Microbial Pathogenesis 1987, 2 : 237-240

Mini-review

Developing a human malaria sporozoite vaccine

James F. Young,' W. Ripley BalIou 2 and Wayne T. Hockmeyer2

'Department of Molecular Genetics, Smith Kline and French Laboratories, Swedeland, PA190406, U.S.A. and 2Department of Immunology, Walter ReedArmy Institute ofResearch, Washington, DC 20307, U .S.A .

Malaria remains as one of the world's great health problems affecting an estimated300 million individuals in tropical areas annually, killing 1 to 2 million of these . Thedisease is caused by a protozoan parasite of the genus Plasmodium which is transmittedto man by mosquitoes . Although four species of the parasite are known to infect man,P._ falciparum is the most common and most dangerous, responsible for nearly allmalaria related deaths. Efforts in the 1 950s and 1 960s to eliminate malaria by vectoreradication and parasite control with novel drugs made a significant impact on reducingthe prevalence of the disease . Unfortunately, this decline was soon met by theemergence of mosquitoes resistant to the insecticides and drug-resistant parasites,leading to a resurgence in the number of cases. Thus, alternative methods of controllingmalaria are being sought and increased attention has now focused on the developmentof a safe and cost-effective vaccine .

The malaria infection begins in humans with the injection of the sporozoite form ofthe parasite into the blood stream by mosquitoes . The sporozoites rapidly migrate tothe liver where they invade the hepatocytes . During the next 5 to 7 days, the sporozoitedifferentiates and multiplies to produce the merozoite form which is released from theliver into the bloodstream . The merozoites invade red blood cells to initiate theerythrocytic replication of the parasite . It is this stage of the parasite growth cycleresponsible for the clinical symptoms of fever, chills, anemia and splenomegalyassociated with the disease .

Recent advances in the identification and characterization of antigens on the variousparasite stages using immunochemical and molecular cloning techniques now providethe first real opportunity to develop vaccines to prevent and control this disease . Themost significant progress in developing a malaria vaccine has been made against themosquito transmitted sporozoite stage of the parasite. An effective vaccine againstthis stage of the parasite would prevent infection, however, since immunity to thesporozoite has no effect on the growth and development of the clinically importantand antigenically distinct blood stages, a single sporozoite escaping the immunesystem could cause disease . Thus, it is imperative that the level of sporozoite immunitybe high in vaccinated individuals .The feasibility of immunizing against sporozoites has been established . Irradiated'

sporozoites have been shown to protect both man" and animals .' This protectionappears to correlate with the presence of antibodies directed to the major surfaceprotein on the sporozoite, the circumsporozoite (CS) protein . Moreover, monoclonalantibodies directed to the CS protein have been shown to passively confer protection

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Tetra- peptide repeats124

288345 357

Rewmnz Region II 42

41 v4 amino acidsFig . 1 . Schematic representation of the circumsporozoite protein of P. falciparum . The protein contains

two conserved domains, region and region II, and a large central repeat domain . The numbers indicate theamino acid number in the protein sequence .'

to animals.' However, because sporozoites cannot be cultured in vitro, it has not beenpossible to develop an an"uated whole organism or subunit vaccine .

Cloning and expression of the P. falciparum CS gene

Reoondy' thogeneenoodingthof!/a/xipm/un/CSprotoinvvaoo!onodandyaquonoed . n

The deduced amino acid sequence for the protein revealed a structure similar to mostsurface glycoproteins, with an amino terminal hydrophobic signal peptide and acarboxy terminal membrane anchor sequence . However, a striking feature of thisprotein was that it contained a large oeniml domain of tetrapeptide repeats (Fig . 1) .This region contained 37 repeats of the sequence asparagine-alanine-asparagine-proline interspersed with four repeats of asparagine-valine-aspartic acid-proline andstudies have shown that these tetrapeptide repeats are conserved in all P. falciparumstrains that have been examined .' The structure of the P. fakiparum CS protein isanalogous to that of the CS proteins the human malaria species, P . vivax,7 those ofthe simian parasites, P. 6nuw/&m/o and P. cynomolgi,' and the mouse malaria, P .berghei,' O although the sequences of the repeats in each species is very different . Infact, the overall sequence homology among these CS proteins of/! fa/ciparum and P.6nuwrles/ is very low with only two regions of approximately 15 amino acids each inthorogionnf!enkingthovepoutdooooinvxhiohareoonoepvod . n Aninterootingobaorvadonwas that the repeat domain of these proteins appears to immunodominant . Allneutralizing monoclonal antibodies raised against sporozoites which have beenexamined react with synthetic peptides corresponding to the repeat domain . Inaddition, po!yo!onol sera from individuals immune to P . falciparum sporozoites alsoreoota!muatuxo!uaivo!yvviththiaregion . ,, Thuo'ituppaamthatthaneutrahzingepitopeis contained within this repeat domain . However, it was hoped that antibodies directedagainntaithorof1heLvvo15aminoaoidonnservodregionamiAhtprovidouroos-reaotivnimmunity against a!l four malaria species known to infect humans. Experimentsdesigned to evaluate this hypothesis were carried out using synthetic peptidesoonempondingiotheoeoonoen/edregionooevve!!aototherepoatdnmain .` z Po!yo/una!sera were raised to these synthetic peptides linked to carrier proteins in both mice andrabbits Although antibodies directed to these regions were produced that reactedyvithau1hentioCSproteinonparunitna'on!ythountibodieodiroutodtnthetetrapopddmrepeats were capable of neuualizing sporozoites in an in vitro human hepatocyteinvasion assay .Attention then focused on producing a molecule encompassing the repeats which

would be suitable for testing in humans . Using recombinant DNA technology, aportion of the CS gene corresponding to the repeat domain of the CS protein wasengineered into an E. coli bacterial expression vector and the encoded protein wasproduced ." This molecule, R32tet32, contains 32 of the CS protein tetrapeptide repeatsfuoedto32aminoooidoonuodedbythotetraoyo!inoreoiotonueregionnf1haplunmid .This protein was purified and tested in mice and rabbits for its ability to induceneutralizing antibodies. Sera were generated which contained high tit*rod antibodies

Human malaria sporozoite vaccine

239

that reacted with sporozoites and inhibited the invasion of human hepatocytes in vitroby live sporozoites . The R32tet32 molecule was highly immunogenic in animals andhigh titered antisera was produced even in the absence of adjuvants, although bothalum and Complete Freunds Adjuvant potentiated the response . Not only did theseantibodies prevent the attachment and penetration of sporozoites into hepatocytes,but they also prevented the intracellular development of the parasite ." This suggestedthat these antibodies in some Way interacted with the extracellular parasite to inhibitsubsequent replication .

Testing of a recombinant sporozoite vaccine in humans

Based on these data, we conducted a Phase I clinical trial with a vaccine based onthis molecule in human volunteers to determine : (11 ) whether the vaccine was safewhen administered to humans, and (2) whether it would induce antibodies directedagainst P. falciparum CS repeat epitopes (the complete details of this study aredescribed by Ballou a t a/") . P. falciparum sporozoite vaccine FSV-1, consisted ofR22tetxz in aqueous saline containing 0 .5 mg aluminum hydroxide gel per 0.5 ml dose .The vaccine was prepared at 5 different concentrations ranging from 10 to 800 gg ofprotein per dose. The vaccine was administered intramuscularly to three volunteers ateach of five doses (10 tg, 30 tg, 100 pg, 300 pg, and 800 tg) on day 0 with boostersat 1 month and 2 months with the same dosage levels. Sera were collected at varioustimes and assayed for the presence of antibodies directed to the repeat region by anELISA assay .

As would be expected for a highly purified protein, the vaccine was well toleratedin all volunteers . Dose-dependent antibody responses to the vaccine were detected asearly as 2 weeks after the primary immunization, and 80% of the vaccine recipientsultimately developed antibodies directed against the repeat when assayed at a serumdilution of 1 :50 or greater . The antibody titers failed to increase following the boosterdoses of FSV-1 except in one volunteer receiving the highest dose . The antibodieswere predominantly !g0I but included a!l four IgGs, IgM and IgA subclasses, inaddition, lymphocyte proliferation assays were also performed on cells from thevaccines. These studies demonstrated significant stimulation of lymphocytes from themajority of volunteers .

In general, good antibody responses were seen during preclinical studies of FSV-1with strains of inbred mice, outbred nAce, rabbits and rhesus monkeys . More recently,etudieyvvithR32tot3,andsynthabupoptidoohoveohoxvnganoticrautriotionofimmuneresponses to the CS repeat epitopes in congenic mice ." Thus failure to boost withthis antigen may reflect an insufficient array of T-helper epitopes in the CS repeat toimpart good memory responses in humans . It may be necessary to generate secondgeneration vaccine molecules which contain additional T cell epitopes, either byproviding more sequences from the natural protein by recombinant DNA technologyor by coupling repeat molecules to unrelated carrier proteins which are highlyimmunogenic in man and may be capable of fulfilling this function .This Phase I study demonstrated the safety and feasibility of immunizing human

volunteers with a highly purified recombinant, subunit CS protein vaccine . It is likelythuthigher!eve!eofantibodionzhanthoyeuohiovedinthiootudyxvi!!beneododforonefficacious vaccine and modifications in dose or immunization schedule may benecessary. Because responses to this vaccine in experimental animals do not predictimmunogenicity in humans, additional o/iniual trials vvi!l be needed to assess theimmunogenicity of this and other CS protein derivatives .

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References

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16. Good MF, Berzofsky JA, Maloy WL et al. Genetic control of the immune response in mice to aPlasmodium falciparum sporozoite vaccine : Widespread nonresponsiveness to single malaria T epitopein highly repetitive vaccine . J Exp Med 1986 ; 164 : 655-60 .