**1. Introduction**

230 Malaria Parasites

Woehlbier, U., Epp, C., Kauth, C.W., et al., 2006. Analysis of antibodies directed against

Wyatt, C.R., Goff, W. & Davis, W.C., 1991. A flow cytometric method for assessing viability

of intraerythrocytic hemoparasites. J Immunol Meth 140, 117-122.

Infect Immun 74, 1313-1322.

merozoite surface protein 1 of the human malaria parasite *Plasmodium falciparum*.

A broadly effective malaria vaccine is a missing component of the practical tools needed to control and eventually eliminate malaria. For more than 50 years malaria researchers have been searching for a strategy that provides long lasting protection against a broad range of parasite strains. Many different approaches have been tested including whole parasite and subunit vaccines composed of one or more parasite surface proteins that are naturally targeted by the host immune response.

Candidates in the malaria vaccine pipeline include surface exposed proteins from each of the morphologically distinct developmental stages of the parasite lifecycle within the human host. Many of these antigens have undergone rigorous developmental and preclinical testing as subunit vaccines [1] but only a few have reached advanced clinical trials: one reason being a lack of funding to carry all promising vaccine candidates to trial [2]. For candidates that have reached Phase II clinical trials in malaria endemic areas, there has been variable success. This is not surprising given that the malaria parasite is a complex and rapidly evolving organism that can quickly adapt to its ever-changing environment and effectively evade human immune responses. Limited understanding of the precise mechanisms and minimal requirements for antimalarial immunity has also hampered vaccine progress [3]. Moreover, the high degree of diversity of parasite surface antigens [4] and the allelic-specificity of the immune response [5, 6] is likely to have contributed significantly to the variable success of malaria vaccines. However genetic diversity is often overlooked in vaccine design with most vaccines formulated on the basis of a single strain. It is now increasingly recognized that to be effective against the worldwide parasite population, a malaria vaccine may need to contain multiple variants of the target antigen [7].

Many studies have investigated the genetic diversity of vaccine antigens that are circulating in natural parasite populations but very little of this knowledge has been applied to malaria vaccine design. Typically, alleles from one or two reference strains (3D7 or FVO) are used in

Using Population Genetics to Guide Malaria Vaccine Design 233

Some 60 years later, the feasibility of a human malaria vaccine received a boost when Sir Ian MacGregor demonstrated that immunity was conferred by transferring hyperimmune sera (IgG) from adults to at-risk African children [10]. Soon afterward, a team at New York University (NYU) including Ruth Nussenzweig and Jerome Vanderberg showed that mice could be vaccinated against malaria with irradiated *Plasmodium bergei* sporozoites [11]. In 1973, Clyde and colleagues at the University of Maryland in collaboration with the NYU team demonstrated that infections of humans via the bite of irradiated sporozoite-positive mosquitoes protected against subsequent homologous challenge, thus providing the first real evidence that man could be vaccinated against *P. falciparum* malaria [12] and subsequently, against *P. vivax* [13]. Later work showed that this approach was also effective in protecting against heterologous strains of *P. falciparum* [14]. These studies demonstrated that a malaria vaccine could elicit sterile protective immunity, and provided the reagents and tools to identify molecules that were important immune targets, such as the circumsporozoite surface protein (CSP) [15]. Unfortunately, at the time it was impractical to vaccinate large numbers of people with irradiated sporozoites isolated from infected

mosquitoes and attempts to culture sporozoites showed only limited success [16].

The increasing availability of molecular cloning and PCR techniques in the early 1980's saw a major shift in focus to the development of synthetic "subunit" vaccines based on individual parasite proteins and peptides and the abandonment of whole-parasite approaches. Several teams around the world had identified parasite surface proteins as targets such as the sporozoite antigens: circumsporozoite surface protein (CSP) [15] and thrombospondin related antigen (TRAP, [17, 18]) ; Liver stage antigen 1 (LSA1,[19]); and the merozoite surface proteins, MSP1 [20], MSP2 [21], S-Antigen [22] and Apical Membrane Antigen 1 (AMA1, [23, 24]). Initial studies in animal models were promising but excitement began to wane as subunit vaccines showed only around half of the candidates had a protective effect in clinical trials. Of the eight malaria vaccine candidate antigens that have reached Phase II trials, only one, "RTS,S" which is based on CSP, has now been tested in

The malaria parasite is a complex organism with 3000 or so known proteins [26, 27] including a few hundred that are immunogenic surface antigens [28]. This makes it a formidable target and therefore a vaccine as complex as the parasite itself may be required to immunise against malaria [29]. Evidence that sporozoite vaccinees were resistant to both homologous and heterologous challenge [14] makes whole-parasite vaccination approaches an appealing option so long as technological challenges can be overcome. New techniques for mass-producing sporozoites are reportedly in development [30] and both irradiation [31] and genetically attenuated [32, 33] parasite vaccines are being tested. Low doses of blood stage parasites have been shown to induce T-cell mediated responses targeted at cytoplasmic proteins with high levels of sequence conservation [34]. In addition, chemicallyand genetically-attenuated blood-stage parasites are being developed (personal communication M. Good, Griffith University and K. Evans, Walter and Eliza Hall Institute). Consequently, whole parasite approaches have been experiencing a renaissance and have

**2.2 Subunit vaccines** 

Phase III clinical trials [25].

**2.3 Whole parasite vaccines** 

vaccine formulations, but this only partially represents the diversity of alleles circulating in the parasite population (Figure 1 [8]). Moreover, if vaccine alleles are found at low frequencies, the power to measure the success of vaccine trials is limited. This chapter reviews the current state of knowledge of the genetic diversity of leading malaria vaccine candidates, including those for both major human malaria parasites, *Plasmodium falciparum* and *Plasmodium vivax.* We examine the relevance of this diversity to malaria vaccines currently in development and how to use this information in determining which alleles should be used to provide broad coverage against the majority of parasite strains. Using leading candidate vaccine antigens as examples, we outline how population genetic tools can be used to characterise genetic diversity and the distribution of alleles and to define potentially immunologically relevant subgroups of alleles and polymorphisms. Finally, we make recommendations for the design of the next generation of malaria vaccines.

Fig. 1. Worldwide frequency of alleles represented in ten leading malaria vaccine candidates (adapted from [8]). MSP119 3D7 and FVO are being developed as a combination vaccine as well as in isolation.

#### **2. Malaria vaccines: A brief history**

#### **2.1 Early studies**

The idea that a malaria vaccine might be possible was raised in the late 1800's with the observations of Robert Koch during his expedition to the Melanesian Island of Papua New Guinea (PNG). He found that while local children frequently displayed symptoms of malarial disease and harboured parasites in their blood, adults remained relatively disease and parasite-free [9]. He also noted that Malay and Chinese workers that had recently immigrated to PNG became ill with malaria more often than their counterparts who had been in residence for longer periods of time. These observations form the basis for our contemporary understanding of the epidemiology of malaria and the theory that immunity to malaria is eventually acquired in areas where people are constantly exposed to infection. In his conclusions, Dr. Koch stated that it would be possible to vaccinate against malaria toxins (and therefore the symptoms caused by infection).

Some 60 years later, the feasibility of a human malaria vaccine received a boost when Sir Ian MacGregor demonstrated that immunity was conferred by transferring hyperimmune sera (IgG) from adults to at-risk African children [10]. Soon afterward, a team at New York University (NYU) including Ruth Nussenzweig and Jerome Vanderberg showed that mice could be vaccinated against malaria with irradiated *Plasmodium bergei* sporozoites [11]. In 1973, Clyde and colleagues at the University of Maryland in collaboration with the NYU team demonstrated that infections of humans via the bite of irradiated sporozoite-positive mosquitoes protected against subsequent homologous challenge, thus providing the first real evidence that man could be vaccinated against *P. falciparum* malaria [12] and subsequently, against *P. vivax* [13]. Later work showed that this approach was also effective in protecting against heterologous strains of *P. falciparum* [14]. These studies demonstrated that a malaria vaccine could elicit sterile protective immunity, and provided the reagents and tools to identify molecules that were important immune targets, such as the circumsporozoite surface protein (CSP) [15]. Unfortunately, at the time it was impractical to vaccinate large numbers of people with irradiated sporozoites isolated from infected mosquitoes and attempts to culture sporozoites showed only limited success [16].

### **2.2 Subunit vaccines**

232 Malaria Parasites

vaccine formulations, but this only partially represents the diversity of alleles circulating in the parasite population (Figure 1 [8]). Moreover, if vaccine alleles are found at low frequencies, the power to measure the success of vaccine trials is limited. This chapter reviews the current state of knowledge of the genetic diversity of leading malaria vaccine candidates, including those for both major human malaria parasites, *Plasmodium falciparum* and *Plasmodium vivax.* We examine the relevance of this diversity to malaria vaccines currently in development and how to use this information in determining which alleles should be used to provide broad coverage against the majority of parasite strains. Using leading candidate vaccine antigens as examples, we outline how population genetic tools can be used to characterise genetic diversity and the distribution of alleles and to define potentially immunologically relevant subgroups of alleles and polymorphisms. Finally, we

make recommendations for the design of the next generation of malaria vaccines.

Fig. 1. Worldwide frequency of alleles represented in ten leading malaria vaccine candidates (adapted from [8]). MSP119 3D7 and FVO are being developed as a combination vaccine as

The idea that a malaria vaccine might be possible was raised in the late 1800's with the observations of Robert Koch during his expedition to the Melanesian Island of Papua New Guinea (PNG). He found that while local children frequently displayed symptoms of malarial disease and harboured parasites in their blood, adults remained relatively disease and parasite-free [9]. He also noted that Malay and Chinese workers that had recently immigrated to PNG became ill with malaria more often than their counterparts who had been in residence for longer periods of time. These observations form the basis for our contemporary understanding of the epidemiology of malaria and the theory that immunity to malaria is eventually acquired in areas where people are constantly exposed to infection. In his conclusions, Dr. Koch stated that it would be possible to vaccinate against malaria

well as in isolation.

**2.1 Early studies** 

**2. Malaria vaccines: A brief history** 

toxins (and therefore the symptoms caused by infection).

The increasing availability of molecular cloning and PCR techniques in the early 1980's saw a major shift in focus to the development of synthetic "subunit" vaccines based on individual parasite proteins and peptides and the abandonment of whole-parasite approaches. Several teams around the world had identified parasite surface proteins as targets such as the sporozoite antigens: circumsporozoite surface protein (CSP) [15] and thrombospondin related antigen (TRAP, [17, 18]) ; Liver stage antigen 1 (LSA1,[19]); and the merozoite surface proteins, MSP1 [20], MSP2 [21], S-Antigen [22] and Apical Membrane Antigen 1 (AMA1, [23, 24]). Initial studies in animal models were promising but excitement began to wane as subunit vaccines showed only around half of the candidates had a protective effect in clinical trials. Of the eight malaria vaccine candidate antigens that have reached Phase II trials, only one, "RTS,S" which is based on CSP, has now been tested in Phase III clinical trials [25].

#### **2.3 Whole parasite vaccines**

The malaria parasite is a complex organism with 3000 or so known proteins [26, 27] including a few hundred that are immunogenic surface antigens [28]. This makes it a formidable target and therefore a vaccine as complex as the parasite itself may be required to immunise against malaria [29]. Evidence that sporozoite vaccinees were resistant to both homologous and heterologous challenge [14] makes whole-parasite vaccination approaches an appealing option so long as technological challenges can be overcome. New techniques for mass-producing sporozoites are reportedly in development [30] and both irradiation [31] and genetically attenuated [32, 33] parasite vaccines are being tested. Low doses of blood stage parasites have been shown to induce T-cell mediated responses targeted at cytoplasmic proteins with high levels of sequence conservation [34]. In addition, chemicallyand genetically-attenuated blood-stage parasites are being developed (personal communication M. Good, Griffith University and K. Evans, Walter and Eliza Hall Institute). Consequently, whole parasite approaches have been experiencing a renaissance and have

Using Population Genetics to Guide Malaria Vaccine Design 235

Below, we describe the most developed malaria vaccine candidates for the two major malaria parasites, *P. falciparum* and *P. vivax* as well as some novel vaccine candidates that have recently been identified for *P. falciparum*. This is not a comprehensive listing, a complete list of preclinical, clinical and inactive or discontinued malaria vaccine projects can be found in the World Health Organisation's Malaria Vaccine Rainbow Tables [51].

*P. falciparum* is responsible for most of the mortality and morbidity associated with malaria, with up to 1 million deaths and around 225 million clinical cases caused by this species [52]. It is the only major human malaria parasite for which an *in vitro* culture system is available thus making it more tractable for investigations to characterise molecular structure and function and interactions with its human and anophelene hosts. As a result, malaria research has focused on developing ways to combat this particular species with less focus on other human infecting malaria parasites. The number of well-developed *P. falciparum* vaccine candidates in advanced stages of clinical development reflects this bias and there are strong candidates for every stage of the *P. falciparum* lifecycle within the human host. Some

CSP, which is found in the outer sporozoite membrane, was shown to be a target of antibodies that prevented hepatocyte infection, thereby interrupting an obligatory stage in the parasite's lifecycle [15]. Cloning of the CSP gene (amongst other antigens) opened the doors to mass production and it was hoped, a malaria vaccine [53]. CSP contains a central (NANP) repeat region containing B-cell epitopes flanked by non-repetitive sequences containing T-cell epitopes [53-55]. In 2004, a considerable protective effect was observed with a CSP-based vaccine known as RTS,S with 49% efficacy against severe malaria for a period of 18 months in children aged 1-4 years [44]. This vaccine is composed of the central repeat and the C-terminal regions of CSP isolated from the reference strain, 3D7, combined with a Hepatitis B surface antigen (which provides protection against Hepatitis B and therefore satisfies ethical requirements). Recently published data from a phase III trial including 6000 African children has shown a 50% reduction in clinical episodes and 35% reduction in severe disease in children aged 5-18 months [25]. Therefore, RTS,S is likely to be the first licensed malaria vaccine and should be available within the next few years [56]. Most would agree though that it is an important proof of principle rather than an end to the

TRAP also known as Sporozoite Surface Protein (SSP) [18, 57] is present within the micronemes of the sporozoite and is essential for gliding motility and host cell invasion in the mosquito and human pre-erythrocyte stages [58-60]. TRAP is also expressed in the blood stages though its function in this stage is not well understood [18, 57]. TRAP is a Type I transmembrane protein consisting of multiple adhesive domains and a central repeat region flanked by unique sequence. Anti-repeat monoclonal antibodies have been shown to at least partially inhibit sporozoite invasion *in vitro* [61] and potent T-cell responses have been identified in individuals vaccinated with irradiated sporozoites [62, 63] as well as people

**3.1** *Plasmodium falciparum*

of these are described in detail below.

**3.1.1 Circumsporozoite Protein (CSP)** 

**3.1.2 Thrombospondin Related Adhesion Protein (TRAP)** 

hunt for a vaccine.

garnered much support from funding organisations [35]. As both subunit and whole parasite approaches are valid approaches [35, 36], they are being pursued in parallel. This review focuses primarily on the former approach.

### **2.4 Malaria vaccine subclasses**

Malaria vaccines fall into three main classes based on the lifecycle stage that they target and their expected biological effects [37-39]. These include:


Vaccine approaches currently being tested include individual candidates from a single lifecycle stage as well as combination vaccines formulated with targets from multiple lifecycle stages.
