**4. Vaccines targeting specific parasite stages**

The malaria parasite has a complex life cycle, alternating between the human and mosquito host. In the human host, the malaria parasite transits across different body compartments and alternates between intracellular and extracellular locations (**Figure 1**). This developmental complexity of the malaria parasite has a profound impact on the study design of the malaria vaccine and assessment efficacy (**Table 1**).

During the pre-erythrocytic stage, the parasites exist as the extracellular motile sporozoite upon injection by the mosquito during feeding, and the intracellular liver parasites. Vaccines

developed to target the pre-erythrocytic stage aim at inducing antibodies that target mainly the sporozoites and/or inducing T cells that will eliminate intracellular hepatic forms, thus

Vaccines targeting the asexual blood stages of the parasites are divided into two categories. The vaccines can be anti-parasite, which aim to control and eliminate parasite development in the blood or anti-disease, which aim to prevent the pathologies induced by the parasite. These vaccines need to induce different types of immune responses targeting different phases of the asexual blood stage. Vaccines targeting the sexual stage parasites aim to prevent transmission of gametocytes to the mosquito and/or gamete mating and ookinete development in

It is worth noting that while the parasite expresses different set of genes at different stages of its life cycle, there are also many antigens that are expressed across the different parasite stages. Vaccination against these shared antigens may have an effect at different phases of the

For anti-parasite vaccines targeting the pre-erythrocytic stage, the assessment of vaccine efficacy is relatively easy. Complete efficacy for this stage is defined as sterile protection, whereby

preventing or controlling the extent of the subsequent blood stage development.

**Targets Induced immunity Mechanisms Readout**

Antibodies against sporozoites

Antibodies

T cells against liver stage

Antibody cell dependent inhibition (ADCI) Cellular immunity

Antibodies neutralizing inflammatory factors Antibodies inhibiting parasite/host interactions

Antibodies blocking gamete mating, ookinete formation or oocyst maturation

Presence of parasites in the

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Blood parasite load

Blood parasite load Severe complications\*

Presence of parasites in the mosquito vector (midgut, salivary glands)

blood

Fever

Inhibition of parasite development and replication/

Inhibition of erythrocyte invasion and parasite replication/survival

Inhibition of pathogenesis

Inhibition of parasite development in the mosquito

**Table 1.** Targets and mechanisms for anti-malaria vaccines.

survival

life cycle [22, 49–52], making them just as attractive for vaccine against malaria.

the mosquito midgut.

**Pre-erythrocytic vaccines** Sporozoite antigens Liver stage antigens

**Blood stage vaccines** Asexual blood stage

Parasite derived

Parasite adhesion

Sexual blood stage

**Transmission-blocking vaccines**

antigens

toxins

ligands

antigens Mosquito stage antigens

**5. Anti-parasite vaccines**

**Figure 1.** *Plasmodium falciparum* life cycle and vaccine strategies. The cycle in humans includes three stages: the preerythrocytic stage, which is asymptomatic; the asexual blood stage, which induces pathology; and the sexual stage, which is transmitted to *Anopheles* mosquitoes. At each of these stages, the parasite expresses various proteins that are targets of vaccine candidates. The different vaccine strategies for each stage are indicated.


**Table 1.** Targets and mechanisms for anti-malaria vaccines.

To assess vaccine efficacy in the field, it is also critical to have epidemiological data concerning the vaccine site. The level of endemicity will have an impact on the surveillance time following the last immunization and also on the size of the cohort. Low endemic conditions will

The malaria parasite has a complex life cycle, alternating between the human and mosquito host. In the human host, the malaria parasite transits across different body compartments and alternates between intracellular and extracellular locations (**Figure 1**). This developmental complexity of the malaria parasite has a profound impact on the study design of the malaria

During the pre-erythrocytic stage, the parasites exist as the extracellular motile sporozoite upon injection by the mosquito during feeding, and the intracellular liver parasites. Vaccines

**Figure 1.** *Plasmodium falciparum* life cycle and vaccine strategies. The cycle in humans includes three stages: the preerythrocytic stage, which is asymptomatic; the asexual blood stage, which induces pathology; and the sexual stage, which is transmitted to *Anopheles* mosquitoes. At each of these stages, the parasite expresses various proteins that are

targets of vaccine candidates. The different vaccine strategies for each stage are indicated.

require longer follow-up and a larger cohort to obtain statistically significant results.

**4. Vaccines targeting specific parasite stages**

vaccine and assessment efficacy (**Table 1**).

342 Towards Malaria Elimination - A Leap Forward

developed to target the pre-erythrocytic stage aim at inducing antibodies that target mainly the sporozoites and/or inducing T cells that will eliminate intracellular hepatic forms, thus preventing or controlling the extent of the subsequent blood stage development.

Vaccines targeting the asexual blood stages of the parasites are divided into two categories. The vaccines can be anti-parasite, which aim to control and eliminate parasite development in the blood or anti-disease, which aim to prevent the pathologies induced by the parasite. These vaccines need to induce different types of immune responses targeting different phases of the asexual blood stage. Vaccines targeting the sexual stage parasites aim to prevent transmission of gametocytes to the mosquito and/or gamete mating and ookinete development in the mosquito midgut.

It is worth noting that while the parasite expresses different set of genes at different stages of its life cycle, there are also many antigens that are expressed across the different parasite stages. Vaccination against these shared antigens may have an effect at different phases of the life cycle [22, 49–52], making them just as attractive for vaccine against malaria.

## **5. Anti-parasite vaccines**

For anti-parasite vaccines targeting the pre-erythrocytic stage, the assessment of vaccine efficacy is relatively easy. Complete efficacy for this stage is defined as sterile protection, whereby no parasite can be detected in blood of immunized individuals after the sporozoite challenge. This is an all or none phenomenon, because a single sporozoite developing in the liver can lead to full-blown blood infections.

These hypnozoites are responsible for relapse up to 18 months after a sporozoite injection [68], thus complicating analysis and may require longer follow-up to detect relapse. Up to now, few challenges with *P. vivax* sporozoite have been performed [69]. There are no standard protocols and many issues need to be addressed [70]. First, the production of *P. vivax* sporozoites is limited since it requires infected blood from infected patients or monkeys to feed mosquitoes. Second, contrary to *P. falciparum*, no *P. vivax* cloned lines are available. Most of the lines available are derived from infected patients [71] or have been maintained in monkeys [72]. These lines contain multiple clones, which are poorly characterized at the molecular level [73]. This makes it difficult to obtain reproducible infection profile after experimental infection with mosquito bites of naïve volunteers and to characterize hypnozoite relapse profile. Moreover, as with anti-malarial drug studies in the field, the absence of validated genetic or serologic tools to distinguish between reinfection and relapse [74–77] may also prevent detecting strain-specific effect. For anti-parasite vaccines targeting the blood stage, efficacy is assessed after sporozoite or asexual blood stage parasite challenge. Sterile protection occurs when no parasite can be detected in blood of immunized individuals. Detection of parasitemia can be monitored either by microscopy or by PCR, the latter providing more information. Due to its higher sensitivity, it allows the detection of at least 3–5 parasite cycles even before the parasite is detectable by microscopy. PCR [78–81] bar-coding methods [82] can also be applied to genotype blood parasites. This allows assessing multiplicity of infection and determines whether the vaccine efficacy observed is strain-specific [83, 84]. Strain-specific vaccines have little interest since they

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To assess the vaccine efficacy of anti-parasite vaccines, a challenge is essential. As mentioned earlier, challenge can be performed using sporozoites or blood stage parasites. However, due to the limited availability of insectaries that can provide infected mosquitoes on a regular basis, and the absence of accepted surrogates of protection, there is a necessity for blood stage challenge in healthy volunteers. Contrary to murine or monkey models where direct challenge with blood stage parasites is common, challenge with blood stage parasites in human has only been performed in limited vaccine studies using naïve volunteers [85, 86]. Because of safety reasons, blood parasites used for challenge need to be fully characterized. For a long time, only 3D7, a clone of the NF54 line, has been used. This line is susceptible to a wide range of antimalarials. Other parasite lines have been recently developed [87, 88]. However, since most of blood stage candidates are polymorphic, it is of utmost importance to assess the effect of polymorphism to have an idea of potential vaccine coverage. In addition, blood cells used for blood stage parasite propagation need to be pre-screened for the presence of a wide range

Despite these limitations, studies have shown that blood stage challenge can be safe and may allow the assessment of anti-asexual blood stage vaccine efficacy [89, 90]. Moreover, as recently reported, blood stage growth *in vivo* could be quantified more accurately after challenge with asexual blood stage parasites than with sporozoites [91], highlighting the advantage of this procedure. However, as it is not possible for safety reasons to let the parasitemia develop to high levels, its application might be limited. Vaccine efficacy may depend on the development of additional immune responses by the host during infection, which requires more time to be active as shown in mouse model [92]. In addition, some immune mechanisms

will select vaccine-resistant parasites.

of potential pathogens [86].

Intuitively, one would expect great success of pre-erythrocytic vaccines since the limited numbers of sporozoites (a mean of 5–50) injected by infected mosquitoes [53] would be easily eliminated by the different arms of the immune system induced by the vaccine. However, this has proved to be the contrary. To date, only one vaccine formulation, RTS,S, an hybrid molecule containing a large segment of the circumsporozoite protein and S antigen of the Hepatitis B virus mixed with the AS02 adjuvant, has been shown to induce sterile protection in a substantial proportion of the naïve volunteers [36, 54] but to a much lower extent in field trials [55–58].

One reason that could contribute to the lack of success stories with pre-erythrocytic antiparasite vaccines is the procedures implemented to assess protection. Immunized volunteers were subjected to five mosquito bites, a dose required to ensure that naïve control volunteers would develop patent parasitemia 7–14 days after challenge [59, 60]. Alternative protocols using purified sporozoites injected either intradermal or intravenously have been developed, and so far, have proven to be safe and reproducible [61, 62]. Detection of parasitemia is performed by microscopy on Giemsa-stained blood smears over a 20–25-day period. Once a positive blood film is confirmed, the volunteers were treated with blood schizonticides to eliminate blood parasites and prevent any blood stage parasite-induced pathologies [63]. One limitation of this method of detection is that the time taken to detect parasites in the blood can differ up to 7 days. Hence, a delay of parasitemia does not necessarily translate in reduction of liver load. Moreover, there might be other confounding factors affecting the ability to detect blood parasitemia that are not related to the vaccination. To address this problem, sensitive PCR methods have been developed to detect the first wave of released liver merozoites and to assess the efficacy of the vaccine against pre-erythrocytic parasites. Using elegant regression methods, quantitative PCR techniques [64–67] allow an estimation of the reduction of the parasite liver load and an accurate measure of the effect on the growth rate of blood stage parasites.

Assessing pre-erythrocytic vaccine efficacy in the field is complicated due to factors mentioned earlier, such as the nature of the infection and presence of other co-infections. Evaluation of pre-erythrocytic vaccine in the field had mostly relied on microscopy and long follow-up (usually 6 to 24 months). As mentioned above, this assay may not be the most suitable to accurately assess the efficacy of any formulation targeting the pre-erythrocytic stage. In field conditions, many of the volunteers have been previously infected and, depending on age and exposure, may have developed some immunity against blood stage parasites. Thus, to eliminate possible confounding effects of a synergistic immunity of on-going blood stage infection with immunity induced by vaccination, it is important that volunteers are cleared by drug treatment of low-level parasitemia during immunization and before the surveillance period. It is also necessary that low-level blood infection occurrence be assessed by PCR. When implemented, this approach has reduced the follow-up time period to 1 month, saving costs and allowing the assessment of new formulations [39].

For vaccines against the pre-erythrocytic stage of *P. vivax*, efficacy assessment is further complicated by the fact that this species may produce non-replicating liver form called hypnozoites. These hypnozoites are responsible for relapse up to 18 months after a sporozoite injection [68], thus complicating analysis and may require longer follow-up to detect relapse. Up to now, few challenges with *P. vivax* sporozoite have been performed [69]. There are no standard protocols and many issues need to be addressed [70]. First, the production of *P. vivax* sporozoites is limited since it requires infected blood from infected patients or monkeys to feed mosquitoes. Second, contrary to *P. falciparum*, no *P. vivax* cloned lines are available. Most of the lines available are derived from infected patients [71] or have been maintained in monkeys [72]. These lines contain multiple clones, which are poorly characterized at the molecular level [73]. This makes it difficult to obtain reproducible infection profile after experimental infection with mosquito bites of naïve volunteers and to characterize hypnozoite relapse profile. Moreover, as with anti-malarial drug studies in the field, the absence of validated genetic or serologic tools to distinguish between reinfection and relapse [74–77] may also prevent detecting strain-specific effect. For anti-parasite vaccines targeting the blood stage, efficacy is assessed after sporozoite or asexual blood stage parasite challenge. Sterile protection occurs when no parasite can be detected in blood of immunized individuals. Detection of parasitemia can be monitored either by microscopy or by PCR, the latter providing more information. Due to its higher sensitivity, it allows the detection of at least 3–5 parasite cycles even before the parasite is detectable by microscopy. PCR [78–81] bar-coding methods [82] can also be applied to genotype blood parasites. This allows assessing multiplicity of infection and determines whether the vaccine efficacy observed is strain-specific [83, 84]. Strain-specific vaccines have little interest since they will select vaccine-resistant parasites.

no parasite can be detected in blood of immunized individuals after the sporozoite challenge. This is an all or none phenomenon, because a single sporozoite developing in the liver can

Intuitively, one would expect great success of pre-erythrocytic vaccines since the limited numbers of sporozoites (a mean of 5–50) injected by infected mosquitoes [53] would be easily eliminated by the different arms of the immune system induced by the vaccine. However, this has proved to be the contrary. To date, only one vaccine formulation, RTS,S, an hybrid molecule containing a large segment of the circumsporozoite protein and S antigen of the Hepatitis B virus mixed with the AS02 adjuvant, has been shown to induce sterile protection in a substantial proportion of the naïve volunteers [36, 54] but to a much lower extent in field trials [55–58]. One reason that could contribute to the lack of success stories with pre-erythrocytic antiparasite vaccines is the procedures implemented to assess protection. Immunized volunteers were subjected to five mosquito bites, a dose required to ensure that naïve control volunteers would develop patent parasitemia 7–14 days after challenge [59, 60]. Alternative protocols using purified sporozoites injected either intradermal or intravenously have been developed, and so far, have proven to be safe and reproducible [61, 62]. Detection of parasitemia is performed by microscopy on Giemsa-stained blood smears over a 20–25-day period. Once a positive blood film is confirmed, the volunteers were treated with blood schizonticides to eliminate blood parasites and prevent any blood stage parasite-induced pathologies [63]. One limitation of this method of detection is that the time taken to detect parasites in the blood can differ up to 7 days. Hence, a delay of parasitemia does not necessarily translate in reduction of liver load. Moreover, there might be other confounding factors affecting the ability to detect blood parasitemia that are not related to the vaccination. To address this problem, sensitive PCR methods have been developed to detect the first wave of released liver merozoites and to assess the efficacy of the vaccine against pre-erythrocytic parasites. Using elegant regression methods, quantitative PCR techniques [64–67] allow an estimation of the reduction of the parasite liver

load and an accurate measure of the effect on the growth rate of blood stage parasites.

allowing the assessment of new formulations [39].

Assessing pre-erythrocytic vaccine efficacy in the field is complicated due to factors mentioned earlier, such as the nature of the infection and presence of other co-infections. Evaluation of pre-erythrocytic vaccine in the field had mostly relied on microscopy and long follow-up (usually 6 to 24 months). As mentioned above, this assay may not be the most suitable to accurately assess the efficacy of any formulation targeting the pre-erythrocytic stage. In field conditions, many of the volunteers have been previously infected and, depending on age and exposure, may have developed some immunity against blood stage parasites. Thus, to eliminate possible confounding effects of a synergistic immunity of on-going blood stage infection with immunity induced by vaccination, it is important that volunteers are cleared by drug treatment of low-level parasitemia during immunization and before the surveillance period. It is also necessary that low-level blood infection occurrence be assessed by PCR. When implemented, this approach has reduced the follow-up time period to 1 month, saving costs and

For vaccines against the pre-erythrocytic stage of *P. vivax*, efficacy assessment is further complicated by the fact that this species may produce non-replicating liver form called hypnozoites.

lead to full-blown blood infections.

344 Towards Malaria Elimination - A Leap Forward

To assess the vaccine efficacy of anti-parasite vaccines, a challenge is essential. As mentioned earlier, challenge can be performed using sporozoites or blood stage parasites. However, due to the limited availability of insectaries that can provide infected mosquitoes on a regular basis, and the absence of accepted surrogates of protection, there is a necessity for blood stage challenge in healthy volunteers. Contrary to murine or monkey models where direct challenge with blood stage parasites is common, challenge with blood stage parasites in human has only been performed in limited vaccine studies using naïve volunteers [85, 86]. Because of safety reasons, blood parasites used for challenge need to be fully characterized. For a long time, only 3D7, a clone of the NF54 line, has been used. This line is susceptible to a wide range of antimalarials. Other parasite lines have been recently developed [87, 88]. However, since most of blood stage candidates are polymorphic, it is of utmost importance to assess the effect of polymorphism to have an idea of potential vaccine coverage. In addition, blood cells used for blood stage parasite propagation need to be pre-screened for the presence of a wide range of potential pathogens [86].

Despite these limitations, studies have shown that blood stage challenge can be safe and may allow the assessment of anti-asexual blood stage vaccine efficacy [89, 90]. Moreover, as recently reported, blood stage growth *in vivo* could be quantified more accurately after challenge with asexual blood stage parasites than with sporozoites [91], highlighting the advantage of this procedure. However, as it is not possible for safety reasons to let the parasitemia develop to high levels, its application might be limited. Vaccine efficacy may depend on the development of additional immune responses by the host during infection, which requires more time to be active as shown in mouse model [92]. In addition, some immune mechanisms may need higher parasite challenge dose to be triggered. Antibody-dependent cell immunity or ADCI has been proposed to be effective with parasitemia approaching level detectable by microscopy [93].

many field trials. Active and passive case detections are undertaken to detect clinical malaria episodes and define rate of the first episode or all episodes. Criteria to define a malaria case include presence of fever (≥37.5°C) and detection of malarial parasites in peripheral blood. Careful clinical assessment of the origin of fever is needed to ensure the fever is due to the parasite but not due to concomitant bacterial or viral infection. It should be mandatory to prevent undermining the vaccine efficacy. It is also crucial to clear any asymptomatic infections prior to vaccine testing. Clearing asymptomatic parasitemia allows a better identification of malaria-attributable fever [38]. Assessment of the reduction of severe symptom occurrence and mortality is more difficult to use as end-point. Because of active intervention (drug treatment and patient management), severity and mortality occur only in small fraction of clinical cases. Thus, in order to have sufficient statistical power to assess the vaccine testing, very large cohort is required, resulting in huge cost. Moreover, there have been concerns that decreasing the level of exposure to the parasites might, in return, results in an increase in mortality in the long–term [96]. It has been suggested that reduced exposure prevents the development of naturally-acquired clinical immunity [93], which is thought to result from constant parasite exposure. Thus, for any vaccines entering in Phase III trials, these end-points need to be assessed. Two types of vaccine strategies aimed at reducing specifically morbidity and mortality are being developed. Anti-sequestration vaccines are based on the assumption that cytoadherence of infected red blood cells leading to parasite sequestration in deep tissues is responsible for most of malaria pathologies. These vaccines are designed to target parasite ligands such as members of the *var.* multigene family encoding the proteins *Plasmodium falciparum* erythrocyte membrane protein-1 (PfEMP-1), which mediates cytoadherence [97, 98]. It has been proposed that parasites sequester to avoid splenic elimination [99]. The more clinically-advanced antisequestration vaccine candidate aims at preventing pregnancy-associated malaria [100]. Few *var.* genes, which encode PfEMP-1 binding to chondroitin sulfate A (CSA), have been implicated in placental sequestration, thus making them attractive vaccine candidates [101, 102]. Anti-sequestration vaccines are designed to produce antibodies, which prevent the interactions between infected red blood cells and their cognate host cells (endothelial cells, syncytiotrophoblast…). This will lead to an increase in the circulation of blood parasites at all development stages and hence their elimination by the spleen. Primary end-point measures for such vaccines are both parasitological and clinical. Efficacy of desequestration as measured by the number of mature blood forms can be evaluated simply by microscopical observation of Giemsa stainedblood smears. However, preventing sequestration may lead to rapid increase of parasitemia and possibly a faster development of fever episodes and faster treatment application. Thus, the time-window for monitoring parasite development might be limited. Ultimately, assessment of severity and mortality are the most relevant measures for desequestration vaccines. A large cohort is needed to assess efficacy, like any blood stage vaccine targeting parasite growth.

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Another type of anti-disease vaccine is targeting parasite moieties behaving as toxins and inducing immune-mediated pathologies [28, 103]. As these vaccines may have no effect on the parasitemia, the assessment of their efficacies will require very large cohorts to assess clinical outcomes with both active and passive case detection. In terms of safety, a strict clinical follow-up starting as early as the last vaccine dose administration is needed since these may

perturb the immune network and induce immunopathology.

In endemic settings, efficacy of blood stage vaccines has been assessed in natural conditions after challenge by mosquito bites. This type of challenge is perfectly suited if the vaccine can induce sterile immunity. However, it might not be the most appropriate when blood parasite multiplication rate must be measured. This rate depends on the numbers of liver merozoites released and timing of their release. As mentioned above, liver merozoite release is not a homogenous phenomenon in terms of quantity and timing. Thus, to obtain parasite growth curve suitable for comparative analysis between individuals and groups, blood sampling must be carefully planned. Indeed, this implies an active and close follow-up of the volunteers to obtain multiple time points. One possibility to obtain more homogenous results would be to perform the challenge with defined number of infected red blood cells at a same time of infection across all groups [86, 94]. However, this requires overcoming a series of hurdles such as the development of standardized inoculums with known number of parasites at the same stage of development and the availability of donor blood, which have to be heavily tested for the presence of any pathogens. In addition, the parasite in the challenge inoculums would also need to be fully characterized and clearly defined in term of parasite clonality.

To accurately assess the efficacy of anti-parasite blood stage vaccine, it is necessary to evaluate any pre-existing immune responses to the antigens in the blood stage vaccine. Individuals leaving in endemic areas acquire immunity over time. The time required to develop this immunity depends on the endemicity level and their genetic background. This immunity may influence growth rate of the parasite. Pre-existing immunity can synergize with the immunity induced by the vaccination. Vaccination may also boost pre-existing antigen-specific immune responses, which would be ideal for any vaccine formulations. On the contrary, pre-existing immune response may inhibit or mask the immune response induced by vaccination. It has been shown that the antibodies to the N-terminal of *P. falciparum* merozoite surface protein 1 can block the inhibitory activity of antibodies recognizing the C-terminal part [95]. Thus, if such an antibody interference mechanism exists for antigen(s) used in vaccine formulation, it would be necessary to evaluate carefully pre-existing immune responses to these antigens.
