**3. Anti-parasite activities in sera from individuals living in malaria-endemic areas**

Protection against malaria mediated by antibodies recognizing the erythrocytic parasite or the parasitized erythrocyte is mediated by several distinct mechanisms: (1) Binding of the antibody to the surface of merozoites can interfere with the invasion of new erythrocytes and opsonize the merozoite, which results in complement activation and/or phagocytosis; (2) Binding to the merozoite may not suffice to block invasion, but antibodies carried into

The Impact of Immune Responses on the Asexual

(Aucan et al., 2000).

severe malaria (Brasseur et al., 1990) has been reported.

given to individuals in Mali (Miura et al., 2011).

responsible for the contradictory outcomes.

of natural immunity.

Erythrocytic Stages of *Plasmodium* and the Implication for Vaccine Development 211

IgM antibodies are characterized by lower affinity for antigen compared to their IgG counterparts because little or no somatic hypermutation and clonal selection has occurred. IgM antibodies form pentamers to compensate for the lower affinity, which results in a higher overall avidity due to increased number of binding sites. Evidence for a protective role of IgM against malaria infection (Wahlgren et al., 1986; Boudin et al., 1993) or against

Evidence from field studies in Ghana (Dodoo et al., 2000), Senegal (Oeuvray et al., 2000) and East Asia (Soe et al., 2004) suggest that cytophilic antibodies are associated with a lower risk for subsequent clinical malaria episodes. These isotypes are also associated with the antibody-dependent cellular inhibition as discussed above. The importance of immunoglobulin isotypes in addition to the antigen specificity of the humoral response is underscored by studies that report an association between the levels of noncytophilic antibodies and clinical malaria incidence. IgG4 antibodies are preferentially induced after repeated exposure (Aalberse et al., 1983a; Aalberse et al., 1983b) and this isotype is associated with enhanced risk of infection and with a high risk of clinical malaria episodes

The attitude towards the use of measuring growth inhibition as an immune correlate has changed over the past five years. Invasion or growth inhibitory (GIA) activity was considered a reliable predictor of vaccine efficacy and the results from these assays were used to down-select potential vaccine candidates. Initially, GIA activity was determined by using purified immunoglobulins from preclinical or clinical trials at high concentrations and from these studies, strong inhibition was frequently observed. However, the same vaccines that generated very "high" GIA activities in rabbits and other preclinical models failed to induce protection in naïve US individuals (Spring et al., 2009; Duncan et al., 2011). In residents from malaria-endemic areas, GIA activity is, however, a common factor associated with protection against clinical disease (Dent et al., 2008). In this study, malaria-infected participants were drug-cured and followed up until their next malaria infection. Participants whose sera exhibited higher growth inhibition prior to drug cure were protected for significantly longer periods of time than those participants whose antibodies mediated only low GIA activities. Moreover, an age-dependent effect was observed in that GIA activity inversely correlated with the age of study participants. Another confounding factor is the historical exposure of study participants as vaccine efficacy differs dramatically between individuals with no prior malaria history *vs*. residents from malaria-endemic areas. For example, an AMA-1 vaccine tested in US naïve study participants resulted in relatively high GIA activities, but the same vaccine failed to induce either GIA activity or protection as defined by time to the next malaria episode when

Review of the scientific literature on blood stage malaria vaccines reveals no straightforward strategy for evaluating a successful blood stage vaccine. A variety of reasons may be

• Differences in the transmission rates (intensity and stability) and the short lifespan of malaria specific antibodies can skew the measurements and conclusions. Therefore, directly comparing study results from different geographic regions that are not matched by transmission rates and seasons should be avoided. Transmission rates may have an impact on the clinical outcome of malaria episodes as well as on the induction

the infected RBC by the merozoite can result in growth inhibition; (3) Binding to pRBC can prevent sequestration, thus exposing the pRBC to conditions that are unfavorable for development, which may lead to increased clearance of pRBC by the spleen; and (4) Binding to pRBC can interfere with rosetting, a prerequisite for invasion. Studying the invasion of erythrocytes by merozoites has resulted in the identification of various invasion pathways, which the parasite can utilize. These pathways can be categorized broadly into: (1) sialic acid dependent and (2) sialic acid independent pathways. Field studies of the anti-parasite activity in sera from individuals with acquired natural immunity have shown that one of the earliest mechanisms to block blood stage parasites is to develop antibodies that interfere with the sialic acid dependent invasion (Baum et al., 2003; Nery et al., 2006; Persson et al., 2008). Antibodies obtained from sera of young African children are more likely to block sialic acid dependent pathways while the ability to block sialic acid independent pathways is acquired after years of exposure to the parasite and with age. Antibodies capable of inhibiting rosetting can mediate protection, which reduces the risk of cerebral malaria (Vigan-Womas et al., 2010). Nevertheless, the actual role of antibodies in protecting against disease in the field remains controversial. While they may reduce the parasite load of individuals living in endemic areas, they do not mediate sterile protection or total suppression of parasites in the blood (Genton et al., 2002). Recently, an apical merozoite antigen (AMA)-1 based vaccine that induces high antibody titers, and high GIA responses *in vitro* in human vaccinees (Ellis et al., 2009) was evaluated for its protective effect in a blood challenge. To this end, the vaccinees were immunized and then challenged with blood infected with the 3D7 parasite clone. While the vaccine was able to reduce the multiplication rate *in vivo*, it was unable to mediate complete elimination of the parasites after a blood challenge (Duncan et al., 2011). This may indicate that either a single antigen such as AMA-1 or blood-stage antigens in general may be able to alleviate the morbidity associated with disease (as seen in the case of natural immunity), but may not be able to induce complete protection against disease.

Longitudinal studies in hyper-endemic malaria transmission areas have revealed factors related to the development (or lack thereof) of malaria-specific antibodies. Due to overlapping malaria infections in high transmission areas, where infections can occur frequently or even daily, it is difficult to study immune responses elicited by and maintained after a discrete infection. Studies in high transmission areas have a limited ability to consider age because participants suffer many unrelated infections during their first 5 years of life, including viral, bacterial and other parasitic diseases. One example is nematode infections, which are quite common in malaria-endemic areas and predispose the host to Th2-type immune responses. Exposure to malaria is an important potential confounder in immune-epidemiological studies. Therefore, the inadequate measurement and adjustment for differences in exposure may lead to the underestimation of the strength of associations between immunological variables and malaria incidence (Kinyanjui et al., 2009). The strongest evidence that antibodies are important mediators of naturally acquired immunity is from passive transfer experiments of antibody from immune adults used to treat children with severe *P. falciparum* malaria (Cohen et al., 1961; McGregor and Carrington, 1963; Bouharoun-Tayoun et al., 1990). IgG and IgM antibodies present in sera of malaria-exposed individuals recognize and bind directly to trophozoites or schizonts. Neutralization and agglutination of merozoites and pRBC by these antibodies are reported as possible protective mechanisms during *Plasmodium* infection (Cohen and Butcher, 1971).

the infected RBC by the merozoite can result in growth inhibition; (3) Binding to pRBC can prevent sequestration, thus exposing the pRBC to conditions that are unfavorable for development, which may lead to increased clearance of pRBC by the spleen; and (4) Binding to pRBC can interfere with rosetting, a prerequisite for invasion. Studying the invasion of erythrocytes by merozoites has resulted in the identification of various invasion pathways, which the parasite can utilize. These pathways can be categorized broadly into: (1) sialic acid dependent and (2) sialic acid independent pathways. Field studies of the anti-parasite activity in sera from individuals with acquired natural immunity have shown that one of the earliest mechanisms to block blood stage parasites is to develop antibodies that interfere with the sialic acid dependent invasion (Baum et al., 2003; Nery et al., 2006; Persson et al., 2008). Antibodies obtained from sera of young African children are more likely to block sialic acid dependent pathways while the ability to block sialic acid independent pathways is acquired after years of exposure to the parasite and with age. Antibodies capable of inhibiting rosetting can mediate protection, which reduces the risk of cerebral malaria (Vigan-Womas et al., 2010). Nevertheless, the actual role of antibodies in protecting against disease in the field remains controversial. While they may reduce the parasite load of individuals living in endemic areas, they do not mediate sterile protection or total suppression of parasites in the blood (Genton et al., 2002). Recently, an apical merozoite antigen (AMA)-1 based vaccine that induces high antibody titers, and high GIA responses *in vitro* in human vaccinees (Ellis et al., 2009) was evaluated for its protective effect in a blood challenge. To this end, the vaccinees were immunized and then challenged with blood infected with the 3D7 parasite clone. While the vaccine was able to reduce the multiplication rate *in vivo*, it was unable to mediate complete elimination of the parasites after a blood challenge (Duncan et al., 2011). This may indicate that either a single antigen such as AMA-1 or blood-stage antigens in general may be able to alleviate the morbidity associated with disease (as seen in the case of natural immunity), but may not be able to induce complete

Longitudinal studies in hyper-endemic malaria transmission areas have revealed factors related to the development (or lack thereof) of malaria-specific antibodies. Due to overlapping malaria infections in high transmission areas, where infections can occur frequently or even daily, it is difficult to study immune responses elicited by and maintained after a discrete infection. Studies in high transmission areas have a limited ability to consider age because participants suffer many unrelated infections during their first 5 years of life, including viral, bacterial and other parasitic diseases. One example is nematode infections, which are quite common in malaria-endemic areas and predispose the host to Th2-type immune responses. Exposure to malaria is an important potential confounder in immune-epidemiological studies. Therefore, the inadequate measurement and adjustment for differences in exposure may lead to the underestimation of the strength of associations between immunological variables and malaria incidence (Kinyanjui et al., 2009). The strongest evidence that antibodies are important mediators of naturally acquired immunity is from passive transfer experiments of antibody from immune adults used to treat children with severe *P. falciparum* malaria (Cohen et al., 1961; McGregor and Carrington, 1963; Bouharoun-Tayoun et al., 1990). IgG and IgM antibodies present in sera of malaria-exposed individuals recognize and bind directly to trophozoites or schizonts. Neutralization and agglutination of merozoites and pRBC by these antibodies are reported as possible protective mechanisms during *Plasmodium* infection (Cohen and Butcher, 1971).

protection against disease.

IgM antibodies are characterized by lower affinity for antigen compared to their IgG counterparts because little or no somatic hypermutation and clonal selection has occurred. IgM antibodies form pentamers to compensate for the lower affinity, which results in a higher overall avidity due to increased number of binding sites. Evidence for a protective role of IgM against malaria infection (Wahlgren et al., 1986; Boudin et al., 1993) or against severe malaria (Brasseur et al., 1990) has been reported.

Evidence from field studies in Ghana (Dodoo et al., 2000), Senegal (Oeuvray et al., 2000) and East Asia (Soe et al., 2004) suggest that cytophilic antibodies are associated with a lower risk for subsequent clinical malaria episodes. These isotypes are also associated with the antibody-dependent cellular inhibition as discussed above. The importance of immunoglobulin isotypes in addition to the antigen specificity of the humoral response is underscored by studies that report an association between the levels of noncytophilic antibodies and clinical malaria incidence. IgG4 antibodies are preferentially induced after repeated exposure (Aalberse et al., 1983a; Aalberse et al., 1983b) and this isotype is associated with enhanced risk of infection and with a high risk of clinical malaria episodes (Aucan et al., 2000).

The attitude towards the use of measuring growth inhibition as an immune correlate has changed over the past five years. Invasion or growth inhibitory (GIA) activity was considered a reliable predictor of vaccine efficacy and the results from these assays were used to down-select potential vaccine candidates. Initially, GIA activity was determined by using purified immunoglobulins from preclinical or clinical trials at high concentrations and from these studies, strong inhibition was frequently observed. However, the same vaccines that generated very "high" GIA activities in rabbits and other preclinical models failed to induce protection in naïve US individuals (Spring et al., 2009; Duncan et al., 2011). In residents from malaria-endemic areas, GIA activity is, however, a common factor associated with protection against clinical disease (Dent et al., 2008). In this study, malaria-infected participants were drug-cured and followed up until their next malaria infection. Participants whose sera exhibited higher growth inhibition prior to drug cure were protected for significantly longer periods of time than those participants whose antibodies mediated only low GIA activities. Moreover, an age-dependent effect was observed in that GIA activity inversely correlated with the age of study participants. Another confounding factor is the historical exposure of study participants as vaccine efficacy differs dramatically between individuals with no prior malaria history *vs*. residents from malaria-endemic areas. For example, an AMA-1 vaccine tested in US naïve study participants resulted in relatively high GIA activities, but the same vaccine failed to induce either GIA activity or protection as defined by time to the next malaria episode when given to individuals in Mali (Miura et al., 2011).

Review of the scientific literature on blood stage malaria vaccines reveals no straightforward strategy for evaluating a successful blood stage vaccine. A variety of reasons may be responsible for the contradictory outcomes.

• Differences in the transmission rates (intensity and stability) and the short lifespan of malaria specific antibodies can skew the measurements and conclusions. Therefore, directly comparing study results from different geographic regions that are not matched by transmission rates and seasons should be avoided. Transmission rates may have an impact on the clinical outcome of malaria episodes as well as on the induction of natural immunity.

The Impact of Immune Responses on the Asexual

(Esen et al., 2009; Belard et al., 2011).

**4.2 Antigens within merozoite organelles** 

assay (ADCI).

Erythrocytic Stages of *Plasmodium* and the Implication for Vaccine Development 213



Antigens found in the apical organelles of the merozoites such as the apical membrane antigen (AMA)-1 or the erythrocyte binding antigen (EBA-175 RII) have long been studied due to their immunogenicity or biological function. While the exact function of AMA-1 is unknown, EBA-175 has been better characterized. EBA-175 is found in the micronemes of the merozoite and is secreted by merozoites in order to bind to erythrocytes that are ready for invasion. This binding facilitates the attachment of merozoites to the coated erythrocytes in a strain-specific manner (Camus and Hadley, 1985). EBA-175 is a member of a family of binding proteins such as EBA-140, EBA-181, MAEBL; all of these antigens share a receptor binding domain (Region (R)-II). The analysis of sera from malaria-endemic areas for the presence of EBA-175 specific antibodies revealed some association with protection in children that have higher antibody titers (reviewed in (Fowkes et al., 2010)). Recently, an

AMA-1 is a highly polymorphic antigen generated in the rhoptries of the merozoites and it appears on the parasite's surface just before invasion occurs. AMA-1 is highly immunogenic and anti-AMA-1 antibody responses are found in sera from individuals living in malariaendemic areas. Another promising feature of this antigen is the fact that AMA-1 is expressed on sporozoites and thus the antigen could act as a pre-erythrocytic as well as an erythrocytic vaccine. Analysis of AMA-1 specific antibodies in growth inhibition assays provided even more promise as the antibody activity is typically very high compared to other blood stage antigens. A clinical study in which volunteers were challenged by mosquito bite revealed that vaccination with AMA-1 was unable to provide sterile protection and only yielded a limited delay in the development of parasitemia in vaccinees compared to challenge control subjects (Spring et al., 2009). Characterization of the AMA-1 gene sequence revealed a fatal characteristic of this antigen which will likely preclude its use as a malaria vaccine in the field: well over 150 allelic variants of the antigen have been reported (Takala et al., 2009) and

EBA-175 based vaccine yielded some clinical efficacy (El Sahly et al., 2010).

