**4. Identification of antigens that are targeted by immune responses in immune individuals**

The merozoite consists of the merozoite surface coat, the micronemes, the rhoptries, apicoplast and a nucleus. From each of these components several antigens have been isolated and their immunological potential and biological function has been evaluated.

## **4.1 Merozoite surface coat antigens**

Several antigens expressed on the merozoite surface coat have been evaluated as vaccine candidates. Highlighted in this section are the most frequently targeted of those antigens:


### **4.2 Antigens within merozoite organelles**

212 Malaria Parasites

• Differences in the kinetics of induction of natural immunity (mainly caused by high

• Differences in the definitions of clinical malaria and study endpoints. In some settings, are the individuals with no signs of malaria truly protected or simply not exposed to

• The quality of plate antigens used for the ELISA: Proteins used for *in vitro* analyses may be of inferior quality due to misfolding or truncation. Recombinant proteins may not represent the native antigen structure. Moreover, for the analysis of MSP-1 specific immune responses induced by natural exposure to the parasite, most investigators have focused on the MSP-1p19 fragment rather than the MSP-1p42 protein, which is initially

• Mismatching of recombinant antigens and parasite phenotype: In many studies, the parasite variant prevalent in the study area is not matched with the recombinant

• Differences in the age of study participants: concentrations of malaria-specific antibodies are age dependent and, therefore, only comparisons of study participants

• Lack of information about study participants' conditions such as the use of bed nets, use of anti-malarial drugs or folk-medicine, co-infection with other parasites, bacteria

• Incompatibility in study design: in some cases study participants are pre-treated with anti-malarial drugs to clear parasites from the circulation prior to immunization while in other studies vaccines are administered while study participants have – in some cases significant numbers of- parasites in the blood. Therefore, it is inappropriate to compare

The merozoite consists of the merozoite surface coat, the micronemes, the rhoptries, apicoplast and a nucleus. From each of these components several antigens have been isolated and their immunological potential and biological function has been evaluated.

Several antigens expressed on the merozoite surface coat have been evaluated as vaccine candidates. Highlighted in this section are the most frequently targeted of those antigens: - MSP-1: the properties and functions of this antigen will be discussed in detail in Section 5 since this antigen represents a major blood stage vaccine candidate (Ockenhouse et al.,


**4. Identification of antigens that are targeted by immune responses in** 

protein used for the *in vitro* analysis (*e.g.*, plate antigen for ELISA assay).

transmission rates) may influence the immune factors mediating protection.

the parasite?

and viruses.

**immune individuals** 

**4.1 Merozoite surface coat antigens** 

2006; Ogutu et al., 2009).

dependent antibodies.

presented on the merozoite surface.

vaccine potency and efficacy between such studies.

with similar ages are valid.

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

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

The Impact of Immune Responses on the Asexual

Erythrocytic Stages of *Plasmodium* and the Implication for Vaccine Development 215

different mechanisms mediate protection: higher levels of IgG1 specific for GLURP and IgG3 for MSP-2 in children correlate with resistance to malaria and high-level parasitemia compared to malaria-susceptible children (Courtin et al., 2009). Higher anti-MSP-1 IgG1 levels were associated with protection against high-density parasitemia. The study also evaluated the *in vitro* anti-parasite activity of the sera from these children and reported an age-dependent decline in the *in vitro* GIA activity of the sera. The GIA activity was

A recent comprehensive meta-analysis of 33 clinical studies investigated the relationship between anti-merozoite antibodies and the incidence rate of malaria (Fowkes et al., 2010). The closest association between antibody titers and reduced risk was observed with IgG specific for the C-terminus of MSP-3 and MSP-1 (MSP-1p19). In contrast, antibodies directed to the N-terminus of MSP-1 and the presence of antibodies to MSP-2 was not significantly associated with protection. The analysis also revealed a positive association between

Our laboratory has focused on the major merozoite surface protein -1 (MSP-1). This antigen was identified in immune complexes from merozoite lysates (gp195), which provided the rationale for developing vaccines against it (Lyon et al., 1997). MSP-1 is first produced as a 195kD precursor that undergoes two successive proteolytic cleavage events (Blackman et al., 1994). The second processing event occurs immediately before invasion, resulting in the cleavage of the p42 molecule into a p33 and a p19 fragment. The p19 fragment remains attached to the merozoite surface through a GPI anchor (Gerold et al., 1996) and is comprised of two epidermal growth factor (EGF)-like domains (Morgan et al., 1999), which may have a role in the invading complex. Serological studies have provided significant evidence suggesting that immune responses directed against the C-terminus of MSP-1 (MSP-1p19 and MSP-1p42) are associated with immunity in preclinical models (Long et al., 1994; Egan et al., 1999; Darko et al., 2005; Parkkinen et al., 2006). Moreover, protective immunity as defined by lower mortality and morbidity of individuals residing in endemic

Various biological factors influencing the function of MSP-1 specific antibodies have been

(1) The role of MSP-1 specific antibody titers, isotype and the association with protection

A meta-analysis of 33 clinical studies revealed that the presence of MSP-1p19 specific antibodies is associated with a lower incidence rate of malaria (Fowkes et al., 2010). Moreover, high levels of anti-PfMSP-1p19 immunoglobulin G were associated with reduced malaria in an age-adjusted multivariate analysis (Perraut et al., 2005). In contrast, other reports failed to show any associations between MSP-1p19 (MSP-1) Abs and clinical outcome (Dodoo et al., 1999; Nebie et al., 2008). At this point we can only speculate about the cause of this discrepancy. As outlined above, the differences in the methodology and/or

dependent on anti-MSP-1, anti-AMA-1 and anti-MSP-2 specific antibody titers.

reduced risk for infection and antibody titers against AMA-1 and GLURP-R0.

areas was also associated with MSP-1p19 (Egan et al., 1999; John et al., 2004).

**5. MSP-1 and its role in immunity and infection** 

**5.1 The role of MSP-1 in natural immunity** 

and/or reduction in morbidity:

reported in individuals with natural immunity:

humoral responses against AMA-1 indicate that immunity is allele-specific and therefore, an AMA-based vaccine would primarily provide strain-specific protection at best. Efforts are underway to develop AMA-1 vaccines that induce allele-cross-reactive responses to overcome this limitation (Remarque et al., 2008; Dutta et al., 2010). However, the success of any AMA-1 based malaria vaccines is also impeded by the fact that sera from malariaendemic areas appear to contain antibodies capable of blocking the activity of AMA-1 specific antibodies (Miura et al., 2008).

### **4.3 Antigens expressed on the surface of infected erythrocytes**

The access of antibodies to merozoite antigens is limited as merozoites quickly invade new erythrocytes. Antibodies have the ability to gain entry to into infected erythrocytes (Bergmann-Leitner et al., 2009), but cannot mediate ADCI and would act independently of phagocytic cells as described above. Therefore, efforts are underway to identify antigens on pRBC which are theoretically always accessible for binding by specific antibodies. The surface localization of the antigens indicates that they are crucial for sequestration of the pRBC in the placenta or post-capillary venules. This warrants their exploration as potential vaccine targets. The variant surface antigens (VSA) (reviewed in (Hviid, 2010)) have been shown to mediate sequestration of the parasite and immune responses towards these antigens confer protection in a strain-specific manner. One of the members, the *P. falciparum* erythrocyte membrane protein (*Pf*EMP)-1, has been reported to be encoded by the *var* gene family and displays varying immunogenicity depending on the variant that is generated (Bull et al., 2005). One of these variants, *var2csa* is expressed by parasites that sequester in the placenta leading to severe malaria attacks in primagravid women often resulting in miscarriage and/or death of the mother (reviewed in (Beeson and Duffy, 2005)). This has led to efforts to develop vaccines that will be administered prior to or early in pregnancy (Avril et al., 2009).

#### **4.4 Multi-antigen responses: Reducing the risk for clinical infection or reducing parasite density**

The analysis of naturally acquired antibodies induced by the malaria parasite in an attempt to identify their antigen-specificity is challenging. Studies have frequently focused on a few select antigens, thus ignoring this complexity and the possibility of synergy in the response to multiple antigens. The complexity of these humoral responses was demonstrated using microarray assays in which 18 recombinant antigen fragments spanning various regions and alleles of four leading vaccine candidates (namely, MSP-1, MSP-2, MSP-3 and AMA-1) were tested (Gray et al., 2007). The results clearly demonstrate complex combinations of specific antibodies leading to an association with some form of protection. Reactivity to individual antigens did not correlate with protection, but combinations of antibodies to AMA-1 and allelic variants of MSP-2 were prevalent in individuals protected against clinical malaria.

A field study in Senegal in which factors such as reappearance of parasites, asymptomatic carriage of parasites, time to first clinical episode, and incidence of clinical episodes were considered led to the observation that antibodies to NANP, MSP-1p19, *Pf*EMP-3, *Pf*EB200 were associated with a lower risk for severe disease (Perraut et al., 2003). Another comprehensive study conducted in Senegal (305 children followed over 1 year) showed that

humoral responses against AMA-1 indicate that immunity is allele-specific and therefore, an AMA-based vaccine would primarily provide strain-specific protection at best. Efforts are underway to develop AMA-1 vaccines that induce allele-cross-reactive responses to overcome this limitation (Remarque et al., 2008; Dutta et al., 2010). However, the success of any AMA-1 based malaria vaccines is also impeded by the fact that sera from malariaendemic areas appear to contain antibodies capable of blocking the activity of AMA-1

The access of antibodies to merozoite antigens is limited as merozoites quickly invade new erythrocytes. Antibodies have the ability to gain entry to into infected erythrocytes (Bergmann-Leitner et al., 2009), but cannot mediate ADCI and would act independently of phagocytic cells as described above. Therefore, efforts are underway to identify antigens on pRBC which are theoretically always accessible for binding by specific antibodies. The surface localization of the antigens indicates that they are crucial for sequestration of the pRBC in the placenta or post-capillary venules. This warrants their exploration as potential vaccine targets. The variant surface antigens (VSA) (reviewed in (Hviid, 2010)) have been shown to mediate sequestration of the parasite and immune responses towards these antigens confer protection in a strain-specific manner. One of the members, the *P. falciparum* erythrocyte membrane protein (*Pf*EMP)-1, has been reported to be encoded by the *var* gene family and displays varying immunogenicity depending on the variant that is generated (Bull et al., 2005). One of these variants, *var2csa* is expressed by parasites that sequester in the placenta leading to severe malaria attacks in primagravid women often resulting in miscarriage and/or death of the mother (reviewed in (Beeson and Duffy, 2005)). This has led to efforts to develop vaccines that will be administered prior to or early in pregnancy (Avril

**4.4 Multi-antigen responses: Reducing the risk for clinical infection or reducing** 

The analysis of naturally acquired antibodies induced by the malaria parasite in an attempt to identify their antigen-specificity is challenging. Studies have frequently focused on a few select antigens, thus ignoring this complexity and the possibility of synergy in the response to multiple antigens. The complexity of these humoral responses was demonstrated using microarray assays in which 18 recombinant antigen fragments spanning various regions and alleles of four leading vaccine candidates (namely, MSP-1, MSP-2, MSP-3 and AMA-1) were tested (Gray et al., 2007). The results clearly demonstrate complex combinations of specific antibodies leading to an association with some form of protection. Reactivity to individual antigens did not correlate with protection, but combinations of antibodies to AMA-1 and allelic variants of MSP-2 were prevalent in individuals protected against clinical malaria.

A field study in Senegal in which factors such as reappearance of parasites, asymptomatic carriage of parasites, time to first clinical episode, and incidence of clinical episodes were considered led to the observation that antibodies to NANP, MSP-1p19, *Pf*EMP-3, *Pf*EB200 were associated with a lower risk for severe disease (Perraut et al., 2003). Another comprehensive study conducted in Senegal (305 children followed over 1 year) showed that

specific antibodies (Miura et al., 2008).

et al., 2009).

**parasite density** 

**4.3 Antigens expressed on the surface of infected erythrocytes** 

different mechanisms mediate protection: higher levels of IgG1 specific for GLURP and IgG3 for MSP-2 in children correlate with resistance to malaria and high-level parasitemia compared to malaria-susceptible children (Courtin et al., 2009). Higher anti-MSP-1 IgG1 levels were associated with protection against high-density parasitemia. The study also evaluated the *in vitro* anti-parasite activity of the sera from these children and reported an age-dependent decline in the *in vitro* GIA activity of the sera. The GIA activity was dependent on anti-MSP-1, anti-AMA-1 and anti-MSP-2 specific antibody titers.

A recent comprehensive meta-analysis of 33 clinical studies investigated the relationship between anti-merozoite antibodies and the incidence rate of malaria (Fowkes et al., 2010). The closest association between antibody titers and reduced risk was observed with IgG specific for the C-terminus of MSP-3 and MSP-1 (MSP-1p19). In contrast, antibodies directed to the N-terminus of MSP-1 and the presence of antibodies to MSP-2 was not significantly associated with protection. The analysis also revealed a positive association between reduced risk for infection and antibody titers against AMA-1 and GLURP-R0.
