**3.1.4 Merozoite Surface Protein 1 (MSP1)**

MSP1 is the most abundant and best studied of the *P. falciparum* merozoite surface antigens. The protein is synthesised as a 190-kDa-precursor protein that is cleaved into four fragments of 83, 30, 38, and 42 kDa [100]. These exist as a non-covalently associated complex tethered by a glycosylphosphatidylinositol (GPI) anchor at the C-terminal [101]. During erythrocyte invasion, the N-terminal fragments are shed when MSP142 undergoes a secondary processing event generating a further 33 kDa (MSP133) and 19 kDa fragment (MSP119) [102- 104] which facilitates parasite entry into the erythrocyte [105]. The MSP119 fragment is retained on the surface of the invading merozoite [106].

The C-terminal end of MSP1 is a leading malaria vaccine candidate. The MSP133 sequence is dimorphic, with diverse alleles clustering within the two families known as K1 and Mad20 [107], however MSP119 is relatively conserved across the two allele families with six nonsynonymous SNPs that are commonly used to describe MSP119 haplotypes [108-113]. Within each of the allele-families, MSP133 also contains several single nucleotide polymorphisms (SNPs) and a 3 bp deletion [108, 111].

The FMP1/ASO2A vaccine, which is formulated with the 3D7 allele of MSP142, has been tested in Phase II trials. The vaccine initially showed promise in safety and immunogenicity trials in 40 Malian adults, with responses generated to parasite clones carrying diverse MSP142 alleles (FVO and Camp/FUP) [114]. Phase II efficacy trials including 400 children in Western Kenya did not protect against infection or lower parasite densities, nor did it reduce clinical episodes [115]. Though the authors stated that the vaccine was no longer a promising vaccine candidate, this was without having investigated how many vaccinees were infected with the vaccine strain. In addition, high throughput genotyping studies in Mali have demonstrated that the 3D7 allele had a prevalence of only 16% [113]. A vaccine containing both MSP142 3D7 and FVO alleles fused together with conserved regions of MSP1 is currently being tested [116].

#### **3.1.5 Merozoite Surface Protein 2 (MSP2)**

MSP2, a 45-52 kDa glycoprotein is tethered to the membrane a glycosylphosphatidylinosital (GPI) anchor and is the second most abundant protein (based on copy numbers) on the merozoite surface [49]. The protein consists of highly conserved N- and C-terminal ends flanking a highly polymorphic central repeat region. MSP2 sequences fall into two distinct allelic families namely FC27 and 3D7 (IC-1) [117-120]. Within these allele-families, the central repeats vary in length, number and sequence among isolates. Allele-specific and length polymorphism in MSP2 has been used as the basis for high-resolution genotyping of *P. falciparum* isolates [121, 122].

Using Population Genetics to Guide Malaria Vaccine Design 239

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

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].

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

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].

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

would need to be combined with other merozoite antigens.

**3.1.8 Pfs25 and Pfs45/48** 

structuring [8, 164, 165].

Further clinical trials are planned [51].

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

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