Alexander Upfill-Brown05407712HUMBIO 153 : Parasites and Pestilence Dr. D Scott Smith

Malaria Vaccine Development: A Case for Attenuated Whole-Cell Pre-Erythrocytic Vaccines for Plasmodium falciparum

Introduction: An unmet need

It is hard to imagine a person alive today who is not aware of the vast prevalence of

malaria as well as the crippling rates of mortality and morbidity that are associated with its

presence. According to the World Health Organization (WHO) there were 247 million

cases of malaria in 2006 that caused nearly one million deaths.1 In countries with high

disease rates, malaria can cut economic growth rates by as much as 1.3%.1 Malaria is a

preventable and curable disease making it primarily a disease of poverty. There are

multiple drugs on the market that can eliminate the parasite from the human host;

however, they are expensive and in short supply in sub-Saharan countries that are the most

burdened by the disease. Current prevention strategies include indoor residual spraying

and long-lasting insecticide-treated bed nets.2 While drugs and prevention strategies have

proven successful in reducing disease burden in some countries, few will argue that

current strategies in these fields are sufficient. Vast amounts of money today is being

directed to malaria vaccine development programs: arguably the most promising public

health measure for the control and eventual eradication of malaria.

Many organizations and government-funded programs are involved in the

development of vaccines. The big funding organizations are the Bill and Melinda Gates

foundation, the Clinton foundation, and the Global Fund to Fight AIDS, Tuberculosis, and

Malaria. Research is spear headed by US Military Malaria Vaccine Program, the Seattle

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Biomedical Research Institute, GlaxoSmithKline (GSK), and the PATH Malaria Vaccine

Initiative.

Background: P. falciparum and vaccines

Necessary to the understanding of the current debates regarding malaria vaccine

strategies is a basic understanding of the parasite and its effect on humans. Currently there

are five know species of malaria that infect humans: Plasmodium falciparum, the most

deadly cause of malaria, Plasmodium vivax, the most prevalent cause of malaria,

Plasmodium ovale, Plasmodium knowlesi, and Plasmodium malariae.3 Because of its

heightened lethal effects, P. falciparum is the overwhelming focus of vaccine development

efforts. The most deadly complication of falciparum infection is cerebral malaria, which

results in a coma due to tissue hypoxia in the central nervous system.3 P. falciparum

parasites insert electron-dense knobs into the surface of the membrane of the red blood

cells they infect; these knobs cause adhesion to the vascular walls causing hypoxia and

infarction.4 These knobs can also cause adhesion between multiple erythrocytes causing

“rosetting” or clumping; this is also responsible for the complicating effects of falciparum.4

When researchers consider vaccine strategies, one criterion assessed is the optimal

site of intervention in the life cycle of P. falciparum. Sexual reproduction occurs in

mosquitoes of the Anopheles genus, which then transmit infective sporozoites to the human

host when taking a blood meal. It has been estimated that 100-300 sporozoites are

transferred to the host when the mosquito is feeding.5 Sporozoites are then carried

through the blood stream to the liver where they infect hepatocytes and develop into

merozoites. This entire process takes five to seven days. Vaccines that stop the malarial

life cycle before the development of merozoites are known as pre-erythrocytic vaccines.

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Upon rupture of the hepatocyte, tens of thousands of merozoites emerge and begin the

erythrocytic cycle—the periodic infection and rupture of erythrocytes. This is the stage

that causes the symptoms associated with malaria. Vaccines that interrupt proliferation of

the virus in the erythrocytic stage are known as blood-stage vaccines. Some merozoites

develop into gametophytes that are then taken up by the mosquito when it feeds. Vaccines

that prevent the uptake or development of gametocytes are known as transmission

blocking vaccines (TBVs). The gametocytes mature and sexually combine in the mosquito

eventually resulting in the formation of infective sporozoites.3

Secondly, modern vaccine approaches provide another point of differentiation

between possible vaccine strategies. In the literature, the biggest division is between

scientists advocating the use attenuated whole parasites and those advocating the use

recombinant antigens in order to induce a host immune system response.6 Whole

attenuated parasites are living non-replicating forms of the parasite that no longer have the

infective characteristics of the wild type parasite. One strategy for attenuation is radiation

and another is genetic engineering (the select knockout of relevant genes). Recombinant

antigen vaccines use proteins expressed by the parasite (often on the surface) that trigger

immune response simulating infection by the whole parasite. Recombinant antigen

vaccines are more selective than whole parasite vaccines.6 They can be synthesized with

multiple antigens and they are always formulated with an adjuvant. Adjuvants have only

been recently understood. They function as secondary signals that greatly amplify the host

immune response by activating different classes of immune cells and processes.7 Toll-like

receptor (TLR) agonists are a type of adjuvant that can increase antigen-specific immune

responses by as much as 5 times to 500 times.8 Other less favored approaches are DNA and

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viral-vectored introductions. The approaches are more complicated and have so far

received the least amount of attention from the research community. With the basic

outline of possible malarial vaccine strategies, let us now look at vaccines in development.

Recombinant Antigen Vaccines: Strength and weaknesses

Based on the two criteria mentioned above, most vaccines for malaria will fit into

one of five vaccine categories: pre-erythrocytic whole-organism, pre-erythrocytic

recombinant antigen, blood-stage whole-organism, blood-stage recombinant antigen,

transmission blocking recombinant antigen (No transmission blocking whole-organism

vaccines were encountered in the literature). Because of the limited scope of this paper I

will focus on the pre-erythrocytic vaccines and blood-stage recombinant vaccines. These

approaches prove to be the most promising. The vaccine strategy with the most potential

is the pre-erythrocytic attenuated whole-organism model and this will be discussed last.

Pre-erythrocytic recombinant antigen (PERA) vaccines have been moderately

successful. One vaccine in particular, RTS,S/AS02, is currently in its Phase III clinical trial:

this makes it the most clinically advanced malaria vaccine in development.9 RTS,S is

derived from the circumsporozoite protein (CSP) that is expressed on the surface of

sporozoites and infected hepatic cells. The vaccine consists of the C-terminus of the CSP

fused to the hepatitis B surface antigen.9 The RTS,S antigen is formulated with adjuvant

AS02, developed by GSK. “The objective [of adjuvants] is to induce tailored immune

responses directed against the pathogen” and AS02 has proved to be very effective at

increasing host immune response.8 RTS,S was developed over twenty years ago;

consequently, it has been put through a variety of trials: it has “reduced the risk of clinical

episodes of malaria in young children by 53 percent over an eight-month follow-up period,

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and provided 65 percent protection against risk of infection in infants over a six-month

follow-up period.”1010 In a randomized phase IIb clinical trial in 2022 Mozambican

children the prevalence of falciparum was 29% lower in the RTS,S/AS02 cohort after 21

months.1111 Although RTS,S is effective, it is by no means comprehensive. Because it is a

recombinant antigen vaccine, RTS,S can be relatively cheaply synthesized. Current

estimates state that the vaccine will be available for use in children aged 5 years to 17

months in 2012.1010

These aspects of RTS,S make it valuable weapon in the fight against malaria, but as

can be gleaned from the data presented above, it has limited effectiveness. One reason for

incomplete coverage by RTS,S originates from the very specific immune antibody response

it generates. There are many surface proteins on sporozoites and infected hepatic cells,

and these may be variably expressed. Furthermore, recombinant antigen vaccines are

developed based upon a single halpotype, and there may be as many 105 different

haplotypes for the gene encoding CSP.1212 Mostly, sporozoite antigen diversity is

determined by location and physical barriers to gene flow, but there is plenty of overlap

(over 21 haplotypes exist in The Gambia alone).1212 In the same article (12) one finds a

very disconcerting result: “The majority of haplotypes upon which current vaccines are

based were found to be present at extremely low frequencies in the global parasite

population.” The implications of this are ominous, especially when using focused antibody

generators like RTS,S/AS02.

RTS,S/AS02 is the main PERA vaccine in development today. An extensive search

through the literature resulted in a select few of other PERA vaccine candidates, but

development had been terminated due to poor performance in trials. While results from

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RTS,S/AS02 trials are significant, it is not potent enough to make possible the eradication

of malaria. Other viable vaccine approaches are improving rapidly and getting ready to

compete with the RTS,S/AS02 vaccine.

The majority of blood-stage recombinant antigen (BSRA) vaccines are derived from

merozoite surface proteins (MSPs), apical membrane antigens (AMAs), and to a lesser

degree various surface antigens (VSAs) as well as many others.6 There are no standout

candidates in this category. Current efforts are focused on finding the most effective

combination of antigens: for example, one combination that has been tested involves MSP-

1 and AMA-1. Antibodies of these antigens have been proven to inhibit the invasion of

erythrocytes in vitro.9 One group at the Walter Reed Army Institute of Research is

developing a multi-antigen, multi-stage vaccine that aims prevent infection and also limit

disease if malaria does develop.1414 In the proposed vaccine, antigens derived from pre-

erythrocytic CSP and liver stage antigen-1 (LSA-1) induce immunity, and antigens derived

from blood stage proteins MSP-1 and AMA-1 will serve to limit disease.1414 This novel

combination will center around RTS,S/AS02, but the added antigens will theoretically

increase its scope an efficacy. More testing is needed regarding the interaction of antibody

responses when multiple antigens are present, especially ones that are not normally

present at the same time during the P. falciparum life cycle.

Like PERA vaccines, BSRA vaccines are more effective when combined with an

adjuvant. A unique adjuvant has been described for use with blood-stage P. falciparum

vaccinations that is worth noting here because of its specificity and its simplicity to

synthesize. The presence of an adjuvant component Hemozoin (HZ) has been discovered

and confirmed in blood stage parasites.1313 HZ is a malarial heme-detoxification

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byproduct, and it is able to bind strongly and specifically to toll-like receptor 9 (TLR9) and

thereby activate macrophages and dendritic cells. 1313 Synthetic Hz (sHZ) is as potent an

adjuvant and wild type HZ making sHZ and ideal candidate for development as an adjuvant.

Even with a highly potent adjuvant effect inducer, BSRA vaccines face major challenges.

More so than PERA vaccines, BSRA vaccines face the daunting challenge of

overcoming antigenic diversity. According to the findings of Barry et al, the diversity of

merozoite antigen expression does not vary geographically. Rather, it is hypothesized that

the immune response of the host is responsible for the antigenic diversity.1212 This

opinion is shared Polley and Conway. In 2001 they conducted a study analyzing the gene

encoding for AMA-1 and discovered a high degree of polymorphism in a single endemic

population. They concluded that naturally acquired protective immune responses in

humans were responsible for the selective maintenance of multiple alleles within the

population.1515 This indicates that antigenic diversity evolved to evade host immune

responses. Designing a vaccine that addresses the unusually high amounts of antigenic

diversity within a single P. falciparum antigen will take a large amount of additional

research. Furthermore, some antigens exist that “are encoded by large multi-gene families

and parasites can switch the expression of different genes to facilitate immune evasion.”6

An example of such a antigen is the VSA PfEMPI, which is expressed on the surface of

erythrocytes; these are the proteins that form the electron rich “knobs” discussed in the

background section.4

Pre-Erythrocytic Whole-Organism Vaccines: the gold standard

The strength of the whole-organism approach lies is the fact that all possible

antigens are presented to the immune system so the full spectrum of antibodies can be

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produced. A test carried out in humans proved that attenuated sporozoites confer

immunity for heterologous strains of P. falciparum originating from geographically distinct

locations.16 Reasons that research focuses on pre-erythrocytic organisms rather than

blood-stage organisms or gametophytes are as follows. First, in the pre-erythocytic stage

the number of infected hepatocytes is very low relative to the number of infected

erythrocytes in blood stage18: 100-300 infected hepatocytes5 relative to tens of thousands

infectious merozoites that emerge from one hepatocyte17 and infect red blood cells.

Another reason the pre-erythrocytic stage is targeted is the parasite is not transmittable

during this stage. By intervening before erythrocytic infection, individuals are unable to

transmit the parasite and they do not experience any symptoms of malaria. Thirdly, it is

believed that pre-erythrocytic stages do not exhibit significant antigenic variation17—the

major drawback of the blood-stage vaccine approach.

The concept of using live attenuated sporozoites as a vaccine for Plasmodium first

arose during the 1970s.16 The recent surge of interest in whole-organism sporozoite

vaccines was initiated by Stephen Hoffman in 2002. Hoffman now runs a biotechnology

company called Sanaria that is working to scale up production procedures for the isolation

of radiation-attenuated Plasmodium falciparum sporozoites from mosquito salivary glands.

Although sporozoites can be attenuated by genetic engineering or chemical

treatment, the process approaching clinical trials most rapidly relies on radiation-induced

attenuation. There has been success in a human trial where irradiated mosquitoes bit

volunteers more than 1,000 times and this conferred almost complete protection to

subsequent challenges (94% were protected) up to 42 weeks.16 This method of vaccination

has an unbeatable efficacy: the main draw back in vaccine development is the production of

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a pure, safe, and effective product. The current method employed is the manual dissection

of mosquito salivary glands; with a team of six, Sanaria is able to produce 500 doses of

vaccine per hour.16 The vaccine, known as Sanaria PfSPZ Vaccine, is currently in clinical

trials following FDA approval: the current trial is a dose escalation study.16 One

complication of using an attenuated whole organism vaccine is the transport to and storage

at immunization sites as the live cells need to remain cryogenically frozen. Without proper

transportation infrastructure, rural and poor populations will be unable to use the vaccine.

Sanaria believes that distribution in the liquid nitrogen vapor phase eliminates

transportation difficulties because there is no need for refrigerated trucks or electricity at

the storage site.16 Sanaria PfSPZ Vaccine is exceedingly promising. The effort now is

focused on engineering a suitable production facility—to me that is as possible, if not more

probable, then comprehending the complexities of antigenic variation and interaction in

Plasmodium falciparum. As gene deletion technology catches up with radiation attenuation,

Sanaria is prepared to take on its production as well.16

Currently there are a handful of P. falciparum sporozoite genes where deletion has

resulted in successful termination of parasite proliferation during the liver stage.

Overviewed by Vaughan et al, the gene knockouts are successful at the following loci:

jointly at p52 and p36, sap1, jointly at uis3 and uis4, and fabb/f.17 P36/p52 and sap1 cause

termination at earlier stages in liver development.17 Questions about the advantages of

having longer liver stage development remain as it allows the host immune system

exposure to a greater number of antigens but also has a high risk of progressing to blood-

stage malaria.

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Ahmed et al showed the power of sap1 deletion in a rodent model using Plasmodium

yoelii. Sap1 plays an essential role in establishing infection in the liver; its specific role is

believed to be post-transcriptional gene expression control.19 The study shows that sap1 is

a very safe choice for vaccine development: even at exorbitantly high concentrations of

intravenously delivered sap1- sporozoites, no infection proceeded past the liver stage.19

Lastly, the deletion of sap1 does not interfere with the expression of important membrane

proteins (such as CSP) that function as antigens stimulating immune response and sterile

protection.19 There is a sap1 analogue in P. falciparum and both forms have similar

expression patterns, however genetic attenuation of the sap1 gene has not been attempted

in P. falciparum.

P. falciparum has been successfully genetically engineered to express deletions at

the p36 and p52 loci by VanBuskirk et al. Parasites that harbor deletions are known as

genetically attenuated parasites or GAPs. Dual deletion of the p36 and p52 loci caused

complete parasite developmental arrest during the liver stages.18 The p36-/p52- GAPs

were introduced to human hepatocytes in vitro as well as humanized livers of mice in vivo.

Both experiments resulted in consistent termination of parasite proliferation in the liver

stages: after four days there was no sign of Plasmodium in the humanized mice liver cells.18

The study presented here confirms the safety of genetic attenuation as a procedure for

creating whole-organism vaccines. The next step in the development of GAP vaccines is the

production of a master cell bank that can support quantities of vaccine necessary for FDA

sanctioned trials.

Conclusions:

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What I have argued in this paper is that the pre-erythrocytic stage is the most

promising for vaccine intervention, and that the preferable method of vaccination is the use

of attenuated whole-organisms, specifically sporozoites.

Attempted intervention during the erythrocytic cycle has many obstacles. Dramatic

variation of the various antigens targeted for vaccine development severely limits the reach

of a vaccine. Also, during this stage of the infection hundreds of thousand of merozoites

can be found circulating through the blood. Complete elimination of merozoites is a lofty

expectation. Furthermore, transmission is harder to control once the infection reaches the

blood.

Transmission blocking vaccines seem to have been abandoned. The vast majority of

deaths caused by malaria occur in children under five years old. Children usually die of

malarial complications that arise because of a relative lack of exposure to the P. falciparum

parasite. With this in mind, a transmission blocking vaccine will not save the lives of those

the malarial vaccine campaigns are aiming to save. It allows the development of the

infection and this can be lethal in P. falciparum naïve children. Perhaps an altruistic

vaccine will be favored in the future when drugs to treat P. falciparum malaria become

more available to children living in endemic areas.

In the malarial vaccine development community there is a race between supporters

of recombinant antigen vaccines and those of attenuated whole-organism vaccines.

Supporters of recombinant antigen vaccines are very close to seeing the first malaria

vaccine approved for general use. The biggest set back to antigen specific vaccine

development is not due to production compexities—authors stress the cost-effectiveness of

this approach—but rather a need for more detailed understanding of antigen variation.

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The whole organism approach lacks the productive capacity as of now, but they are in

possession of a far more powerful and reliable vaccine. I trust that engineers the world

over are up to the challenge of designing production and purification processes that will

dramatically increase the availability of these incredibly valuable vaccines.

1 References

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22 Bill & Melinda Gates Foundation. “Malaria Strategy Overview.” <http://www.gatesfoundation.org/global-health/Documents/malaria-strategy.pdf>

33 John, David and William Petri. Medical Parasitology 9th ed. Sauders Elsevier, St. Louis, 2006. 79-98.3

44 Hviid, Lars. “The role of Plasmodium falciparum variant surface antigens in protective immunity and vaccine development.” Human Vaccine 6.1 (2010): 84-89. Web. 20 Feb. 2010.4

55 Jin, Yamei, Chahnaz Kebair, and Jerome Vanderberg. “Direct Microscopic Quantification of Dynamics of Plasmodium berghei Sporozoite Transmission from Mosquitoes to Mice.” Infectious Immunology 75 (2007): 5532-9. Web. 25 Feb. 20103

66 Richards, Jack and James Beeson. “The future for blood-stage vaccines against malaria.” Immunology & Cell Biology 87 (2009): 377-390. Web. 20 Feb. 2010.6

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1111 Alonso, Pedro et al. “Duration of protection with RTS,S/AS02A malaria vaccine in prevention of Plasmodium falciparum disease in Mozambican children: single-blind extended follow-up of a randomized controlled trial.” Lancet 366 (2005): 2012-18. Web. 20 Feb. 2010.10

1212 Barry, Alyssa et al. “Contrasting Population Structures of the Genes Encoding Ten Leading Vaccine-Candidate Antigens of the Human Malaria Parasite, Plasmodium falciparum.” PLoS ONE 4.12 (2009): e8497. Web. 20 Feb. 2010.12

613 Coban, Cevayir et al. “Immunogenicity of Whole-Parasite Vaccines against Plasmodium falciparum Involves Malarial Hemozoin and Host TLR9.” Cell Host & Microbe 7 (2010): 50-61. Web. 12 Feb. 2010.9

1414 Heppner, D. Gray et al “Towards an RTS,S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research.” Vaccine 23 (2005):

2243-2250. Web. 25 Feb. 2010.14

1315 Polley, Spencer and David Conway. “Strong Diversifying Selection on Domains of the Plasmodium falciparum Apical Membrane Antigen 1 Gene.” Genetics 158 (2001): 1502-1512. Web. 20 Feb. 2010.

16 Hoffman, Stephen et al. “Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria.” Human Vaccines 6.1 (2010): 97-106. Web. 20 Feb. 2010.

17 Vaughan, Ashley M et al. “Genetically engineered, attenuated whole-cell vaccine approaches for malaria.” Human Vaccines 6.1 (2010): 107-113. Web. 24 Feb. 2010

18 VanBuskirk, Kelley M et al. “Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design.” PNAS 106.31 (2009): 13004-9. Web. 20 Feb. 2010.

19 Aly, Ahmed S I et al. “Targeted deletion of SAP1 abolishes the expression of infectivity factors necessary for successful malaria parasite liver infection.” Molecular Microbiology 69.1 (2008): 152-163. Web. 20 Feb. 2010.13

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