**3. Testing malaria vaccines in the field**

Vaccine development in malaria have employed a composite of rational and empirical approaches and depended on multiple epidemiological and experimental studies. Individuals living in endemic regions acquire immunity over time after repeated exposure to the parasites. Such immunity, also called premonition, is partial, species-specific and biphasic [8, 9]. In the first phase, the hosts still get infected but do not develop clinical symptoms. On the contrary, the second phase, which is the prevention or limitation of parasite multiplication, takes long to develop. This second phase is heavily dependent on parasite exposure - more the exposure the host gets, lesser the time this immunity takes to develop [9, 10]. Hence, the goal of vaccine strategies is to reduce the time needed to acquire protective immunity and to

The use of experimental models is critical to vaccine development. Many researchers advocate the use of human parasites in human hosts as it is the optimal experimental model for malaria [11]. However, field studies are inherently limited by the inability to control multiple experimental parameters such as the number of infective mosquito bites, the number of parasite per infective dose, and the genetic background of the host and parasite. In addition, there are numerous ethical considerations, which restrict access to peripheral blood samples for antibody and T cell studies, important for investigating long-term protection. Thus, many researchers have turned to more controllable models, such as monkey or human *Plasmodium* in monkeys [12–14] or rodent *Plasmodium* in mice [15]. Using these models, there have been numerous vaccination studies using genetically-attenuated parasites [16], irradiated parasites [17, 18], chemically-attenuated parasites [19, 20], live parasites under drug prophylaxis [21–23], and defined antigenic formulations [24–30]. These studies have demonstrated that vaccination can reduce parasite development, prevent pathology in infected animals, prevent transmission to mosquitoes, and even induce sterile immunity. Another major advantage of these models is that the outcomes following vaccination is well-defined and easily measurable, such as development of sterile immunity, inhibition of parasite development in the liver or in the blood, and/ or prevention of certain pre-defined clinical signs or of pathologies. So far, only whole parasite formulations using irradiated sporozoites [31, 32] or live parasite immunization under chloroquine [33–35], and a limited number of sporozoite antigen formulations, such as RTS,S [36], have been shown to induce sterile immunity in significant proportion of the human volunteers.

Vaccine clinical testing in humans involves multiple phases. Phase IA involves a small group of naïve volunteers (<100), from non-endemic regions, with no previous experience of malaria, while Phase I involves malaria-exposed individuals from endemic regions. In both phases, vaccine safety and immunogenicity are assessed. Only after the vaccine has shown a good safety profile with encouraging immunogenicity data, phase IIa test study can be initiated with a larger set of volunteers (>100–1000) from non-endemic regions. In Phase IIa, vaccine efficacy is assessed by subjecting the volunteers to a challenge with mosquito bites or intravenous injections of infected red blood cells. Phase IIb involves assessing the vaccine efficacy in a larger set of volunteers from endemic regions. Promising Phase II results qualify moving the

make the immunity long-lasting.

340 Towards Malaria Elimination - A Leap Forward

**2. Vaccine developmental phases**

A malaria vaccine could potentially target many different stages of the infection. It could work by: (1) preventing *de novo* infection (either in the liver or the blood), (2) controlling parasite levels in the blood and duration of the blood infection, (3) preventing pathology induced by the infection and thus preventing or reducing morbidity or mortality, and (4) preventing or reducing transmission to mosquitoes. However, not all of these outcomes can be assessed accurately in the field. Only the first and fourth outcomes mentioned can be assessed accurately and experimentally with reproducible results, mainly due to the standard operating procedures that have been implemented over the years.

Outcome assessment in the field is complicated due to the nature of the infection itself. In endemic regions, malaria infections are usually chronic [37]. Low-level parasite persistence may affect immune reactivity by amplifying or down-regulating vaccine-induced immune responses. It may also confuse diagnostics such as fever detection [38]. Occurrence and extent of chronicity may vary according to age, endemicity and host genetics. Thus, in many trials, antimalarial treatments are applied to the tested cohorts to clear prior malarial infections to reduce confounding factors [39].

Presence of co-infections is another factor that makes outcome assessment in the field difficult. Often, endemic cohorts are also infected with other pathogens, such as worms, bacteria or viruses, without being overtly sick [40–44]. Immune responses to these pathogens may either potentiate or inhibit the development of the protective response induced by infection or vaccination [45]. Due to cost constraints, it is rarely possible to make a full analysis for all possible pathogens, but it is advisable to perform retrospective studies to assess their possible influence on the malaria vaccination outcomes.

Another major roadblock for malaria vaccine development is the absence of correlates or surrogate markers of protection. These markers are crucial as they would facilitate the testing of large sets of vaccine formulations and would reduce costs and organization constraints [46]. As an example, vaccine development against Hepatitis B was greatly simplified when it was shown that concentration of Hepatitis B S antigen antibodies over 10 UI/ML level was a surrogate marker of protection (for review Plotkin et al.) [47]. This greatly accelerated the testing of multiple new formulations in a limited number of volunteers and also helped in the development of subsequent improved formulations. There is clearly a gap in our knowledge of the immune correlates of protection against malaria. It is still not clearly known what defense mechanisms are crucial in humans for mediating protection against malaria. This severely handicaps our progress towards effective vaccine development [48].

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