Malaria is a life-threatening infectious disease in humans that is caused by a single-celled parasite called Plasmodium. The parasite is carried between people by mosquitos; when an infected mosquito bites a human, the parasite is injected into the bloodstream with the mosquito's saliva. Plasmodium first infects liver cells but then re-enters the bloodstream, where it infects red blood cells leading to symptoms of disease. If another mosquito bites the infected individual at this so-called ‘blood-stage’, the parasite can be passed to this mosquito and the cycle of transmission continues.

Currently there are no vaccines available that can effectively protect against malaria. Although an experimental vaccine containing a weakened form of the parasite can protect against the liver-stage parasites, it fails to prevent the parasite from multiplying in the red blood cells. Therefore, the individuals remain susceptible to severe malaria.

Recently, researchers have developed a new strategy for immunization that provides exposure to both liver-stage and blood-stage parasites. Human volunteers taking an anti-malarial drug were deliberately exposed to mosquitos carrying the parasite on three separate occasions. Although the volunteers were infected with the parasite, the anti-malarial drug killed the parasites inside the red blood cells. After the end of the drug treatment, the volunteers were exposed to mosquitos carrying the parasite and they were still protected from infection. These results are promising, but it is not clear if the volunteers have acquired immunity to liver-stage or blood-stage parasites, or even both.

To answer this important question, Nahrendorf et al. developed a similar immunization strategy in mice. Just like the human volunteers, the mice were treated with an anti-malarial drug and exposed to mosquitos carrying Plasmodium on three separate occasions. Although the immunizations did not protect the mice against early infection in the liver, they did provide long-term protection against parasites multiplying in the red-blood cells.

The immunity generated by this immunization strategy also protected the mice against another strain of Plasmodium, different to the one used in the immunizations. The experiments also show that prolonged exposure to the blood-stage parasites can even lead to immunity against the liver-stage parasites.

Nahrendorf et al.'s findings show that this immunization strategy can protect individuals against both the liver-stage and blood-stage parasites. The next challenges are to find out how the immunity generated by one stage of infection can protect against the other stages, and to discover which molecules on the parasite the immune system targets.

Protective immunity against microorganisms is developed after repeated infection and recovery (Mutapi et al., 2013). Vaccines are usually successful if they mimic these naturally acquired immune responses (Fox, 1984; Mielcarek et al., 2006). While protection against viruses and bacteria can be induced by vaccination with killed (inactivated) or live-attenuated pathogens (Delany et al., 2014), there is no licensed vaccine for human parasitic diseases like malaria that pose a major global health burden.

The apicomplexan malaria parasite Plasmodium is transmitted by bites of female anopheline mosquitoes. In the vertebrate host, sporozoites injected into the dermis migrate to the liver, where they establish a clinically silent infection of hepatocytes. Merozoites are then released from the liver and invade erythrocytes, leading to an exponential asexual replication cycle that is entirely responsible for the clinical signs and symptoms associated with malaria. Immunity against severe disease can be acquired following repeated infection, but sterile parasite clearance is rarely achieved (Goncalves et al., 2014). Clinically immune adults in endemic areas still harbor parasites in their blood-stream (Okell et al., 2009). These asymptomatic carriers also develop gametocytes, the form transmissible to mosquitoes, thereby allowing the parasite to complete its life cycle.

To achieve malaria control and eventually eradication, transmission must be blocked (Kappe et al., 2010). A vaccine that protects against pre-erythrocytic parasites, and thus outperforms naturally acquired immunity, would greatly facilitate this aim. Parasites that arrest during the liver stage, either because of irradiation (Nussenzweig et al., 1967) or targeted gene deletion (Mueller et al., 2005), can provide immunity against challenge infection. Indeed, immunization of human volunteers with irradiated sporozoites can induce sterile protection in experimental settings (Clyde et al., 1973; Seder et al., 2013). However, in the absence of acquired immunity to the blood-stage parasite a pre-erythrocytic vaccine that is only partially effective, and therefore permits breakthrough erythrocytic infections, will provide no protection against severe malaria (Bejon et al., 2011). The inclusion of a blood-stage component together with an effective pre-erythrocytic vaccine is therefore preferred to provide a multi-stage malaria vaccine that minimizes both transmission and disease (Ellis et al., 2010; Goodman and Draper, 2010).

A recently described experimental malaria immunization protocol using chemoprophylaxis and sporozoites (CPS) (Roestenberg et al., 2009) ensures exposure to pre-erythrocytic and blood-stage parasites, and hence has the unique potential to induce protection against all Plasmodium life cycle stages in the vertebrate host. Three immunizations with bites of 10–15 Plasmodium falciparum-infected mosquitoes under chloroquine chemoprophylaxis are sufficient to elicit sterile protection against homologous challenge in human volunteers (Roestenberg et al., 2009; Bijker et al., 2013, 2014). Although it is not possible to measure liver parasite burden in human volunteers directly, it appears that immunity exclusively targets pre-erythrocytic parasite life cycle stages, as there is no protection against direct blood challenge (Bijker et al., 2013). CPS immunization is thus substantially more effective than immunization with irradiated sporozoites, which requires 1000 mosquito bites (Clyde et al., 1973) or five intravenous (iv) injections of more than 100,000 sporozoites for sterile protection (Seder et al., 2013). We therefore hypothesize that transient blood-stage parasitemia, before abrogation by chloroquine, may contribute to immunity following CPS immunization.

In this study, we have investigated the stage- and strain-specificity of protection in a novel mouse model of CPS immunization using Plasmodium chabaudi. P. chabaudi establishes a chronic, non-lethal blood-stage infection, which has been used extensively to characterize the immune response to blood-stage parasites in vivo (Stephens et al., 2012). A recently optimized protocol for P. chabaudi mosquito transmission (Spence et al., 2012) allows us now to also study pre-erythrocytic stages of this rodent parasite. Heterologous protection can readily be assessed since many genetically distinct P. chabaudi isolates displaying a variety of virulence phenotypes are available (Mackinnon and Read, 1999; Otto et al., 2014). We immunized C57BL/6 mice three times with bites of P. chabaudi-infected mosquitoes under oral chloroquine chemoprophylaxis similar to human clinical trials (Roestenberg et al., 2009; Bijker et al., 2013, 2014). This approach is unique amongst all published animal models of CPS immunization (Beaudoin et al., 1977; Golenser et al., 1977; Orjih et al., 1982; Belnoue et al., 2004; Friesen and Matuschewski, 2011; Inoue et al., 2012; Nganou-Makamdop et al., 2012b; Doll et al., 2014; Lewis et al., 2014; Peng et al., 2014), which have (without exception) used iv injection of high numbers of Plasmodium berghei or Plasmodium yoelii sporozoites for immunization. Furthermore, rather than evaluating effector mechanisms by challenging shortly after immunization (Beaudoin et al., 1977; Belnoue et al., 2004; Friesen and Matuschewski, 2011), we performed the challenge 100 days after the final immunization to test the generation and maintenance of long-term immunological memory.

CPS immunization with P. chabaudi by mosquito bite does not generate pre-erythrocytic immunity, which is acquired only after immunization with high doses of sporozoites. Instead, immunization by bite elicits blood-stage immunity that is effective against the immunizing strain and also a more virulent, genetically distinct P. chabaudi. Moreover, extended exposure to blood-stage parasitemia elicits robust pre-erythrocytic immunity, comparable to protection afforded by high dose sporozoite immunization. Exposure to blood-stage parasites thus elicits heterologous blood-stage immunity and can contribute to the pre-erythrocytic efficacy of this immunization regimen. Therefore, these findings add significantly to advances from previous CPS immunization mouse models by evaluating the generation of immune memory after immunization with P. chabaudi by mosquito bite. This is relevant for our understanding of acquired immunity in a malaria endemic setting, and can inform multi-stage malaria vaccine development.

We investigated the stage- and strain-specificity of protection in a novel mouse model of CPS immunization (Figure 1). C57BL/6 mice were immunized three times at 2-week intervals with P. chabaudi AS-infected mosquito bites. For certain experimental questions, it was necessary to deviate from the natural route of infection and inject sporozoites iv to control the dose. Starting on the day of infection, mice were then treated orally with 100 mg per kg chloroquine for 10 days following each immunization. To assess the long-term efficacy of acquired immunity, mice were challenged approximately 100 days after the last immunization. Protection against pre-erythrocytic or blood-stage parasites was evaluated after mosquito bite or direct blood challenge.

Figure 1

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CPS immunization leads to a transient blood-stage infection

Transient blood-stage parasitemia is a key feature of CPS immunization. We used quantitative RealTime (qRT) PCR to measure erythrocytic parasite burden after each immunization with P. chabaudi AS-infected mosquito bites under chloroquine cover. After the first immunization, approximately 50,000 parasites per ml whole blood were detected within the first erythrocytic cycle (Figure 2). The amount of blood-stage parasites within the first erythrocytic cycle varied extensively (median 47,596, range 67–222,699), reflecting the stochastic inoculation of sporozoites during mosquito bite (Beier et al., 1991; Ponnudurai et al., 1991; Medica and Sinnis, 2005). Thereafter, chloroquine reduced parasitemia by 86–96% every 24 hr. After the fourth cycle, the majority of erythrocytic parasites were cleared. Similarly, after the second and third immunization mice experienced a substantial number of circulating blood-stage parasites for 48–72 hr. However, although blood-stage parasites were detected in all but one mouse, parasitemia in the first erythrocytic cycle was reduced by 5- and 13-fold after the second and third immunizations, respectively, when compared to infection controls (Figure 2). Consequently, one CPS immunization is sufficient to reduce blood-stage parasite burden within the first erythrocytic cycle, which indicates either pre-erythrocytic or blood-stage immunity.

Figure 2

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Pre-erythrocytic immunity requires high doses of sporozoites during CPS immunization

In order to assess directly whether pre-erythrocytic immunity was generated by this CPS immunization protocol, liver parasite burden was analyzed after mosquito bite challenge. Surprisingly, there was no difference in the liver parasite burden between mice given three CPS immunizations with P. chabaudi AS-infected mosquito bites and infection controls (Figure 3A). Therefore, immunization by mosquito bite under chloroquine cover according to this protocol failed to elicit pre-erythrocytic immunity. This is in contrast to results from other animal models of CPS immunization where pre-erythrocytic immunity is induced after high numbers of sporozoites are injected iv (Belnoue et al., 2004; Friesen and Matuschewski, 2011; Inoue et al., 2012; Nganou-Makamdop et al., 2012b; Lewis et al., 2014; Peng et al., 2014). To test if pre-erythrocytic immunity can be induced after CPS immunization with large numbers of sporozoites, we used P. chabaudi CB, since mosquitoes infected with this parasite strain harbor an increased number of sporozoites in their salivary glands compared to P. chabaudi AS (Spence et al., 2012), making these experiments technically feasible. In agreement with previous studies (Belnoue et al., 2004; Friesen and Matuschewski, 2011; Inoue et al., 2012; Nganou-Makamdop et al., 2012b; Lewis et al., 2014; Peng et al., 2014), mice immunized iv three times with 10,000 P. chabaudi CB sporozoites under chloroquine cover did show reduced liver parasite burden (up to 90%) after mosquito bite challenge, compared to infection controls (Figure 3B). Conversely, mice immunized iv three times with a low dose of 100 P. chabaudi CB sporozoites (representative of the estimated number of P. chabaudi sporozoites that initiate infection via mosquito bite, Spence et al., 2012) do not acquire pre-erythrocytic immunity (Figure 3B). It also appears that CPS immunization with 10,000 live sporozoites was more effective at inducing pre-erythrocytic immunity than immunization with 10,000 irradiated P. chabaudi CB sporozoites, which arrest during hepatic development (Suhrbier et al., 1990) and do not establish a blood-stage infection (Figure 3B). This suggests that complete liver-stage maturation and the increased blood-stage parasitemia that accompanies immunization with 10,000 sporozoites (as compared to 100 sporozoites or mosquito bite) could contribute to pre-erythrocytic protection.

Figure 3

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CPS immunization by mosquito bite elicits blood-stage immunity

To test whether the transient blood-stage infection resulting from CPS immunization by mosquito bite (Figure 2) is sufficient to induce protection against erythrocytic parasites, we challenged mice approximately 100 days after the last immunization and measured blood-stage parasitemia (Figure 4). Mice that were CPS immunized with P. chabaudi AS had similar numbers of parasitized erythrocytes as compared to mock immunized controls within the first five erythrocytic cycles following mosquito bite challenge (Figure 4A). However, from erythrocytic cycle 6 parasitemia was significantly reduced and blood-stage parasites were cleared more rapidly in immunized mice. This was reflected in a 6-fold reduction of total area under the curve (AUC) (right inset, Figure 4A). Protection against erythrocytic parasites was also evaluated by direct blood challenge, using blood-stage parasites obtained from a donor mouse infected by mosquito bite. Similar to the results of mosquito bite challenge, blood-stage parasitemia was significantly reduced (Figure 4B), but the infection was still chronic in some mice (Figure 4—figure supplement 1). However, blood-stage protection was abrogated when CPS immunized mice were challenged with serially blood passaged (SBP) P. chabaudi; blood-stage parasites with increased virulence following multiple passages through naive mice (Spence et al., 2013, Figure 4C). In this case, CPS immunization reduced blood-stage parasite burden only between erythrocytic cycle 6 and 8, as compared to mock immunized controls. This was reflected in only a 1.2-fold reduction in total AUC (right inset, Figure 4C). Therefore, CPS immunization by mosquito bite elicits homologous blood-stage immunity, which is most effective in the context of mosquito transmission.

Figure 4 with 1 supplement see all

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CPS immunization elicits heterologous blood-stage immunity

CPS immunization has so far not been shown to induce protection against challenge with genetically distinct strains of Plasmodium; a situation that would be encountered in human malaria-endemic areas. The genetic diversity amongst strains of P. chabaudi (Mackinnon and Read, 1999; Otto et al., 2014) allows us to investigate heterologous immunity in this model of CPS immunization. Mice that were CPS immunized with P. chabaudi AS-infected mosquito bites had reduced peak parasitemia, and blood-stage parasites were cleared faster, when compared to mock immunized mice after homologous (P. chabaudi AS) and heterologous (P. chabaudi CB) mosquito bite challenge (Figure 5A–D). A direct comparison between P. chabaudi AS and CB challenge revealed nonetheless that homologous blood-stage immunity is more effective than heterologous immunity. In this experiment, CPS immunization reduced total AUC by 25-fold following homologous challenge, as compared to 6-fold following heterologous challenge (Figure 5E). Nevertheless, CPS immunization elicits blood-stage protection against a robust heterologous challenge with the genetically distinct, and more virulent, CB strain of P. chabaudi.

Figure 5

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Extended exposure to blood-stage parasites elicits robust pre-erythrocytic immunity

Blood-stage parasites appear to be both the source and target of protection following CPS immunization with P. chabaudi AS-infected mosquito bites. It was shown that immunization with serially blood passaged P. yoelii parasites with prophylactic chloroquine treatment can elicit pre-erythrocytic immunity (Belnoue et al., 2008). We wanted to assess whether blood-stage parasites have the potential to induce pre-erythrocytic protection also in the context of mosquito transmission. We therefore asked whether a fulminant blood-stage infection could elicit cross-stage immunity against pre-erythrocytic parasites. We infected mice with P. chabaudi AS by mosquito bite or intraperitoneal (ip) injection of recently mosquito transmitted blood-stage parasites. The two groups of mice were not drug-treated, and therefore experienced a low-grade chronic, recrudescing blood-stage infection for up to 90 days (Spence et al., 2013). After mosquito bite challenge, both groups of mice demonstrated reduced liver parasite burden (up to 85%), compared to infection controls (Figure 6). Cross-stage immunity is therefore a powerful mechanism for protection against pre-erythrocytic parasites, which may be absent during CPS immunization with small sporozoite numbers as the blood-stage infection is curtailed by the use of chloroquine.

Figure 6

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Timing, route of infection, and antigen dose play major roles in determining the initial priming of the antimalarial immune response (Legorreta-Herrera et al., 2004; Elliott et al., 2005; Belnoue et al., 2008; Guilbride et al., 2012; Nganou-Makamdop et al., 2012a). We incorporated many of these aspects from human clinical trials (Roestenberg et al., 2009; Bijker et al., 2013, 2014) in a new P. chabaudi mouse model of CPS immunization to investigate the stage- and strain-specificity of CPS-induced protection against malaria. Our results highlight the complexity of immunity against the different life cycle stages of the malaria parasite (Table 1). Pre-erythrocytic immunity appears to depend on the number of immunizing sporozoites. In this study, we find no evidence of pre-erythrocytic immunity after CPS immunization with P. chabaudi infected mosquito bites, which inoculate an estimated maximum of 100 sporozoites per immunization (Spence et al., 2012, 2013). Sterile pre-erythrocytic protection was however reported in human CPS immunization trials (Roestenberg et al., 2009; Bijker et al., 2013, 2014). Anopheles stephensi mosquitoes experimentally infected with P. falciparum 3D7 or NF54 habor 50–200 times more sporozoites in their salivary glands (Bijker et al., 2013) compared to P. chabaudi AS (Spence et al., 2012). However, only few sporozoites are injected into the skin during mosquito bite and this number is independent of salivary gland sporozoites load (Beier et al., 1991; Ponnudurai et al., 1991; Medica and Sinnis, 2005). Since the number of sporozoites establishing a liver-stage infection can further be influenced by inherent sporozoite infectivity (Khan and Vanderberg, 1991), our best estimate of immunizing sporozoite dose is the number of infected erythrocytes observed directly after egress from the liver. Assuming that 10,000 merozoites are released from one infected liver cell (White et al., 2014), we estimate approximately 400 infected hepatocytes (95% CI 137–1250) in human volunteers (Bijker et al., 2013) compared to 5 in P. chabaudi AS immunized mice (95% CI 1–31). Therefore, the number of infected hepatocytes after CPS immunization by mosquito bite in the P. chabaudi mouse model is approximately 100-fold lower than in CPS immunized humans. We show that this differences in infected hepatocyte numbers by a factor of 100 can be significant for the development of pre-erythrocytic immunity: three immunizations with 10,000 P. chabaudi sporozoites iv induce long-lasting protection against mosquito bite challenge, while three immunizations with 100 P. chabaudi sporozoites fail to do so. This is in general agreement with other rodent malaria studies using P. berghei (Beaudoin et al., 1977; Golenser et al., 1977; Orjih et al., 1982; Friesen and Matuschewski, 2011; Nganou-Makamdop et al., 2012b; Lewis et al., 2014) or P. yoelii (Belnoue et al., 2004; Inoue et al., 2012; Doll et al., 2014; Peng et al., 2014), in which sterile pre-erythrocytic immunity is observed after immunization with high sporozoite doses (typically 10,000–50,000 sporozoites per immunization), while a reduction in sporozoite dose or the number of immunizations leads to breakthrough blood-stage infections upon challenge (Belnoue et al., 2004; Inoue et al., 2012). Reduction of the number of P. falciparum-infected mosquitoes also reduces the frequency of sterilely protected volunteers (Bijker et al., 2014), indicating that the number of sporozoites establishing a liver-stage infection fails to surpass the protective threshold to elicit sterile pre-erythrocytic immunity. This may also explain why in malaria-endemic areas pre-erythrocytic immunity is thought to be absent (Tran et al., 2013). In addition to maximizing specific responses against immunodominant antigens, CPS immunization with high numbers of sporozoites may broaden the immune repertoire by including protective responses against subdominant antigens, which could enhance heterologous pre-erythrocytic protection (Trieu et al., 2011). This may further be enhanced by the longer liver-stage development of P. falciparum (egress 6.8 days after mosquito bite, Roestenberg et al., 2012) compared to P. chabaudi (egress after 52 hr, Stephens et al., 2012). Longer liver stage development may positively influence the generation of pre-erythrocytic immunity by allowing time for protective immune responses to develop.

A key feature of CPS immunization is that it permits exposure to all Plasmodium life cycle stages in the vertebrate host, including parasitized erythrocytes. It is well known that repeated exposure to blood-stage parasites, for example, in rodent models (Jarra and Brown, 1985; Legorreta-Herrera et al., 2004; Elliott et al., 2005), in humans exposed to ultra-low doses of parasitized erythrocytes while receiving drug treatment (Pombo et al., 2002), during malaria-therapy of neurosyphilis patients (Collins and Jeffery, 1999), and in people living in malaria-endemic areas (Bull et al., 1998), induces blood-stage immunity. Our results also clearly show that during CPS immunization repeated transient blood-stage infection (less than 0.01% parasitemia for 48–72 hr) elicits long-lasting blood-stage immunity. Protection against challenge infection was only apparent after multiple erythrocytic replication cycles and patent blood-stage parasite densities, which could indicate that blood-stage protection was not observed in CPS immunized human volunteers after direct blood-challenge because drug treatment is required at low parasite densities as soon as patency is reached (typically between the third and fourth erythrocytic cycle, Bijker et al., 2013). Blood-stage parasites are however recognized in human volunteers, which was demonstrated by an earlier increase of IFNγ and monokines induced by IFNγ (MIG) concentrations in CPS immunized volunteers compared to infection controls after direct blood challenge (Bijker et al., 2013). It will be of value to investigate whether the observed blood-stage protection in this mouse model is also detected in CPS immunized primates, where a longer blood-stage infection than that allowed in human clinical trials is possible.

Despite the reduced peak parasitemia and faster clearance of blood-stage parasites during the acute phase of infection in CPS immunized mice, recrudescent parasitemia could still be observed in the chronic phase of infection after challenge, suggesting a parasite variant escapes the protective immune response (McLean et al., 1982). There are very few reports on protective efficacy of CPS immunization (or indeed any sporozoite-based vaccine) against direct blood-challenge (Belnoue et al., 2004; Inoue et al., 2012; Peng et al., 2014). Doll et al. (2014) reported that sustained, subpatent blood-stage infection after treatment with a commonly used dose of chloroquine can induce partial blood-stage protection. Low-grade transient blood-stage parasitemia, achieved by attenuation of blood-stage parasites using an antimalarial drug (Pombo et al., 2002; Elliott et al., 2005), a DNA alkylating agent (Good et al., 2013) or genetic tools (Ting et al., 2008; Aly et al., 2010) similarly provides protection against homologous and heterologous blood-stage challenge.

We could show that CPS-induced blood-stage immunity is effective against heterologous mosquito bite challenge with a more virulent and genetically distinct strain of P. chabaudi (Mackinnon and Read, 1999; Lamb and Langhorne, 2008). In agreement with the observed cross-species protection after CPS immunization with mefloquine (Inoue et al., 2012), and one study using chemically attenuated sporozoites for immunization (Purcell et al., 2008), heterologous protection is less effective than homologous immunity. Nevertheless, CPS immunization can elicit long-lived protection against both homologous and heterologous blood-stage parasites, which will be important to minimize disease severity in the case of breakthrough blood-stage infections. This is essential for the development of an effective multi-stage malaria vaccine (Ellis et al., 2010; Bejon et al., 2011).

In stark contrast to the observed heterologous blood-stage protection after mosquito bite challenge infection, protection was almost completely abrogated following direct blood-challenge with virulent parasites obtained after continuous serial blood passage. This suggests that blood-stage parasites immediately after mosquito transmission express antigens not present on serially blood passaged parasites and that these antigens may be the target of protective immunity following CPS immunization. Serially blood passaged parasites can hence escape from CPS-induced blood-stage immunity. One group of Plasmodium genes, whose expression is altered by mosquito transmission during blood-stage infection is the Plasmodium interspersed repeat gene family (pir); termed cir in P. chabaudi (Lawton et al., 2012). Transcription of more than half of all cir genes is increased in blood-stage parasites after mosquito transmission compared with their transcription after serial blood passage. This diversification of cir transcription is associated with a more effective host immune response, which in turn attenuates parasite virulence (Spence et al., 2013). The cir genes could also be candidate targets for cross-stage immunity, as P. berghei pir genes are also transcribed during the liver stages (personal communication BM Franke-Fayard and CJ Janse, Leiden University Medical Center, The Netherlands). An investigation into shared PIR proteins between liver and blood-stage parasites may hence provide valuable information for multi-stage malaria vaccine development. Furthermore, the absence of blood-stage protection in previous CPS models may have been due to challenge with serially blood passaged rather then recently mosquito transmitted blood-stage parasites. It is therefore always essential to evaluate blood-stage immunity in the context of mosquito transmission.

As shared antigenic targets between liver- and blood-stage parasites have been described (Tarun et al., 2008), the exciting possibility of cross-stage protection has been considered but only rarely assessed. Genetically modified fabb/f- sporozoites that arrest late in liver-stage development protect against iv challenge with 100 blood-stage parasites (Butler et al., 2011). On the other hand, a blood-stage infection with serially blood passaged P. yoelii, drug-treated with chloroquine after 4–5 days, reduces liver parasite load upon iv challenge with 35,000 sporozoites (Belnoue et al., 2008). In our model of CPS immunization by mosquito bite, it is likely that repeated transient blood-stage infection during immunization elicits the observed blood-stage protection, although we cannot yet exclude that responses acquired against pre-erythrocytic antigens, which are shared with blood-stage parasites (Tarun et al., 2008), contribute as well. Because of the low number of infected hepatocytes after CPS immunization with P. chabaudi-infected mosquito bites this seems however unlikely. Nevertheless, we demonstrate unequivocally that extended exposure to blood-stage parasites is an effective stimulator of pre-erythrocytic immunity. Exposure to blood-stage parasites during CPS immunization may thus significantly contribute to the observed pre-erythrocytic protection in human volunteers (Roestenberg et al., 2009; Bijker et al., 2013, 2014). Indeed, cross-stage immunity could be responsible for the unprecedented efficacy of CPS immunization compared to immunization with irradiated sporozoites (Clyde et al., 1973; Seder et al., 2013), which arrest early during liver-stage development and never establish a blood-stage infection. While extending exposure to replicating blood-stage parasites by delayed drug administration is not possible in humans, the incorporation of chemically (Good et al., 2013) or genetically (Ting et al., 2008; Aly et al., 2010) attenuated blood-stage parasites should be considered to further enhance the generation and maintenance of both pre-erythrocytic and blood-stage immunity. This makes CPS immunization a powerful tool for the development of an effective multi-stage malaria vaccine.

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Author details

1. Wiebke Nahrendorf

   1. Division of Parasitology, MRC National Institute for Medical Research, London, United Kingdom
   2. Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, Netherlands

   Present address

   1. Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, United Kingdom
   2. Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   WN, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   Wiebke.Nahrendorf@ed.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

2. Philip J Spence

   Division of Parasitology, MRC National Institute for Medical Research, London, United Kingdom

   Present address

   1. Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, United Kingdom
   2. Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   PJS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Irene Tumwine

   Division of Parasitology, MRC National Institute for Medical Research, London, United Kingdom

   Present address

   Mill Hill Laboratories, Francis Crick Institute, London, United Kingdom

   Contribution

   IT, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Prisca Lévy

   Division of Parasitology, MRC National Institute for Medical Research, London, United Kingdom

   Present address

   Mill Hill Laboratories, Francis Crick Institute, London, United Kingdom

   Contribution

   PL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. William Jarra

   Division of Parasitology, MRC National Institute for Medical Research, London, United Kingdom

   Contribution

   WJ, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Robert W Sauerwein

   Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, Netherlands

   Contribution

   RWS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

7. Jean Langhorne

   Division of Parasitology, MRC National Institute for Medical Research, London, United Kingdom

   Present address

   Mill Hill Laboratories, Francis Crick Institute, London, United Kingdom

   Contribution

   JL, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   jlangho@nimr.mrc.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

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