**6. Future direction**

218 Malaria Parasites

Characterizing immune responses induced by MSP-1 vaccines revealed that several factors impact the immunogenicity and functional activity of the induced antibodies (vaccine platforms will be discussed in detail in Section 6): (1) the expression system used for the production of the recombinant protein (*i.e., E. coli*, baculovirus, or yeast) (Arnot et al., 2008; Reed et al., 2009), (2) the amino-acid sequence used for vaccine development (*i.e.* full length gp195, MSP-1p42 or MSP-1p19) (Stowers et al., 2001; Woehlbier et al., 2006) and (3) the vaccine platform used to deliver the MSP vaccine (*i.e*, recombinant protein, recombinant

Our laboratory has intensively studied the anti-parasite effects induced by MSP-1p42 specific antibodies. Early observations indicated that MSP-1 specific antibodies impacted the various parasite strains differently depending on their classification as MAD20 or Wellcome/K1-like. These two alleles differ markedly in their p33 fragments while only by four amino acids (E-TSR *vs.* Q-KNG, respectively) in the p19 portion of the molecule. To this end, MSP-1 specific antibodies were able to significantly delay the intra-erythrocytic development of the 3D7, but not the FVO parasite clone (Bergmann-Leitner et al. 2009). In the case of FVO parasites, anti-MSP-1p42 antibodies prevented schizont rupturing by stalling or arresting intra-erythrocytic parasite development likely through direct interactions with intra-erythrocytic parasites within the parasitophorous vacuole, which is putatively connected to the surface of the pRBC by the parasitophorous duct. This duct gives antibodies, but not larger immune components access to the parasite inside the vacuole (Bergmann-Leitner et al., 2009). In contrast, the same antisera tested on the 3D7 parasite clone were unable to interfere with the release of the merozoites. These antibodies were still able to agglutinate merozoites and interfere with invasion. We expanded our analysis to the parasite clone CAMP/FUP that has a p33 and a p19 EGF-like domain 1 identical to the sequence of the 3D7 parasite clone, and an EGF-like domain 2 identical to the FVO parasite clone. We observed the same response pattern as reported for the FVO parasite indicating that antibodies within the EGF-like domain might be responsible for stalling the rupture of the schizonts. In contrast, no significant growth inhibition was observed following successful invasion indicating that EGF-like domain 1 specific antibodies may be mediating this particular biological effect. To test this working hypothesis, the activity of affinity purified antibodies specific to the entire p19 or each of the EGF-like domains were compared to the source material (antibodies induced by immunization with the MSP-1p42 vaccine representing either the FVO or the 3D7 allele). We concluded that only antibodies that bound to regions within the p42 or to the p19 subunit, but not the EGF-like domain 1 or 2 subunits, displayed growth inhibitory activities. This was surprising given previous observations suggesting that responses directed to the p19 were associated with reduced parasite density or clinical disease (see above). However, the proper folding of the recombinant fragments used, which represent the two EGF-like domains has not yet been confirmed. This result may indicate that protective epitopes may depend on proper tertiary structure of the molecule. In support of this theory, it has been reported that inhibitory anti-MSP-1 specific antibodies map to epitopes formed through the "properly" folded p19 subunit and not to its sub-domains (McBride and Heidrich, 1987;

**5.3 Biological effect of MSP-1 specific antibodies on parasite growth and function** 

viral vectors or DNA vaccines).

Uthaipibull et al., 2001).

The current body of literature clearly supports the development of a blood stage vaccine. Although such a vaccine would not prevent infection, it can reduce morbidity and mortality associated with malaria infection and therefore such a vaccine would save the lives of many residents of malaria-endemic areas. The experience and knowledge gained from these studies should be used to rationally design new vaccine formulations and future clinical trials. Factors that need to be considered for their success are:

*(1) Vaccine platform.* Most erythrocytic vaccines tested so far are based on recombinant, soluble proteins. When using recombinant proteins it is paramount to assure proper, thus native-like, protein folding. Sera from malaria-endemic areas are a useful tool to establish the degree of cross-reactivity between the recombinant vaccine and the "native" antigen. Alternative vaccine platforms to those primarily described here, soluble proteins plus adjuvant, are particle-based approaches. When using this approach it is important to assure proper orientation of the protein on the particle. Some proteins that are inherently unstructured such as MSP-2 could benefit from particle formation because the particle provides a stabilizing scaffold. Moreover, the distance between the epitopes and the density may be crucial in order to induce proper immune responses. Such particle presentation could be achieved by using either recombinantly expressed antigen on whole-killed bacteria or viruses. For example, mouse studies using recombinant adenovirus encoding MSP-1 demonstrated "protection" (defined by the authors as delayed and lower parasitemia) (Draper et al., 2009).

*(2) Development of preclinical and clinical models that better predict human anti-malarial responses.* When using preclinical animal models, the parasite growth kinetic is frequently different between those *Plasmodium* species that are suitable for the respective animal model and *P. falciparum* thus failing to simulate the clinical situation. Testing vaccine candidates in animals can be challenging when testing *P. falciparum* antigens: except for one confirmed antigen, *Pf*CelTOS, (Bergmann-Leitner et al., 2010), malaria antigens are relatively speciesspecific, *i.e.,* immunization with *P. falciparum* antigens does not confer protection against a heterologous *Plasmodium* species. Thus, investigators often make their decisions based solely on immunogenicity in the animal model (cellular and/or humoral responses notionally thought to be important). Alternatively, investigators have searched for orthologs of the *P. falciparum* antigen in the respective *Plasmodium* species relevant for their preclinical model to conduct immunization and challenge studies. A caveat of this approach is that the ortholog may have a different function than the *P. falciparum* antigen in human malaria or there simply may not be a valid ortholog (*e.g*. *P. falciparum* LSA-1 does not have an ortholog in

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rodent *Plasmodia*). Another important consideration for the establishment of relevant models of human malaria is the immunization and challenge routes. Challenge routes should take into consideration the natural inoculation route. Our work with pre-erythrocytic antigens has shown that vaccine efficacy can vary significantly if the challenge route is changed (intravenous *vs*. mosquito bite) and thus the efficacy of a vaccine could be over- or underestimated if an artificial challenge method is used (Leitner et al., 2010) which may bypass vaccine-induced effector mechanisms (Vanderberg et al., 2007). For erythrocytic antigens this has been an issue as well because - until recently - it was ethically inconceivable to challenge human volunteers with malaria-infected blood due to the risk of transmitting life threatening blood borne diseases. However, extensive testing of the blood source used for the challenge has allowed a limited challenge study with the understanding that significant improvements are needed before blood challenges can be performed routinely similar to the mosquito bite challenges (Moorthy et al., 2009). A final issue to consider is that blood stage challenge in humans may not fully predict the situation where an individual receives a blood stage vaccine followed by mosquito bite challenge, as the vaccine-induced immune responses may be edited (*i.e.*, altered) by the sporozoite and the liver-stage infection.

*(3) Improving surrogate readout assays to down-select vaccine candidates.* At this time, different assays are being used as surrogate markers for down-selecting vaccine candidates. Without an immune correlate of protection, the predictive value of these readout methods remains questionable. Therefore, identifying immune correlates for the various target antigens is necessary to 'validate' the readout methods and allow their use for rational down-selection of vaccine candidates

#### **7. Acknowledgments**

This work was supported by the U.S. Agency for International Development under project number 936-3118, award number GHA-T-00-08-00007-01, and by the United States Army Medical Research and Materiel Command.

#### **8. Disclaimer**

The authors' views are private and are not to be construed as official policy of the Department of Defense or the U.S. Army or the Centers for Disease Control.

#### **9. References**


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*(3) Improving surrogate readout assays to down-select vaccine candidates.* At this time, different assays are being used as surrogate markers for down-selecting vaccine candidates. Without an immune correlate of protection, the predictive value of these readout methods remains questionable. Therefore, identifying immune correlates for the various target antigens is necessary to 'validate' the readout methods and allow their use for rational down-selection

This work was supported by the U.S. Agency for International Development under project number 936-3118, award number GHA-T-00-08-00007-01, and by the United States Army

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**1. Introduction** 

antigen [7].

targeted by the host immune response.

**12** 

*3Burnet Institute, 4Monash University,* 

*Australia* 

**Using Population Genetics to Guide** 

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

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

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

Alyssa E. Barry1,2, James Beeson3, John C. Reeder3,4,

*1Walter and Eliza Hall Institute for Medical Research, 2Department of Medical Biology, University of Melbourne,* 

**Malaria Vaccine Design** 

Freya J.I. Fowkes3 and Alicia Arnott3

