Malaria Vaccine Researchat the

Naval MedicalResearch Center

CAPT Daniel J. Carucci, MC, USN

At a time in bioscience when many people assume that (NMRU) working throughout Southeast Asia (NMRU-vaccines are available for most infectious diseases, it 2), South America (NMRC-DET), Africa (NMRU-3)often comes as a surprise that the ancient disease, and most recently in Ghana, West Africa. In addition,malaria, is still the most important parasitic infectious NMRC has established the first Navy Clinical Trialsdisease in the world today. It also poses one of the Center at the National Naval Medical Center, Bethesda,greatest threats to the U.S. military operational forces MD, the only clinical trials center dedicated for malaria

than any other naturally-occurring infectious disease. In vaccine testing and development.fact, in every campaign this century fought where malariawas present, more casualties resulted from malaria than Malaria Life Cyclefrom bullets. During the war in Vietnam, entire divisions So why is there no vaccine against a disease that causeswere rendered ineffective due to large numbers of such an incalculable costs in terms of human lives andmalaria cases.(l) Even more importantly for the world suffering and results in the death of more than a millionpopulation there are between 300 and 500 million cases children each year? Many feel that the answer lies in theeach year and between 1.5 and 2.7 million deaths complexity of the parasite itself. Unlike most viruses andannually, mostly in children living in Sub Saharan bacteria, pathogens such as malaria live a complex life.Africa.(2) The single cell para~te Plasmodium starts its life in

Drugs used to prevent malaria infection, though humans as "sporozoite" forms injected by the bite of anuniversally effective until the 1960's and 1970's, are infected female Anopheles mosquito. Hundreds ofeither no longer effective or are becoming less effective sporozoites enter the blood stream with the mosquito'sin many parts of the world due to the development of drug saliva and within minutes, invade individua:l cells in theresistance. And yet despite over 15 years of research, liver. Inside these liver cells the parasites multiply to over

there is still no licensed vaccine against malaria. 10,000 in 1 week.Navy malaria researchers are developing approaches As the parasites at this stage appear to cause no signs of

to preventing and treating malaria through drug and illness, many feel this is an ideal time to attack thevaccine development at the Naval Medical Research parasite with drugs and vaccines. If left unchecked, theCenter (NMRC) core Malaria Program located in Silver parasites (now called "merozoites" and numbering overSpring, MD, and also through a network of overseas one million) burst out of the liver cells into the bloodresearch commands-Naval Medical Research Units stream and attach themselves to red blood cells.

September-October 2001 23

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Individual parasites enter the red blood cells and multiply salivary glands become weakened but are not killed.to 16-20 parasites in approximately 2 days. All When the mosquitoes are allowed to feed on an animal ormanifestations of disease occur at this stage of the human, the "irradiated sporozoites" invade liver cellsparasite's life cycle. normally, but they do not divide and do not develop

Approximately, every 2 days parasites burst from the further. The immune system recognizes the parasiteinfected red blood cells, invade new red blood cells, and proteins present in the liver cells and mounts an attack onmultiply an additional 1 0-20 times. Within a few days the these cells killing the parasite. This "irradiated sporozoitenumbers of parasites in the body reach many billions and vaccine" is comprised of allowing 150 mosquitoes tocause fevers, chills, fatigue, and lethargy. In severe cases, feed on the arm of an individual once a month for 6especially in children, the parasites destroy so many red months. Individuals immunized by this method arecells and suppress the production of new red cells that completely protected against infection with malaria for atsevere anemia develops. In some cases, the infected red least 9 months. Unfortunately, this effective vaccine iscells become "sticky" and attach to the inner surface of completely impractical except as an experimental model.capillaries, especially in the major organs, causing A second vaccine model is that of"naturally acquired"blockages. immunity. Children raised in malarious areas who are

When the parasites block the blood flow in the brain continually exposed to the bites of infected mosquitoes"cerebral malaria" develops resulting in seizures and and who survive to the age of about 10 years old,coma. These two syndromes, severe anemia or cerebral generally develop immunity to malaria. This immunitymalaria, are the predominant causes of death in children does not result in sterile protection, but does protectand non-immune travelers such as military personnel. against severe disease. In fact, in many areas of the world,

In order to reproduce, some parasites in the red cells do it is not uncommon to find nearly all adolescents andnot divide and multiply, but transform into male and adults with malaria parasites in their blood yet they arefemale "gametocytes." These gametocytes are taken up apparently quite healthy. In these individuals the immuneby feeding mosquitoes and sexually reproduce in the system keeps the number of parasites in the blood low andmosquito's stomach. The resulting sporozoites travel to prevents the manifestations of severe disease. Interest-the mosquito salivary glands ready to be injected on the ingly, when these individuals travel to non-malariousnext blood meal and to infect another individual. areas, they often lose a substantial part of their immunity

and are at risk for more severe malaria when they return.Malaria Vaccine Development Armed with these two models of malaria vaccines, it is

Recognizing the complexity of the parasite, many convenient to approach vaccine development in one ofscientists feel that an effective vaccine may need to attack three ways. The fIrst is to assume that there are one or twomultiple stages of the parasite's life cycle. In addition, the "key" proteins that the parasite expresses and can bevaccine will have to mount different immune responses targeted for vaccine development. One would construct ato attack the parasite at its different stages. For example, vaccine that produces a very strong immune responsesoluble circulating antibodies may be needed to attack the against these one or two proteins and hope that this will besporozoites while in the blood stream, killer immune cells sufficient for killing the malaria parasite. For example,(T -cells) may be needed to attack the parasites within the one protein expressed at the liver stage and one proteinliver cells, and a combination of antibodies and T -cells expressed at the blood stage could be targeted. In fact, themay be required to attack the parasites inside the red cells. best experimental malaria vaccine to date is based on aFurther, antibodies may be needed to prevent the single protein expressed at the sporo~oite and liverreproduction of the parasites within the mosquito stages. This vaccine, RTS,S, is a recombinant proteinstomach. vaccine (a protein produced in the laboratory that is

No vaccine to date has been shown to provide the similar to a protein actually made by the parasite) and hasbreadth of immune responses that may be required for been shown to provide 30 percent protection for severalpreventing malaria. The challenge is daunting, though weeks in studies.(3) It is hoped that this vaccine can bethere are two models that suggest that a protective malaria improved upon sufficiently, perhaps by adding additionalvaccine is feasible. If Plasmodium infected mosquitoes recombinant proteins, to provide long-term protection.are bombarded with a precise amount of gamma However, it may be that the parasite is sufficientlyirradiation, the parasites living inside the mosquito complex and has the ability to evade the host's immune

24 NAVY MEDICINE

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system as to require an attack at many targets responses between different strains of mice,( 5) can besimultaneously. This second approach targets all known combined with recombinant protein or recombinant poxproteins (10 to 15) expressed at multiple stages of the viruses to increase protection in mice,(6) are immuno-parasite life cycle. Unfortunately, it may not be practical genic in monkeys( 7) and can protect monkeys from lethalto produce the multiple recombinant proteins required infection with malaria (Rogers, et aI, submitted).due to their expense, complexity of production and Based on these findings and others, NMRC's Malariadifficulty in purification. Program, conducted the flfst-ever clinical trial in healthy

To develop this type of multistage vaccine, a humans using a DNA vaccine.(8) This vaccine, thoughnontraditional approach may be needed. Researchers at not designed to be protective, was shown to be safe, wellthe Malaria Program, Naval Medical Research Center, tolerated and to produce strong cellular immunity.(9) Ahave been developing novel DNA-based vaccines used second trial evaluated this same DNA vaccine in a novelalone or in combination with recombinant proteins and vaccine delivery system, the needle-less Biojectorviruses to target multiple stages of the parasite life cycle system (Epstein/Wang, et al submitted).as well as exploiting the two models of immunity to To assess the safety and immune responses to multiplemalaria, irradiated sporozoites and naturally-acquired genes introduced simultaneously a clinical trial wasimmunity. A third approach would target all expressed conducted using a combination of five malaria genes,proteins from a particular stage, in essence reproducing targeted to the liver stage of the parasite life cycle. Duringthe "whole organism" immunity seen in both the the development of this five-gene vaccine, researchers atirradiated sporozoite vaccine and the naturally-acquired NMRC's Malaria Program, demonstrated that if theseimmunity models. To do this would require the malaria genes were reengineered in the laboratory eachidentification and pattern of expression of all known had the potential to function significantly better than thegenes and proteins from each of the stages of the parasite original native malaria genes. Since Plasmodium DNAthat are the targets of these vaccines, as well knowing uses a very different balance in its genetic code aswhich are those that generate protective immune compared to humans, by changing the genetic code in theresponses, an approach that is actively being developed at malaria genes to be more like that of human genes, theythe Navy's malaria program. were able to show these genes could produce 10-20 times

more foreign protein than the native malaria genes.DNA-based vaccines In studies in mice, these "synthetic genes" stimulated

DNA vaccines are different from any other licensed an immune response in mice 3-20 times greater than thevaccine available. Instead of immunizing with a foreign native genes. Clinical trials are now being planned toprotein, DNA vaccines immunize with the genetic assess the effectiveness of these synthetic vaccines.blueprint that encodes for that foreign protein. Cells from Although DNA-based vaccines hold tremendousimmunized individuals take up a "ring of DNA" promise, not only for malaria vaccines, but for a varietycontaining the foreign gene and uses its own cellular of other diseases, based on results to date, they may not bemachinery to translate the injected genetic material to able to simulate sufficiently strong immune responses inproduce the foreign protein. The immune system then monkeys and humans on their own to confer completeresponds to the foreign protein much in the same way that protection against malaria. Researchers at NMRC andit would if the protein was introduced intact. elsewhere have shown that combining a DNA vaccine

Thus DNA vaccines offer significant advantages over "prime" followed with the same genes expressed by aother types of vaccines. They are relatively easy to modified virus, similar to the smallpox vaccine, or toproduce and purify, and thus are easy to modify; recombinant protein, provides better immune responsesstimulate strong T-cell responses; may not require and protection in animal models than either alone.( 1 0-12)refrigeration; and have the potential for combination A major effort, therefore, is underway focusing onallowing the targeting of multiple foreign proteins improving malaria vaccine development by this "prime-simultaneously. During the past 6 years the NMRC boost" approach.. Because DNA vaccines on their ownMalaria Program has developed extensive experience offer such a tremendous potential advantage over allwith DNA-based vaccines demonstrating that DNA other types of vaccines available today, a major area ofvaccines can protect mice from infection with malaria,( 4) research is focusing on improvements in vaccine deliverycan be combined to overcome the differences in systems to make DNA vaccines on their own as effective

September-October 2001 25

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as the prime-boost vaccines. This includes novel means species of malaria parasite will be completed by the endof getting the DNA into the cells, using absorbable micro of 2002, including the second most important malariaparticles, coated gold-beads, specialized polymers, and parasite in humans, P. vivax-.others; improving the DNA sequences in the vaccinesused by the host cell machinery; and adding additional Functional GenomicsDNA vaccines expressing proteins helpful in recruiting NMRC researchers have also been on the forefront inimmune cells to the area of vaccine delivery. developing and utilizing novel technologies to exploit the

enormous amounts of data generated from the malariaGenomics genome project toward drug and vaccine development.

The future of vaccine development for malaria and a Working broadly on several fronts, the NMRC team hasrange of other pathogens may actually lie within the produced the fIrst chromosome-specific DNA microarray,genetic code itself. In order to duplicate whole organism enabling the study of the expression of thousands ofimmunity, such as occurs in both the irradiated sporozoite genes simultaneously, for example in response tomodel and the naturally acquired immunity model, it may antimalarial drugs and from various stages of the parasitebe necessary for the immune system to mount multiple life cycle.types of immune responses simultaneously against In collaboration with Scripps Research Institute (SRI)multiple targets. Herein lies that power of the genetic they have employed a method to identify hundreds tocode. thousands of parasite proteins from various stages of the

In 1996, Malaria Program researchers in an life cycle using high-throughput liquid chromatographyinternational collaboration with The Institute for coupled with tandem mass spectrometry, have partneredGenomic Research (TIGR), the Sanger Centre, and with San Diego Supercomputing Center and the U.S.Stanford University, with funding from the Department Navy High Performance Computing CenterofDefense, the NIH, the Burroughs Well come Fund, and (supercomputers) to dramatically improve computerthe Well come Trust, embarked on a project to determine performance used in protein identification, deployedthe entire genetic sequence of the human malaria recombinational cloning systems to produce hundreds ofparasite, P.falciparum.(13) With the complete genomic plasmid clones in a fraction of the time by traditionalsequence of the malaria parasite in hand, researchers methods, and developed in-house relational databasewould have the ability to identify every potential drug and capabilities needed to handle the enormous volumes ofvaccine target, elucidate complex biochemical pathways, informat:ion generated from these functional genomicsand be able to develop tools to study fundamental parasite projects.( 18)biological processes. DNA-based vaccines offer a unique opportunity to

Within 18 months of starting the project, NMRCI transform genomic sequence data into deployableTIGR researchers published the first complete genetic vaccines. Although "whole genes" are currently beingsequence of a malaria parasite chromosome.(14) A investigated as potential DNA vaccines, researchers atsecond chromosome was published shortly after.(15) the NMRC malaria program are also constructingPlans are now underway to publish a series of articles by "minigene" vaccines, comprised of a series ofearly 2002 including the complete genomic sequence of "epitopes," small regions of individual genes predicted toP. falciparum. be important in stimulating immune responses,

As part of the DNA sequencing consortium's effort, all particularly T-cell responses.( 19-21) The next genera-of the genomic sequence data has been released to the tion oroNA-based vaccines that will result from the datapublic during the 4-year history of the project. This early from the Malaria Genome Project will likely bereleased data has enabled researchers to 'jump start" their composed of dozens to hundreds of epitopes strungresearch already leading to the identification of two ,together like beads on a string.potentially important targets for new antimalarialdrugs.(16,17) The consortium has made such progress Conclusionwith the completion of the P. falciparum project, By the end of 2002, researchers will have at theirincluding overcoming several technical hurdles as well as disposal the complete genomic sequence of P.developing improved and more cost effective means of falciparum, and its two hosts: humans(22) and thesequencing, that the genomes from at least two additional mosquito, Anopheles gambiae. In this nascent field of

26 NAVY MEDICINE

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genomics, it is as yet unclear how these data will be best 10. Sedegah M JonesTR Kaur M t I B t. .thdt 1 .d th 1 . . ' , ,e a. oosmgwI

use.o e UCI ~te e comp ex InteractIons between the r~combinant vaccinia increases immunogenicity and protec-parasIte and Its host(s). However, combined with tIve efficacy of malaria DNA vaccine. Proc Natl A cad Sci UStechnical advances in functional genomics and rational A 1998; 95:7648-53.drug and vaccine design, including DNA -based vaccines, ~ I: Rob~son ~, Montefiori DC, Johnson RP, et al. Neu-these genomic data may provide the foundation for n:allZmg. antIbody-mdependent co~~inment of i~unodefi-entirely novel approaches to drug and . c~ency VIrUS c~alleng~s b.y DNA pnmmg and recombmant poxd vaccme VIrUS booster ImmunIzatIons [see comments] Nat Med 1999'evelopment, and may lead to new strategies to combat 5:526-34. .,

n?t only malaria but many other infectious and emerging 12. Amara RR, Villinger F, Altman ill, et al. Control of adIseases. The challenge over the next decade will be to mucosal challenge and prevention of AIDS by a multiproteinuse these novel approaches to better understand the DNAlMVA vaccine. Science 2001;292:69-74.malaria parasite and its interactions with its hosts to 13. Hoffinan SL, Bancroft WH, Gottlieb M, et al. Fundingdevelop strategies to intervene and reduce 'the for the malaria genome sequencing project. Nature 1997;tr d h 11 d . 387:647.

emen ous uman to , an to provIde effective vaccines 14 Gardner MJ TettelI.n H C .DJ t I Chrd dru t DOD ~. ." aruCCI , ea. omosome

an ~s 0 ~rotect J.orces agamst the threat of this 2 sequence of the human malaria parasite plasmodiumdestructIve dIsease. Navy researchers are at the cutting falciparum. Science 1998;282:1126-1132.edge in this 21st century era of biomedicine. 15. B.owman S, Lawson D, Basham D, et al. The complete

nucleotIde sequence of chromosome 3 of PlasmodiumReferences falciparum. Nature 1999;400:532-538.

1. Beadle C, Hoffinan SL. History of malaria in the United 16. Waller RF, Keeling PI, Donald RG, et al. Nuclear-en-State~ Naval Forces at war: World War I through the Vietnam coded pr?teins ~get to the plastid in Toxoplasma gondii andconflIct. Clin.Infect.Dis. 1993;16:320-329. PlasmodIum falcIparum. Proceedings of the National Acad-

2. Anonymous. World malaria situation in 1992. Weekly emy of Sciences of the United States of America 1998;Epidemiological Record 1994;69:309-314. 95:12352-12357.

3. Stoute JA, Kester KE, Krzych U, et al. Long-term effi- 17. Jomaa H, Wiesner J, Sanderbrand S, et al. Inhibitors ofcacy and immune responses following immunization with the t!te no~evalonate pathway of isoprenoid biosynthesis as an-RTS,S malaria vaccine. J:Infect.Dis. 1998;178:1139-1144. tImalartal drugs [see comments]. Science 1999;285:1573-6.

4. Sedegah M, Hedstrom R, Hobart P, Hoffinan SL. Protec- 18. Carucci DJ. Malaria research in the post-genomic Eration against malaria by immunization with plasmid DNA en- [In Process Citation]. Parasitol Today 2000;16:434-8.coding circumsporozoite protein. Proceedings of the National 19. Doo~an DL, Hof!man SL, Southwood S, et al. Degener-Academy of Sciences of the United States of America 1994. ate CytOtOXIC T cell epItopes from P. falciparum restricted by91:9866-9870. ' HLA-A and HLA-B supertypes alleles. Immunity 1997;7:97-

5. Doolan DL, Sedegah M, Hedstrom RC Hobart P 112.Charoenvit Y, Hoffinan SL. Circumventing genetic restrictio~ 20. Dool~ DL, Hoffinan SL, Southwood S, Wentworth P A,~f protection against malaria with multigene DNA immuniza- Sette A. I?esIgn of a subunit malaria vaccine incorporatingtIon: CD8+ cell-, interferon gamma-, and nitric oxide-depen- HLp:-restrIcted .cD8+ T-cell epitopes of multiple Plasmodiumdent immunity. J Exp Med 1996;183:1739-46. falcIparum antIgens. In: Brown F, Burton D, Doherty P,

6. Sedegah M, Jones TR, Kaur M, et al. Boosting with re- Mekalanos J, Norrby E, eds. Vaccines 97: Molecular Ap-combinant vaccinia increases immunogenicity and protective pro~ches to the Control of Infectious Diseases. New York: Coldefficacy of malaria DNA vaccine. Proceedings of the National Spnng Harbor Labora~ Press, 1997:207-211.Academy of Sciences of the United States of America 1998. 21. Doolan DL, WIzel B, Hoffman SL. Class I HLA-re-95:7648-7653. ' stricted cytotoxic T lymphocyte responses against malaria--

7. Wang R, Doolan DL, Charoenvit Y, et al. Simultaneous elucidation on the basis of HLA peptide binding motifs.~nduction of multiple antigen-specific cytotoxic T lymphocytes Immunol Res 1996;15:280-305.m nonhuman primates by immunization with a mixture offour 22. Venter JC, Adams MD, Myers EW, et al. The sequencePlasmodium falciparum DNA plasmids. Infect Immun 1998; of the human genome. Science 2001; 291:1304-51. I:J

66:4193-202.8. Le TP, Coonan KM, Hedstrom RC, et al. Safety, toler-

ability and humoral immune responses after intramuscular ad-ministration of a malaria DNA vaccine to healthy adult volun-teers. Vaccine 2000;18:1893-1901. ,

9.. Wang R, Doolan DL, Le TP, et al. Induction of antigen-spec~fic cyt~toxic T lymphocytes in humans by a malaria DNA Dr. Carucci is Director of the Malaria Program at Naval Medicalvaccme. SClence 1998;282:476-480. Research Center, Silver Spring, MD.

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