Background
Whole sporozoite immunization approaches, such as chloroquine chemoprophylaxis and sporozoites (CPS-CQ) and radiation-attenuated sporozoites (RAS), efficiently induce protection in murine malaria models [
1-
3]. In humans CPS-CQ is about 20 times more efficient than RAS, requiring bites from a total of 45
versus 1,000 mosquitoes, respectively [
4-
6]. Moreover, long-lasting immune responses after CPS-CQ immunization in studies with mice [
7] and healthy human volunteers [
8] go together with protracted protection. Several murine studies have demonstrated the essential role of CD8
+ T cells in sporozoite-induced pre-erythrocytic immunity [
9-
14,
2]. Generation of these CD8
+ T cells against pre-erythrocytic antigens requires cross-priming by dendritic cells [
15].
CQ has since its discovery in 1934 been used widely and successfully as anti-malarial, until resistance developed [
16], and has more recently been explored for treating cancer and viral infections [
17,
18]. Interestingly, CQ was also shown to enhance cross-presentation of soluble antigens and non-replicating influenza virus
in vitro [
19,
20]. Moreover,
in vivo cross-priming of naïve CD8
+ T cells with soluble ovalbumin was more effective in CQ–treated compared to untreated mice [
21], and CQ improved the induction of influenza-specific cytolytic T cells in mice [
20]. In humans, co-administration of CQ with a hepatitis B vaccine booster significantly increased the number of virus-specific IFNγ-producing CD8
+ T cells [
19].
This study was based on the hypothesis that CQ, which affects endosomal acidification and the degradation and transport of antigens to the cytosol [
22,
23], could favour cross-presentation of pre-erythrocytic
Plasmodium antigens and thereby contribute to the efficient induction of immune responses and protection by CPS-CQ immunization. The first topic of investigation was the effect of CQ on immunization by RAS, an established immunization model relying on CD8
+ T cell responses, thus potentially benefiting from improved cross-presentation. CD8
+ T cells recognizing the immunodominant circumsporozoite (CS) protein can mediate protective immunity [
24]. Therefore, the effect of CQ on the induction of CS-specific CD8
+ T cells by immunization with
P. berghei parasites that express a mutant CS protein containing the model SIINFEKL H-2K
b epitope was investigated next. Finally, a direct comparison was performed betweeen CPS-CQ and CPS with mefloquine (MQ), an anti-malarial with little known immune modulating effects [
25-
27], not including improvement of cross-presentation. Akin to CQ, MQ induces arrest of early blood-stage parasites without an effect on pre-erythrocytic parasite stages, allowing full liver-stage development and brief exposure to early blood stages. By performing experiments in these selected complimentary
P. berghei murine models, the aim of this study was to explore the effect of CQ on protection and T cell responses after whole sporozoite immunization.
Methods
Mice and parasites
Balb/cByJ and C57BL/6j mice (6 to 8 weeks old) were purchased from Elevage-Janvier (Le Genest Saint Isle, France). These mouse strains were selected based on extensive experience with these strains for malaria immunization studies [
28]. The following parasites were used:
P. berghei (ANKA strain) wild type parasites and
P. berghei CS5M parasites in which the endogenous CS gene had been replaced with a modified circumsporozoite gene expressing the H-2Kb restricted SIINFEKL [
15]. Sporozoites were obtained by dissection of the salivary glands of infected female
Anopheles stephensi mosquitoes 21–29 days after a blood meal on infected mice. All animal studies and procedures performed in the Netherlands were approved by the Ethical Committee on Animal Research of the Radboud University Nijmegen (RU-DEC 2009–179, 2009–225, 2010–115, 2010–135). Mice were housed at the Central Animal Facility in Nijmegen and received a standard diet and water
ad libitum. All animal procedures in the United States of America were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University (Protocol Number MO10H167) and followed the National Institutes of Health guidelines for animal housing and care.
Immunization schedules, sporozoite challenge and assessment of protection
Mice were immunized with one (Balb/cByJ; Additional file
1A) or two to three (C57BL/6j; Additional file
1B) intravenous (iv) injections of
P. berghei RAS (16krad, Gammacel 1000
137Cs) at weekly intervals or with one intradermal injection of
P. berghei CS
5M RAS (Additional file
1: Figure S1C). Dose de-escalation of RAS immunization was performed in order to obtain a suboptimal RAS dose to detect possible beneficial effects of CQ (Additional files
1A and 1B). In RAS experiments, RAS-CQ groups received either CQ prophylaxis (chloroquine diphosphate, Sigma-Aldrich) for 10 days (Balb/cByJ –1040 μg base/day oral) or 17 days (C57BL/6j –800 μg base/day intraperitoneal). Efficacy of these prophylactic regimens was established in pilot studies, and they were chosen because of their closest resemblance to the human CPS-protocol. Alternatively, mice were given two subcutaneous injections of 500 μg CQ base, 2 h before and 6 h after each immunization, because this particular regimen was previously shown to improve cross-presentation (Additional file
1B) [
21,
20].
Furthermore, mice under CQ or MQ prophylaxis were immunized three times at weekly intervals by intravenous administration of 20,000 wild-type
PbSPZ (CPS immunization; Additional file
1D). For CPS immunization, CQ (diluted in PBS) and MQ (diluted in DMSO/water for injection) were given orally for 24 consecutive days at dosage 1040 μg base/day (CQ) or 350 μg base/day (mefloquine hydrochloride, Sigma-Aldrich) starting from the first day of
PbSPZ administration.
Challenge infections were performed by intravenous injection of 10,000 or 50,000 sporozoites around four or eleven weeks after the end of CQ/MQ prophylaxis. Giemsa-stained blood smears were screened for parasitized red blood cells every other day from days 3–14 and finally on day 21 after challenge. Protection was defined as the absence of blood-stage parasites until day 21 post-challenge (Additional files
1A, B and C).
Ex vivo memory phenotyping and in vitro IFNγ responses against sporozoites and blood-stage parasites
Mice were euthanized by isoflurane inhalation after intravenous injection of 50 units heparin. Spleen and liver were collected after perfusion of the liver with 10 ml PBS. Cell suspensions of liver and spleen were made by passage of the organs through a 70-μm nylon cell strainer (BD Labware). Liver cells were re-suspended in 35% Percoll (GE Healthcare) and centrifuged at 800 g for 20 min. Liver and spleen erythrocytes were lysed using 5 min incubation on ice in a lysing solution of ammonium chloride. After erythrocyte lysis, hepatic mononuclear cells (HMC) and splenocytes were re-suspended in RPMI 1640 medium.
Five-colour staining of HMC and splenocytes was performed using monoclonal antibodies purchased from Biolegend: Pacific blue-conjugated anti CD3 (17A2), Peridinin Chlorophyll Protein (PerCP)-conjugated anti CD4 (RM4.5), Alexa fluor 700-conjugated anti CD8a (53–6.7), fluorescein isothiocyanate (FITC)–conjugated anti-CD44, allophycocyanin (APC)– or phycoerythrin-Cy7 (PE-Cy7)-conjugated anti-CD62L (MEL-14). Briefly, 106 cells were re-suspended in cold assay buffer (PBS supplemented with 0.5% bovine serum albumin (Sigma-Aldrich)) and incubated for 30 min at 4°C with the monoclonal antibodies. Cells were fixed with Fix & Perm medium A (Invitrogen) and collected in an assay buffer for measurement.
For the detection of parasite-specific cytokine production, HMC and splenocytes (5x10
5 cells/well) were co-cultured in complete RPMI 1640 culture medium [
29] in the presence of
P. berghei cryopreserved sporozoites (
PbSPZ - 5×10
4/ml) or infected red blood cells (
PbiRBC - 5×10
6/ml). Exposure to salivary gland preparations from uninfected mosquitoes and uninfected red blood cells (uRBC - 5×10
6/ml) were used as respective negative controls. Cells were stimulated at 37°C/5%CO2 for 24 hours and Brefeldin A (Sigma-Aldrich) was added during the last four hours (10 μg/ml final concentration). As positive control, PMA (100 ng/ml) and Ionomycin (1.25 μg/ml) (Sigma-Aldrich) were added simultaneously along with Brefeldin A. Cells were harvested after 24-hours
in vitro stimulation and stained with monoclonal antibodies against CD3, CD4, CD8a and CD44 as indicated above. Fixed and permeabilized cells were stained with PE-conjugated anti-IL-2 (JES6-5H4) and APC-conjugated anti-IFNγ (XMG1.2) in Fix & Perm medium B (Invitrogen) at 4°C for 30 min. Flow cytometry was performed on a 9-color Cyan ADP (Beckman Coulter) and data analysis was performed using FlowJo software (version 9.1; Tree Star), using a gating strategy as described previously [
7].
Quantification of SIINFEKL specific CD8+ T cells
Prior to intradermal immunization with 20.000
P. berghei CS
5M RAS, C57BL/6j mice received 2*10
3 CD45.1 + OT-1 cells and the RAS-CQ group received a 10-days CQ treatment (1040 μg base/day – oral). Expansion of CD45.1
+CD8
+ SIINFEKL cells in liver and spleen was assessed by flow cytometry ten days after immunization as described previously [
15] (Additional file
1C).
Data analysis and statistics
Difference in protection between two groups was tested with a Fisher’s exact test. Overall comparisons between immunized and naïve groups were performed using the Kruskal-Wallis test. Direct comparisons between two groups (RAS versus RAS-CQ or CPS-CQ versus CPS-MQ) were performed by Mann–Whitney U test. For the analysis of cytokine production, background responses to salivary glands and uRBC were subtracted from PbSPZ and PbiRBC responses, respectively, for each individual mouse. All statistical analyses were performed using PRISM software version 5.0 (Graphpad, San Diego, CA). A p-value of ≤0.05 was considered statistically significant.
Discussion
Addition of CQ to a
P. berghei RAS immunization protocol improves neither protection nor parasite-specific CD8
+ T cells responses. Only slightly reduced T cell responses and similar protective efficacy are found when CPS-CQ is compared to CPS-MQ. This study did not involve an investigation of the effect of CQ on cross-presentation
in vitro, nor detailed mechanistic antigen presentation studies [
31]. Alternatively, more functional readouts were examined such as parasite-specific CD8
+ T cell responses and protection from challenge infection to assess potential immune enhancing effects of CQ. These combined
P. berghei data do not provide evidence for significant improvement of whole sporozoite immunization in the presence of CQ and, therefore, indicate that CQ is not responsible for the strikingly higher efficiency of CPS-CQ compared to RAS in humans.
Improved cross-presentation resulting in increased IFNγ production by CD8
+ T cells has been shown in
in vitro studies where dendritic cells were pulsed with soluble viral antigen in the presence of CQ [
19]. In mice, CQ was shown to enhance cross-presentation of soluble OVA to OT-I cells both
in vitro and
in vivo and to improve specific CD8
+ T cell responses after alum-OVA immunization [
21]. An effect of CQ on OVA cross-presentation was observed upon administration of 20 μg, but not 200 μg protein [
21], suggesting that immunomodulatory effects of CQ are only beneficial under suboptimal immunization conditions. In the current study, RAS immunization both with and without CQ induces strong cellular responses with similar contribution of CD8
+ T cells, which translates to equal protection levels. Even after down-titration of RAS immunization dose, which is associated with decreasing protection, clear improvement by CQ remains undetected.
In humans, a single administration of CQ during Hepatitis B booster vaccination significantly improved CD8
+ T cell response [
19]. Despite several reports of enhanced immune responses by CQ in mice and men [
21,
23,
32,
19], only one study has reported improved protection; mice immunized with a heat-inactivated influenza virus showed improved survival rates after challenge infection [
20]. The significance of improved immune responses for protection thus remains to be further explored.
One cannot assume that improvement of cross-presentation by CQ is applicable to any soluble protein or peptide, or antigens presented in the form of a whole sporozoite. Improvement of cross-presentation of SIINFEKL peptide (OVA
257–264) and inactivated influenza virus by CQ in mice have been demonstrated [
21,
20]. Here however, CQ administration during sporozoite immunization with SIINFEKL expressing
P. berghei sporozoites showed no increase of SIINFEKL-specific CD8
+ T cells, suggesting that presentation pathways and effects of CQ might differ between pathogens or antigens.
CPS-CQ immunization of C57BL/6j mice requires relatively high and lengthy drug prophylaxis to prevent development of
P. berghei infection (K. Nganou-Makamdop, unpublished data). As a result, CPS-CQ mice cumulatively receive much more CQ than the two doses of 800 μg chloroquine diphosphate salt that were previously shown to improve cross-presentation [
21]. Therefore, the effect of this low CQ-dose regimen on RAS immunization was assessed in a separate experiment, but protection levels were not higher than compared to RAS alone, indicating that the choice of CQ dose was not crucial.
Immune modulating effects of MQ have been reported but do not include cross-presentation [
33,
25-
27]. It cannot be formally excluded that MQ in CPS immunization regimes may have similar properties as CQ. Both CQ and MQ are lysosomotropic agents that limit endosomal acidification [
22], which for CQ is known to result in inhibition of lysosomal enzymes that require an acidic pH to function and the fusion of endosomes with lysosomes [
34]. As such CQ, but not MQ, has been widely used to study the role of endosomal acidification in cellular processes [
35]. CQ has also been studied extensively for its inhibitory effect on autophagy [
17], but a recent publication suggests that MQ has similar effects on autophagy [
36]. Because both endosomal acidification and autophagy might influence antigen presentation, the effect of MQ on these processes may result in immune modulating effects just as is the case for CQ.
In the absence of evidence for a direct immune-modulating effect of CQ during whole sporozoite immunization, it cannot be ruled out that both CQ and MQ might instead contribute to the efficient induction of protection in an indirect way. A review of rodent sporozoite immunization studies demonstrates the importance of optimal exposure to the entire repertoire of liver stage antigens as occurs during CPS-CQ and CPS-MQ, with reduced protective efficacy if liver stage development is halted by drugs or in the case of RAS or genetically attenuated parasites [
37]. Furthermore, some reports show a negative effect of blood-stage parasites on induced pre-erythrocytic CD8
+ T cell responses by interfering with dendritic cell function [
38,
1]. By limiting exposure to blood stages during CPS immunization, CQ and MQ might thus have an indirect positive effect on pre-erythrocytic immunity.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
EB, KN, IC, FZ and RS conceived the experiments; EM, KNM, GvG and IC carried out the experiments and analysed the data. EM, KNM and RS drafted the manuscript. All authors read and approved the manuscript.