**6. Anti-disease vaccines preventing or reducing morbidity and mortality**

Anti-disease vaccines aim to prevent the pathologies induced by the parasite. Hence, to assess the efficacy of these vaccines, it is important to clearly define the symptoms. Symptomatic malaria infections are characterized by recurrent fever and if not treated could develop into more severe complications (i.e. anemia, multi-organ dysfunctions affecting the lungs, kidneys, liver and brain…), and ultimately leading to death. These different clinical occurrences can be considered as end-points when assessing vaccine efficacy. For safety and ethical reasons, these end-points are looked for in experimental clinical trials. However, they are not measured in 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.

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

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.

**6. Anti-disease vaccines preventing or reducing morbidity and** 

Anti-disease vaccines aim to prevent the pathologies induced by the parasite. Hence, to assess the efficacy of these vaccines, it is important to clearly define the symptoms. Symptomatic malaria infections are characterized by recurrent fever and if not treated could develop into more severe complications (i.e. anemia, multi-organ dysfunctions affecting the lungs, kidneys, liver and brain…), and ultimately leading to death. These different clinical occurrences can be considered as end-points when assessing vaccine efficacy. For safety and ethical reasons, these end-points are looked for in experimental clinical trials. However, they are not measured in

microscopy [93].

346 Towards Malaria Elimination - A Leap Forward

**mortality**

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.

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.

### **7. Transmission blocking vaccines**

Transmission blocking vaccines (TBV) are designed to prevent or reduce the development of the sporogonic stage inside the mosquito host. This leads to a reduction in the numbers of infected mosquitoes and hence lesser malaria transmission in the population. As such, experimental clinical trials cannot provide straightforward answers of vaccine efficacy since the effect of such vaccine is at the population level and not at individual level. Assessment of TBV efficacy is done using *in vitro* assays and the membrane-feeding assay. Mosquitoes are fed *in vitro* with *Plasmodium* gametocytes mixed with serums from immunized individuals and the level of sporogonic development is assessed by counting the oocysts in dissected mosquito midguts [104] or, more rarely, the sporozoites in the mosquito salivary glands. Although this assay has been instrumental in identifying target antigens, it remains to be seen whether it might help to define correlate of protections for TBV development.

between the two assays has been described in few studies [110] but the membrane-feeding assay still awaits definitive validation [111]. In summary, it would be relevant for future TBV trials to perform feeding directly on gametocyte carriers using local mosquitoes. These mosquitoes would have to be raised in local insectaries and tested for the absence of any other human pathogens. Measures of TBV efficacy should not be limited to development of oocysts but also to salivary glands sporozoites since the latter are the infectious forms to humans. Hence, future studies should aim at measuring salivary gland sporozoite loads and

Assessing Malaria Vaccine Efficacy

349

http://dx.doi.org/10.5772/intechopen.77044

Here we discussed the different types of malaria vaccines and the different ways to access the vaccine efficacy. We also highlighted the limitations involved and the difficulties encountered by researchers aiming to develop an efficacious vaccine against a complex parasite such as *Plasmodium*. Despite decades of research efforts in vaccine development, no efficient malaria vaccine (i.e. with an efficacy >50%) has been developed. The most clinically-advanced RTS,S, which has been tested in Phase III, conferred at best 30–40% protection against clinical malaria [112]. Modeling studies have been proposed that, together with other malaria interventions, RTS,S vaccination may reduce the incidence of clinical malaria and deaths in many sub-Saharan African countries [113, 114]. Thus, this has led to the approval of licensure for the RTS,S vaccines by the European Union. However, the World Health Organization has not recommended its use in the extended program of immunization for children due to its discouraging vaccine efficacy data. Thus, the future of this vaccine for mass deployment remains uncertain. One of the major reasons of the limited efficacy of RTS,S vaccines and the discontinuation of various other vaccine development efforts is certainly due to antigen polymorphism [83]. In addition to antigen polymorphism, the malaria parasite utilizes many other immune escape mechanisms [115], which have severely hampered the development of malaria vaccines. With the renewed interest in malaria eradication, the development of an effective malaria vaccine is high on the agenda. Diverse strategies are being proposed to develop better vaccines: identification of new vaccine candidate [116], combinations of different antigens targeting the same stage or different stages [117]; new delivery systems and prime-boost strategies using different modalities [118]; and new adjuvants to induce stronger and longer lasting efficient immune responses [119–122]. However, for all vaccine types described, the absence of validated surrogates of protection to help select and prioritize different vaccine formulations is a major roadblock, which should be given priority to

This work was supported by the Agency for Science Technology and Research (A\*STAR),

sporozoite infectivity.

**8. Concluding remarks**

accelerate vaccine testing.

**Acknowledgements**

Singapore.

Currently, none of the TBV has progressed to clinical trials in the field. As the principal outcome of TBV is to reduce the number of infected mosquitoes at the population level, methods for assessing their number in field conditions should be implemented in a timely manner. There are, to date, no standardized methods to estimate the number of infected mosquitoes in the field and estimation would require large sampling size. Moreover, it seems that infected mosquitoes can cluster in discrete locations [105], thus requiring extensive studies on the distribution of infected mosquitos before and after vaccine trials. Since the number of infected mosquitoes depend on the number of circulating gametocytes [106], defining the number of gametocyte carrier prior to vaccine implementation is also a pre-requisite. In addition, defining transmission intensity of the vaccine site is important since it may influence the outcome of the vaccination. This can be defined by seroepidemiology and geographical information system (GIS) applications. For the latter, GPS mapping of mosquitoes and infected humans needs be done. It must be noted that one major limitation of these trials is that they assume that the human and mosquito populations tested are not mobile, which is often not true. An influx of infected individuals can modify the outcome by creating new reservoirs, and an influx of external infected mosquitoes would maintain transmission. It has been suggested that TBV can be tested with accuracy only in enclaved locations such as islands. Ultimately, the main expected outcome is that TBV will reduce transmission and thus reduce morbidity and mortality. The effect of such vaccine is at the population level, a large and costly cohort will be needed to be assessed over a long period of time. However, recent advances in modeling might facilitate TBV assessment by identifying end-point measures, which may serve as correlates of protection [107, 108]. Different end-point measures have been developed to assess TBV efficacy. For TBV that targets gametocytes, numbers of gametocytes and duration of gametocytes are important measures since it is expected that reduction in the number of gametocyte-carriers (reservoir) will decrease transmission. Microscopic determination of gametocytes on Giemsa-stained smears has long been used but they must be complemented with PCR methods since they have shown to underestimate gametocyte load [109]. Gametocyte infectivity to mosquitoes can be measured with the membrane-feeding assay mentioned earlier or with feeding of mosquitoes directly on the skin of gametocyte carriers, which reproduces the natural situation. An honest correlation between the two assays has been described in few studies [110] but the membrane-feeding assay still awaits definitive validation [111]. In summary, it would be relevant for future TBV trials to perform feeding directly on gametocyte carriers using local mosquitoes. These mosquitoes would have to be raised in local insectaries and tested for the absence of any other human pathogens. Measures of TBV efficacy should not be limited to development of oocysts but also to salivary glands sporozoites since the latter are the infectious forms to humans. Hence, future studies should aim at measuring salivary gland sporozoite loads and sporozoite infectivity.
