drug metabolic stability, multiple dose drug regimen and differences in the stringency of protection are some potential issues in CPS-immunization that need to be addressed (Bijker et al. 2015; Goh et al. 2019). Despite the availablity of numerous registered antimalarial drugs with proven chemoprophylactic efficacy (vary in efficacy, safety and mechanisms of action), only a handful of them have been evaluated in clinical or animal studies for CPS-immunization. Thus, to advance CPS immunization, evaluation of new antimalarial drugs and a direct comparison of them is urgently required.

LMF drug is popularly known as “co-artemether”, a lipophilic synthetic antimalarial drug that is used in combination as antimalarial therapies to treat acute uncomplicated and CQ resistant malaria (Ehrhardt and Meyer 2009). The LMF drug is well tolerated (does not appear to have any significant adverse effects) without showing evidence of a teratogenic risk in the first trimester (Aronson 2016; Padberg 2015; Ajayi et al. 2008). The drug is also cost effective and have moderate half-life of ~ 6 days that make it appropriate combination drug to provide greater post-treatment prophylaxis than the other short half-life drugs (Padberg 2015; Ezzet et al. 2000b; Ajayi et al. 2008). Moderate half-life of the LMF drug also provides an additional advantage, since longer half-life of the drug may prone of resistant parasites spread more quickly (Padberg 2015; Blasco et al. 2017). In addition, clinical resistance to LMF has not been reported officially yet, but some studies suggested that LMF may inhibit parasite development in the mosquito (Adjalley et al. 2011; van Pelt-Koops et al. 2012).

On these metrics, we have examined the potential of LMF drug for prophylactic cover in CPS-LMF immunization and explored the role of cytokines expression during CPS-LMF immunization procedure. For that purpose, we first determined the curative dose of LMF drug in the Swiss Albino mice under our experimental conditions. We found that LMF at a dose of 20 and 40 mg/kg/day per mice from the day of P. yoelii infection to the 3 following days (0 to + 3) showed full protection against blood stage infection. These curative doses of LMF drug were in close agreement with previous studies accomplished with P. yoelii parasite (Sidhu et al. 2006; Dama et al. 2017; Basco et al. 1998a). Next, we performed the immunization procedure under LMF drug cover and used the pre-erythrocytic liver stage parasite load and erythrocytic stage infection as an endpoint to study the sterile immunity. In our results, we noted that after the initial LMF treatment cycles (1st and 2nd challange studies), significant changes were not observed in pre-erythrocytic stage parasite load and erythrocytic stage patency, when compared to control LMF untreated parasites. However, after 3rd sporozoites challenge, we observed a decline of pre-erythrocytic liver and blood stage parasite load and an absolute sterile protection was observed after the 5th sequential sporozoites challenge. Furthermore, the repetitive immunization with CPS-LMF cover not only develops sterile protection against infectious sporozoites challenge, but also induces partial immunity against iRBCs challenge (blood stage infection) in the mice. However, the CPS-LMF immunization does not protect mice against iRBCs infection as the mice developed delayed blood stage patency and eventually died.

In previous studies, it has been observed that the presence of transient low-level parasitemia during CPS immunization is sufficient to induce immune recognition of asexual forms (Nevagi et al. 2021; Bijker et al. 2013; Bhardwaj et al. 2016). However, in case of CPS-LMF immunization probably the elicited immune response seems insufficient to confer complete functional blood-stage immunity. Moreover, these results are encouraging and consistent with previous CPS immunization approaches where sterile protection was conferred against pre-erythrocytic liver stage parasites under various drug treatment cover using different host-parasite model system (Bhardwaj et al. 2016; Bijker et al. 2013; Tran et al. 2019; Zenklusen et al. 2018; Belnoue et al. 2004; Peng et al. 2014). Next, we assessed the prime mediators of this protective immunity in CPS-LMF immunized mice by evaluating the mRNA-based pro and anti-inflammatory cytokines expression in liver and spleen before and during the sequential CPS-LMF immunization procedure. After the primary sporozoites inoculation, we found a significant down-regulation in the mRNA expression of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-12) and iNOS response while up-regulation of anti-inflammatory cytokines (IL-4, IL-10 and TGF-β) in comparison with control uninfected group mice. These results were in agreement with our previous studies in which significant reduction in the effectors immune cells population (CD8+ T cell, F4/80+ macrophage, plasmacytoid dendritic cells, i.e., CD11c+ B220+) was noted (Siddiqui et al. 2020b). It has been suggested that during pre-erythrocytic development, sporozoite impaired the antigen presentation, and pro-inflammatory cytokines, while elevated the anti-inflammatory cytokines to assist the establishment of infection and subsequent development in the liver and spleen (Siddiqui et al. 2020b; Schneider et al. 2011; Zheng et al. 2014; Schneider and Higgs 2008; Klotz and Frevert 2008).

In addition, during the CPS-LMF immunization, we observed a reverse trend in cytokines mRNA expression and noted that pro-inflammatory cytokines level slowly goes up, while anti-inflammatory cytokines level slowly went down with a maximum following 5th sporozoites challenge, when compared with both control sporozoites-uninfected and sporozoites-infected LMF-untreated groups. Cytokines associated immune response is a well-known phenomenon during the WSV approach and also connected to the susceptibility of mice to other parasite strains (Burrack et al. 2019; Bhardwaj et al. 2016; Sato et al. 2019). Previous studies using whole sporozoites vaccination approach in human and mouse models have suggested a potential role of liver-stage directed pro-inflammaotry immune responses (CD4+/CD8+ T cell) in developing the protective immunity (Goh et al. 2019; Keitany et al. 2014b; Tarun et al. 2007; Moita et al. 2021). In agreement with this, further interrogation of the T cell responses in animals and human subjects revealed the persistent induction of IFN-γ, TNF-α, NO and abundance of sporozoite-specific central and effector memory CD8+ T cells, and IFN-γ producing CD8+ T cells (Belnoue et al. 2004; Butler et al. 2011; Trimnell et al. 2009; Tarun et al. 2007). Moreover, in vitro studies further demonstrated that the treatment of sporozoite-infected hepatocytes with IFN-γ showed eradication of parasites (P. berghei and P. falciparum malaria parasites) (Doll and Harty 2014; Ferreira et al. 1986; Tweedell et al. 2018; Lelliott and Coban 2016; Gun et al. 2014). In addition, in vivo treatment studies with IFN-γ also suggested a partial protection in the mice and monkey against P. berghei and P. cynomolgi sporozoites challenge (Keitany et al. 2014a; Lelliott and Coban 2016; Gun et al. 2014).

Similarly, numerous other studies also documented the connection between the IFN-γ, TNF-α, IL-12 and iNOS induction and sterile protection following sporozoite challenge (Blanchette et al. 2003; Salim et al. 2016; Romero et al. 2007; Frevert and Krzych 2015; Ferreira et al. 1986; Percário et al. 2012; Mendes et al. 2018). Besides this, it has also been suggested that anti-inflammatory cytokines (such as IL-10 and TGF-β) reduces the expression levels of MHC class II and co-stimulatory molecules on the surface of liver resident dendritic cells (DCs), thus reducing their capability to activate T cells (Osii et al. 2020; Racanelli and Rehermann 2006). Knolle et.al have shown that secretion of IL-10 by kupffer cells inhibit T cell activation, impaired pro-inflammatory cytokines and elevated the anti-inflammatory cytokines (Knolle et al. 1998).

In our study, we found a significant down-regulation of CD4+ T cell-derived IL-4 following 5th sporozoites challenge. It has been reported that IL-4 plays an important role in stimulating effector CD8+ T cell responses against infected hepatocytes (Carvalho et al. 2002; Kurup et al. 2019; Overstreet et al. 2011). IL-4 has also been shown to be involved in preserving the Plasmodium-specific memory CD8+ T cell populations (Kurup et al. 2019). In this view, it is difficult to assume the exact role of IL-4 in sterile protection. It seems that IL-4 expression is context-specific during CPS-LMF immunization and further work is required to illustrate the specific role of this cytokine.

Taken together, our study validated the lumefentrine as safe, effective chemoprophylactic drug and immunization with sporozoites under LMF chemoprophylaxis results in robust sterile protective immunity against subsequent sporozoites challange. In addition, we also observed a noteworthy change in the mRNA expression of cytokines (up-regulation of pro-inflammatory cytokines and down-regulation of anti-inflammatory cytokines) during the course of immunization. In this view, our study also supports the hypothesis that the liver stage immunity developed during the CPS immunization is elicted primarily by highly dominated pro-inflammatory cytokines response (CD4+/CD8+ T cell) and suppressive anti-inflammatory cytokines response. However, to advance our understanding of the immune mechanisms involved in CPS-LMF immunization future studies such as cytokine measurement at protein level, phenotypic characterization of different subsets of immune cells population and antibody-mediated depletion of CD4+ or CD8+ T cells just prior to challenge are needed to further validate these observations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Table 1 (DOCX 15 KB) (12.9KB, docx)
Supplementary Table 2 (DOCX 15 KB) (13.1KB, docx)
Supplementary Table 3 (DOCX 15 KB) (13.8KB, docx)
13205_2021_3022_MOESM4_ESM.tif (346.1KB, tif)

Supplementary Figure 1 (TIF 346 KB) Residual effect of LMF drug on prophylaxis control. To check the residual activity of LMF drug, blood-stage parasitemia was monitored in mice treated with only LMF (curative dose) without sporozoites inoculation similar to as with CPS-LMF immunization group. Naïve Control and Prophylaxis LMF treated control mice were subsequently infected intravenously with 1×104 P. yoelii sporozoites to check the residual activity of LMF. The y-axis shows the Mean (n=5) % parasitemia of mice from each group, as determined by microscopy of Giemsa-stained thin blood smears, and the x-axis corresponds to days post sporozoites infection. Error bars represent Standard Deviation. Statistical significances between different groups were determined using student’s t test.

Acknowledgements

I would like to thank and appreciate all the support and technical assistance provided by Dr. Puri lab at CSIR-CDRI, Lucknow, India during this study. I further extend my gratitude to UoH for graciously providing numerous contributions and making all endeavors possible.

Author contribution

Conceptualization, AJS, JB, and MG; methodology, WSH, AJ, and SAA; validation, MA MS, RB, SJ, and PS; formal analysis, MA, MG RB, and AJS; investigation, AJ, SAA and JB; data curation, MA, AJ, WSH, SAA and MS; writing—original draft preparation, AJS JB, and MA; writing—review and editing, MG, MA, and PS; visualization, AJ, and WSH; supervision, AJS, MA, and JB; project administration, AJS and JB. All authors have read and agreed to the published version of the manuscript.

Funding

This research has been funded by Scientific Research Deanship at University of Ha’il, Saudi Arabia through project number RG-20128.

Declarations

Conflict of interest

The authors have declared no conflict of interest.

Footnotes

Arif Jamal Siddiqui and Jyoti Bhardwaj have contributed equally to this work and share first authorship.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table 1 (DOCX 15 KB) (12.9KB, docx)
Supplementary Table 2 (DOCX 15 KB) (13.1KB, docx)
Supplementary Table 3 (DOCX 15 KB) (13.8KB, docx)
13205_2021_3022_MOESM4_ESM.tif (346.1KB, tif)

Supplementary Figure 1 (TIF 346 KB) Residual effect of LMF drug on prophylaxis control. To check the residual activity of LMF drug, blood-stage parasitemia was monitored in mice treated with only LMF (curative dose) without sporozoites inoculation similar to as with CPS-LMF immunization group. Naïve Control and Prophylaxis LMF treated control mice were subsequently infected intravenously with 1×104 P. yoelii sporozoites to check the residual activity of LMF. The y-axis shows the Mean (n=5) % parasitemia of mice from each group, as determined by microscopy of Giemsa-stained thin blood smears, and the x-axis corresponds to days post sporozoites infection. Error bars represent Standard Deviation. Statistical significances between different groups were determined using student’s t test.

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