**4.1 Identifying targets of human immunity**

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 varies across populations.

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) magnitudes of a protective effect.

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 parasites.

ongoing.

the importance of sequence diversity.

exposure [211, 215]

Using Population Genetics to Guide Malaria Vaccine Design 243

major allelic types is in development [132]. A more recent example of the challenge of antigenic diversity in malaria vaccines is AMA1. A phase II trial of a vaccine containing a single allele of *P. falciparum* AMA1 (3D7-allele) had no significant clinical efficacy when all malaria episodes were considered [46]. However, as was mentioned earlier, the vaccine demonstrated 68% efficacy against malaria episodes caused by AMA1 alleles that were similar in sequence to the vaccine allele. The development of approaches to overcome diversity in AMA1 and generate responses that give broad population coverage are

The most advanced vaccine in development, RTS,S, is based on a region of *P. falciparum* CSP [25]. Because CSP is polymorphic, an obvious question was whether diversity in CSP may explain the partial efficacy of the RTS,S vaccine. Analyses of infecting genotypes in one phase II trial of RTS,S suggested that this was not the case as there was no evidence of selection for infections containing non-vaccine genotypes among vaccinated versus control subjects [197, 198]. These findings highlight that although antigen polymorphism needs to be considered in vaccine development, it may not be a major issue for all vaccines. Clearly, there is a lot more we need to understand about the nature and specificity of responses and

There are a number of other promising antigens in pre-clinical development or early clinical trials where issues of polymorphisms have been explored. The leading *P. vivax* vaccine candidate, DBP [199] has significant polymorphism that appears to impact on the efficacy of inhibitory antibodies [200], and protective immunity shows a degree of strainspecificity [201]. The erythrocyte binding region of the *P. falciparum* vaccine candidate EBA175, also has significant polymorphism [149, 202]. Interestingly, studies suggest that these polymorphisms have little impact on the efficacy of vaccine-induced inhibitory antibodies *in vitro* [203, 204]. However, the ability of *P. falciparum* to vary the expression and use of EBA175 during invasion may be an important means of immune evasion [150], which presents different challenges in vaccine design. Another example is the PfRh invasion ligand family. One member that appears to be an important target of acquired immunity, PfRh2, has significant polymorphism in the N-terminal region [171], but studies suggest it is possible to target a C-terminal conserved stretch of the protein in vaccine design [172]. The challenge of addressing diversity in vaccine development is greatest for PfEMP1, which is expressed on the surface of infected RBCs. PfEMP1 shows extreme sequence diversity (e.g. [205, 206]) and is encoded by a family of around 60 *var* genes per genome that enables clonal antigenic variation through expression of different PfEMP1 variants [207]. A vaccine trial in non-human primates showed strain-specific protective efficacy [208] and it is not clear how this extensive antigenic diversity can be overcome. However, one specific PfEMP1 variant, known as *var2csa*, plays a key role in placental infection [209] and may be a suitable target for a vaccine that helps protect pregnant women from the complications of malaria during pregnancy. Although this single variant has a high level of polymorphism [210], recent studies suggest the extent of antigenic diversity may not be high as there appear to be many shared epitopes that are common to different variants [211, 212], and antibodies that cross-react to different variants have been induced by vaccination [213, 214] and are acquired through natural

It is clear that the importance of allele-specific immunity to malaria has been understudied in sero-epidemiological studies. There is a real need to incorporate strain-specific responses and endpoints in immuno-epidemiological studies to address whether protective responses against particular antigens are strain-transcending or strain-specific. This research is imperative so we can elucidate whether sub-unit vaccines including only one allelic type might protect against a small subset of the parasite population and thus select for parasites bearing heterologous alleles.

## **4.2 Vaccine trials and antigen polymorphism**

Significant sequence polymorphism is present in most of the vaccine antigens that are currently in clinical trials, as well as in many of the lead candidate antigens that are in preclinical development. This includes *P. falciparum* antigens such as CSP (RTS,S vaccine), MSP1-42, MSP2, MSP3, AMA1, and *P. vivax* antigens, such as DBP [36]. Presently, there is only a very limited understanding of how sequence polymorphisms relate to antigenic diversity or the potential for polymorphisms to mediate vaccine escape.

Different approaches can be taken to overcome antigenic diversity present in vaccine candidates. One approach is to include multiple alleles, or variants, of an antigen such that the vaccine-induced responses cover most of the different alleles circulating in a population; referred to as multivalent vaccines. This approach has been widely used in the development of vaccines for viral and bacterial pathogens. Examples include vaccines for *Streptococcus pneumonia*, *Neisseria meningitidis*, and influenza. Among malaria vaccines, multivalent vaccines are in development for several antigens including MSP2 [132] and AMA1 [195] of *P. falciparum*. The level of polymorphism seen among different candidate antigens varies substantially, being high for some candidate antigens, such as AMA1, compared to others, such as EBA175 [8]. Antigens that have a lower level of diversity may be more attractive candidates for vaccine development; however, multiple factors need to be considered in prioritising the many potential antigens for development [36]. An alternative approach is to design vaccine constructs that include a conserved region of the antigen so that vaccineinduced responses would target epitopes common to all variants of the antigen. This approach is being taken for *P. falciparum* MSP3, whereby the vaccine antigen includes a conserved stretch of sequence (known as MSP3-LSP) towards the C-terminal end of MSP3 and reportedly omits the highly polymorphic epitopes [196]. Interestingly however, recent population genetic surveys have shown that T-cell epitopes within MSP3-LSP are somewhat polymorphic [138].

A well-known example of the importance of antigenic diversity in vaccine development is the phase I/IIb trial of the Combination B vaccine for *P. falciparum* malaria [6], which was mentioned earlier. The vaccine had significant efficacy in reducing parasite densities during follow-up and the anti-malarial effect of the vaccine is thought to be mediated primarily by responses to MSP2, rather than other components of the vaccine. Although MSP2 is polymorphic, nearly all sequences can be grouped into two allelic families, 3D7 and FC27. The Combination B vaccine contained only the 3D7 form of MSP2 and, consequently, infections in vaccinated individuals were over-represented by FC27 allelic types. This implies that the vaccine only had substantial efficacy against infections containing the vaccine allele, 3D7. To address this, an MSP2 vaccine containing both of the

It is clear that the importance of allele-specific immunity to malaria has been understudied in sero-epidemiological studies. There is a real need to incorporate strain-specific responses and endpoints in immuno-epidemiological studies to address whether protective responses against particular antigens are strain-transcending or strain-specific. This research is imperative so we can elucidate whether sub-unit vaccines including only one allelic type might protect against a small subset of the parasite population and thus select for parasites

Significant sequence polymorphism is present in most of the vaccine antigens that are currently in clinical trials, as well as in many of the lead candidate antigens that are in preclinical development. This includes *P. falciparum* antigens such as CSP (RTS,S vaccine), MSP1-42, MSP2, MSP3, AMA1, and *P. vivax* antigens, such as DBP [36]. Presently, there is only a very limited understanding of how sequence polymorphisms relate to antigenic

Different approaches can be taken to overcome antigenic diversity present in vaccine candidates. One approach is to include multiple alleles, or variants, of an antigen such that the vaccine-induced responses cover most of the different alleles circulating in a population; referred to as multivalent vaccines. This approach has been widely used in the development of vaccines for viral and bacterial pathogens. Examples include vaccines for *Streptococcus pneumonia*, *Neisseria meningitidis*, and influenza. Among malaria vaccines, multivalent vaccines are in development for several antigens including MSP2 [132] and AMA1 [195] of *P. falciparum*. The level of polymorphism seen among different candidate antigens varies substantially, being high for some candidate antigens, such as AMA1, compared to others, such as EBA175 [8]. Antigens that have a lower level of diversity may be more attractive candidates for vaccine development; however, multiple factors need to be considered in prioritising the many potential antigens for development [36]. An alternative approach is to design vaccine constructs that include a conserved region of the antigen so that vaccineinduced responses would target epitopes common to all variants of the antigen. This approach is being taken for *P. falciparum* MSP3, whereby the vaccine antigen includes a conserved stretch of sequence (known as MSP3-LSP) towards the C-terminal end of MSP3 and reportedly omits the highly polymorphic epitopes [196]. Interestingly however, recent population genetic surveys have shown that T-cell epitopes within MSP3-LSP are somewhat

A well-known example of the importance of antigenic diversity in vaccine development is the phase I/IIb trial of the Combination B vaccine for *P. falciparum* malaria [6], which was mentioned earlier. The vaccine had significant efficacy in reducing parasite densities during follow-up and the anti-malarial effect of the vaccine is thought to be mediated primarily by responses to MSP2, rather than other components of the vaccine. Although MSP2 is polymorphic, nearly all sequences can be grouped into two allelic families, 3D7 and FC27. The Combination B vaccine contained only the 3D7 form of MSP2 and, consequently, infections in vaccinated individuals were over-represented by FC27 allelic types. This implies that the vaccine only had substantial efficacy against infections containing the vaccine allele, 3D7. To address this, an MSP2 vaccine containing both of the

diversity or the potential for polymorphisms to mediate vaccine escape.

bearing heterologous alleles.

polymorphic [138].

**4.2 Vaccine trials and antigen polymorphism** 

major allelic types is in development [132]. A more recent example of the challenge of antigenic diversity in malaria vaccines is AMA1. A phase II trial of a vaccine containing a single allele of *P. falciparum* AMA1 (3D7-allele) had no significant clinical efficacy when all malaria episodes were considered [46]. However, as was mentioned earlier, the vaccine demonstrated 68% efficacy against malaria episodes caused by AMA1 alleles that were similar in sequence to the vaccine allele. The development of approaches to overcome diversity in AMA1 and generate responses that give broad population coverage are ongoing.

The most advanced vaccine in development, RTS,S, is based on a region of *P. falciparum* CSP [25]. Because CSP is polymorphic, an obvious question was whether diversity in CSP may explain the partial efficacy of the RTS,S vaccine. Analyses of infecting genotypes in one phase II trial of RTS,S suggested that this was not the case as there was no evidence of selection for infections containing non-vaccine genotypes among vaccinated versus control subjects [197, 198]. These findings highlight that although antigen polymorphism needs to be considered in vaccine development, it may not be a major issue for all vaccines. Clearly, there is a lot more we need to understand about the nature and specificity of responses and the importance of sequence diversity.

There are a number of other promising antigens in pre-clinical development or early clinical trials where issues of polymorphisms have been explored. The leading *P. vivax* vaccine candidate, DBP [199] has significant polymorphism that appears to impact on the efficacy of inhibitory antibodies [200], and protective immunity shows a degree of strainspecificity [201]. The erythrocyte binding region of the *P. falciparum* vaccine candidate EBA175, also has significant polymorphism [149, 202]. Interestingly, studies suggest that these polymorphisms have little impact on the efficacy of vaccine-induced inhibitory antibodies *in vitro* [203, 204]. However, the ability of *P. falciparum* to vary the expression and use of EBA175 during invasion may be an important means of immune evasion [150], which presents different challenges in vaccine design. Another example is the PfRh invasion ligand family. One member that appears to be an important target of acquired immunity, PfRh2, has significant polymorphism in the N-terminal region [171], but studies suggest it is possible to target a C-terminal conserved stretch of the protein in vaccine design [172]. The challenge of addressing diversity in vaccine development is greatest for PfEMP1, which is expressed on the surface of infected RBCs. PfEMP1 shows extreme sequence diversity (e.g. [205, 206]) and is encoded by a family of around 60 *var* genes per genome that enables clonal antigenic variation through expression of different PfEMP1 variants [207]. A vaccine trial in non-human primates showed strain-specific protective efficacy [208] and it is not clear how this extensive antigenic diversity can be overcome. However, one specific PfEMP1 variant, known as *var2csa*, plays a key role in placental infection [209] and may be a suitable target for a vaccine that helps protect pregnant women from the complications of malaria during pregnancy. Although this single variant has a high level of polymorphism [210], recent studies suggest the extent of antigenic diversity may not be high as there appear to be many shared epitopes that are common to different variants [211, 212], and antibodies that cross-react to different variants have been induced by vaccination [213, 214] and are acquired through natural exposure [211, 215]

Using Population Genetics to Guide Malaria Vaccine Design 245

CSP sporozoite C-terminal 3 13 604 (9-143) 71 (3-20)

TRAP sporozoite N-terminal 2 3 100 (8-48) 84 (8-37)

LSA1 liver stage N-terminal 3 4 74 (10-22) 13 (3-7)

AMA1 merozoite Domain I 3 11 572 (8-162) 181 (6-68)

EBA175 merozoite Region II 2 3 135 (30-48) 51 (15-23)

MSP4 merozoite Full length 2 4 142 (12-42) 47 (9-23)

Pfs48/45 gametocyte Full length 3 4 55 (9-15) 19 (2-8)

Pfs28 ookinete no population data available

MSP3/6 merozoite 1 2 117(51-66) n.r. [218, 219]

*var2csa* trophozoite DBL3 2 3 124 (15-54) n.r. [211]

AMA1 merozoite Full lengthc 2 2 96 (23-73) n.r. (15-18) [223] [224] MSP1 merozoite Full lengthc 2 7 196 (40-116) n.r. (12-31) [187, 225] DBP merozoite Region II 2 8 675 (11-123) n.r. (9-73) [226] Pvs28 ookinete All 1 1 20 2 [227] Pvs25 ookinete All 1 1 30 4 [227] a.Total includes both natural populations and other isolates, range includes only natural populations; b. Total (range) includes only natural populations; c.Individual domains also analysed, data not shown;

Table 1. Summary of population genetic data for leading malaria vaccine candidates

gametocyte Region 0 3 3 48 (9-11) 22 (2-9)

**Total isolates sampled (range)**

2237 (18-

3 2 3 392 (n.d) 275 (n.r.)

region 1 1 33 (15) n.r. (13) [171]

repeat 2 2 168 (31-137) n.r. (13-25) [220, 221]

IV 2 2 74 (37-37) 54 (n.r.) [222]

region 2 2 124 (75-86) 21 (9-12)

region 1 1 23 (12) 9 (4)

1368) 20 (1-15)

**Total haplotypes** 

**(range) References** 

reviewed in [8]

reviewed in [8]

reviewed in [8]

reviewed in [8]

reviewed in [8]

reviewed in [8]

reviewed in [8]

reviewed in [8]

reviewed in [8]

reviewed in [8]

Reiling *et al*. unpublished data

reviewed in [8]

**Antigen Expression Domain Continents Countries**

MSP1 merozoite MSP119 3 11

Blocks 2 and

Repeat

Binding

Binding

Central

Regions II-

*Plasmodium falciparuma*

GLURP

sporozoite/

MSP2 merozoite

MSP3 merozoite

Rh2 merozoite

Rh4 merozoite

CSP pre-eryth

TRAP pre-eryth

n.r. result not available.

*Plasmodium vivaxb*
