**3.1.6 Merozoite Surface Protein 3**

MSP3, also known as Secreted Polymorphic Antigen associated with Merozoites (SPAM) is associated with the merozoite surface. It is secreted into the parasitophorous vacuole of the mature parasite (schizont) where it undergoes proteolytic cleavage [133-135]. Upon rupture of the infected erythrocyte, some MSP3 protein remains associated with the merozoite [133]. MSP3 contains three blocks of a polymorphic heptad repeat flanked by conserved sequences [133]. While initial studies reported limited polymorphism [134, 136] analysis of several isolates has revealed that the sequence is primarily dimorphic in nature with both point mutations and repeat variations in the N-terminal half of the protein with different alleles demonstrating variable antibody binding activity [137]. Antibodies produced in mice can inhibit parasite growth *in vitro* in co-operation with monocytes [135] but are allele-specific [138]. Furthermore, antibodies to different alleles in endemic human populations are individually associated with protection against clinical episodes [138]. Sequence data from natural parasite populations has confirmed that this antigen is under strong balancing selection and therefore a natural immune target [138].

A MSP3 vaccine based on a long synthetic peptide (MSP3-LSP) covering a relatively conserved region (amino acids 181-276) from strain FC27 has been tested in Phase I trials in Switzerland and later in Burkina Faso and shown to be safe and immunogenic with a strong cytophilic response [45, 139]. Although the second trial was not designed specifically to test efficacy the number of clinical episodes was measured as a means to monitor safety. Comparison of two MSP3-LSP vaccinated groups (different adjuvants) compared to individuals that received the alternative vaccine (Hepatitis B) demonstrated that the incidence of clinical malaria was three to four-fold lower in the MSP3-LSP vaccine groups [45]. Interestingly however, the degree of protection wanes substantially during the follow up period of 60 days, suggesting that protection may be short-lived. The GMZ2 vaccine comprising a fusion protein containing conserved regions of MSP3 and Glutamate Rich Protein (GLURP) has also been confirmed as safe and immunogenic in Phase I trials [140] and Phase II trials are currently underway [51].

### **3.1.7 175kDa Erythrocyte Binding Antigen (EBA175)**

The 175 kDa Erythrocyte Binding Antigen, EBA175, is found in the micronemes, which are located at the apical end of the merozoite [141, 142]. It is a parasite ligand that directly associates with its receptor, Glycophorin A on the surface of uninfected erythrocytes. This occurs via an interaction between sialic acids and the Glycophorin A backbone [143, 144]. The cysteine-rich binding region (RII) is a 616 amino acid region consisting of two regions – F1 and F2, which are known as Duffy binding-like (DBL) domains named so as they are homologous to *P. vivax* Duffy binding protein, DBP. These domains are found in several other adhesion ligands of *P. falciparum* including Erythrocyte Membrane Protein 1 (PfEMP1). Antibodies to EBA175-RII can be induced by immunization in animal models by recombinant EBA175 protein [143] and are acquired in humans naturally exposed to malaria [36, 145-147] and these antibodies can inhibit parasite invasion *in vitro* [142, 148]. Studies have suggested that EBA175 alleles are maintained by immune selection [149] and high levels of haplotype diversity are present, most likely as a result of recombination [8]. However, because *P. falciparum* parasites can vary the use of EBA175 to evade antibodies [150], EBA175 could not be used alone but would need to be combined with other merozoite antigens.

Vaccination of primates with the EBA175 vaccine candidate, EBA175-RII-NG, which is based on the 3D7 allele of RII, resulted in a significant decrease in parasite density after homologous challenge [151]. Clinical trials have proven the vaccines safety and shown that it produces antibody responses in vaccinated individuals. Furthermore, the serum of vaccinated individuals was shown to inhibit the binding of recombinant EBA175-RII to erythrocytes [152].

## **3.1.8 Pfs25 and Pfs45/48**

238 Malaria Parasites

MSP2 is a target of naturally acquired antibodies [123, 124] and antibodies are associated with protection against clinical malaria in some studies [125-128]. Longitudinal studies have suggested allele-specific antibody responses, with encountered strains not being observed in subsequent infections [129, 130], while others have shown that individuals can be re-infected with homologous strains [131]. The Combination B vaccine, which was composed of the 3D7 alleles for MSP1, MSP2 and ring-associated erythrocyte surface antigen (RESA) was tested in Phase II trials in Papua New Guinea in the early 1990's. This vaccine, which is discussed in more detail later, was shown to be efficacious, reducing parasite densities significantly with most of the activity attributed to MSP2 [6]. A combination vaccine, MSP1-C1 containing both the 3D7 and FC27 alleles has recently been tested but showed unacceptable

MSP3, also known as Secreted Polymorphic Antigen associated with Merozoites (SPAM) is associated with the merozoite surface. It is secreted into the parasitophorous vacuole of the mature parasite (schizont) where it undergoes proteolytic cleavage [133-135]. Upon rupture of the infected erythrocyte, some MSP3 protein remains associated with the merozoite [133]. MSP3 contains three blocks of a polymorphic heptad repeat flanked by conserved sequences [133]. While initial studies reported limited polymorphism [134, 136] analysis of several isolates has revealed that the sequence is primarily dimorphic in nature with both point mutations and repeat variations in the N-terminal half of the protein with different alleles demonstrating variable antibody binding activity [137]. Antibodies produced in mice can inhibit parasite growth *in vitro* in co-operation with monocytes [135] but are allele-specific [138]. Furthermore, antibodies to different alleles in endemic human populations are individually associated with protection against clinical episodes [138]. Sequence data from natural parasite populations has confirmed that this antigen is under strong balancing

A MSP3 vaccine based on a long synthetic peptide (MSP3-LSP) covering a relatively conserved region (amino acids 181-276) from strain FC27 has been tested in Phase I trials in Switzerland and later in Burkina Faso and shown to be safe and immunogenic with a strong cytophilic response [45, 139]. Although the second trial was not designed specifically to test efficacy the number of clinical episodes was measured as a means to monitor safety. Comparison of two MSP3-LSP vaccinated groups (different adjuvants) compared to individuals that received the alternative vaccine (Hepatitis B) demonstrated that the incidence of clinical malaria was three to four-fold lower in the MSP3-LSP vaccine groups [45]. Interestingly however, the degree of protection wanes substantially during the follow up period of 60 days, suggesting that protection may be short-lived. The GMZ2 vaccine comprising a fusion protein containing conserved regions of MSP3 and Glutamate Rich Protein (GLURP) has also been confirmed as safe and immunogenic in Phase I trials [140]

The 175 kDa Erythrocyte Binding Antigen, EBA175, is found in the micronemes, which are located at the apical end of the merozoite [141, 142]. It is a parasite ligand that directly associates with its receptor, Glycophorin A on the surface of uninfected erythrocytes. This

reactogenicity due to the adjuvant used and the trial was terminated [132].

**3.1.6 Merozoite Surface Protein 3** 

selection and therefore a natural immune target [138].

and Phase II trials are currently underway [51].

**3.1.7 175kDa Erythrocyte Binding Antigen (EBA175)** 

The transmission blocking candidates Pfs25 and Pfs45/48 are found in the zygote/ookinte and gametocyte stages respectively. These antigens, which migrate as single and double bands respectively, are targets of antibodies that have been shown to block transmission of *P. falciparum* to the mosquito vector [153-156]. Pfs25 was cloned well before Pfs48/45 [157, 158] and therefore is the only candidate for which a vaccine trial has been carried out. Preclinical development studies are underway for the latter antigen. The attraction of Pfs25 as a vaccine candidate is that it is not expressed in the human host and has relatively limited polymorphism [159, 160]. It is therefore unlikely to be under the same immune pressures as other antigens, however one downside of this is that natural boosting may not occur unless long lived T-cell responses are elicited by the vaccine. Pfs48/45 gene knock out experiments demonstrate a key role in male fertility [161]. Anti-Pfs48/45 antibodies in individuals naturally exposed to malaria are associated with transmission blocking activity [162] and given that it is expressed within the human host it is likely to allow natural boosting of antibody responses. Pfs25 is relatively conserved [163] while Pfs48/45 shows high levels of diversity worldwide with evidence of diversifying selection and strong geographic structuring [8, 164, 165].

The only vaccine trial of these two antigens that has been conducted is a phase I trial of the Pfs25 vaccine candidate. However it was halted due to unexpected adverse effects [166]. Preclinical studies have demonstrated a significant increase in the immune response of animal models, and immune-sera had a significant transmission blocking effect [167]. Further clinical trials are planned [51].

#### **3.1.9 Other** *Plasmodium falciparum* **vaccine candidates**

There are several other malaria vaccine candidates currently under development which are based on well-known antigens, such as GLURP, which as mentioned above is being tested in

Using Population Genetics to Guide Malaria Vaccine Design 241

Only two *P.vivax* vaccine candidates, the circumsporozoite surface protein (CSP) and the gametocyte antigen, Pvs25, have been tested in clinical trials to date [38]. However, a host of additional *P.vivax* proteins are currently under investigation as potential vaccine candidates, including PvDBP [38], PvTRAP [186], PvMSP-1 [187], PvAMA-1 [188]; and the transmission blocking candidate, Pvs28 [38]. Additional antigens identified as potential *P.vivax* vaccine candidates include PvMSP3, PvMSP4, PvMSP5 and PvRBPs I and II [38]. PvMSP9 has also

Naturally acquired immunity to malaria develops only after years of exposure to infection by Plasmodium parasites. The extensive genetic diversity that is characteristic of malaria surface antigens provides one explanation for this, especially because immune responses are variant-specific. Eventually, antibodies to all of the variants in the parasite population are acquired or reach a threshold whereby protection against clinical episodes is achieved

Numerous epidemiological studies have investigated the role of malarial antigens as natural targets of human immunity with conflicting estimates of their protective effect. While differences in study methodology, transmission intensity, and the rate of natural immunity acquisition [190, 191] will account for some of the discrepancies, parasite genetic diversity is likely to play a major role. This is because the prevalence of the major allelic types of specific antigens and subsequent acquisition of allele-specific immunity

A recent systematic review and meta-analysis investigating the protective effect of antibody responses to merozoite antigens, highlighted the issues of genetic diversity in immunoepidemiological studies [192]. This review pooled all the published evidence for the association between anti-merozoite antibodies and the incidence of *P. falciparum* for each antigenic allele. For each allele, individual study estimates often showed large degrees of heterogeneity and comparing pooled estimates across alleles for the same antigen either showed similar (e.g. MSP1, MSP2) or very different (e.g. MSP119, MSP3, AMA1, GLURP)

A major contributor to the heterogeneity in protective estimates is the fact that allele-specific antibody response to the strain causing the malaria episode was not measured in these studies. If antibody-mediated protection is largely allele-specific then the true causal protective effect will be underestimated in studies that do not use allele-specific *P. falciparum* outcomes. For example, meta-analysis of studies investigating the protective effects of anti-MSP23D7 and MSP2FC27 responses showed no evidence of a reduced risk of symptomatic *P. falciparum* (all strains combined) [192]. Four studies included allele-specific endpoints; two studies in PNG showed protective MSP2 responses to homologous strains [125, 128] whereas studies in South America and Africa [193, 194], show no evidence of a protective effect of pre-existing MSP2 allele-specific immunity on clinical episodes with homologous

been revealed as a promising vaccine candidate in recent studies [189].

**4. Variant-specific immunity** 

**4.1 Identifying targets of human immunity** 

(reviewed in [190]).

varies across populations.

magnitudes of a protective effect.

parasites.

combination with MSP3 [140], Liver Stage Antigen 1 (LSA1) being developed as a component of the ME-TRAP vaccine [168] and as a single component vaccine [169]; and MSP4 [170]. More recently discovered antigens demonstrate significant potential as vaccine candidates including members of the Rh family of proteins (Rh1, 2a, 2b, 4 and 5) [171, 172] and the Rh-interacting protein, RIPr [173], Serine repeat antigen 5 (SERA5, reviewed in [174]); MSP6 [175], MSP7 [176] and the pregnancy-associated malaria vaccine candidate, *var2csa* [177-179]. For some of these antigens, diversity is a significant issue that needs to be evaluated.

Combinations of antigens from different *P. falciparum* lifecycle stages have also been tested. Trials for Spf66, a synthetic combination of peptides from including CSP, MSP1 and two others of unknown origin have now been halted after it was shown to have no effect on malaria [180], NMRC-M3V-Ad-PfCA which is a combination of CSP and AMA1 and is currently in phase 2 trials in the USA [51], GMZ2 which was mentioned above (MSP3 and GLURP, [140]) and the EBA/Rh vaccine candidate, which consists of EBA175 (RIII-V) and Rh2a/b, Rh5 and RIPr binding regions [181]. This vaccine candidate is still undergoing preclinical testing and aims to target different invasion pathways used by the merozoite [172].
