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Review ArticleTHE NATIONAL MEDICAL JOURNAL OF INDIA VOL. 9, No.3, 1996

Development of vaccines against falciparum malariaS. RAMALINGAM, E. MATHAI

ABSTRACTFalciparum malaria is a leading cause of death in manycountries. Drug resistance has emerged as a major cause forconcern, increasing the pathogenic potential of the parasite.So far vaccines have been an elusive option. We discuss heresome of the antigens and vaccines that show promise and onwhich studies are in progress.Natl Med J India 1996;9:120-4

INTRODUCTIONAbout 2.2 billion people are exposed to malaria every year inoverone hundred countries. Of these. 300 million people get infected,and 120 miIlion develop the disease. Africa accounts for 90% ofinfections and 80% of cases-leading to the deaths of a millionchildren annually. More than one-third of the remaining 10%infections occur in India. In 1989 and 1990, the reported caseswere 2.04 and 1.78 million respectively. India alone accounts for80% of cases from middle South Asia.'

The incidence of malaria is increasing due to resistance ofvectors to insecticides and drug resistant parasites. Other contrib-uting factors include mass population movement and environ-mental alteration. Attention has now been focused towards thedevelopment of vaccines against malaria.'

There are a number of reasons to believe that vaccines will beuseful. People in endemic areas develop partial protective immu-nity and are less likely to become severely ill.' Also the mortalityrate in these areas is low. Further. early experiments in animalswith irradiated sporozoites have shown protective immunity.

The development of a malarial vaccine is. however, not easy asthe antigens are multiple, and are also specific to species, strainand stage." The immunity induced has the same characteristics.

Though Plasmodium vivax accounts for most cases of malaria.95% of the deaths are caused by Plasmodium Jalciparum. Hencestudies on vaccine production have centred around P.Jalciparum.The recent epidemics of P. falciparum malaria in the northwestern and north eastern states oflndia prompted us to review thesubject of vaccines for P.falciparum.

CLASSIFlCA TIONThe antigens of P.Jalciparum which have been tested as potentialvaccine candidates are classified according to the stage of devel-opment at which they are expressed. The list in Table I includesmost of the important antigens, but is by no means complete.

Christian Medical College. Vellore 6J2004. Tamil Nadu, IndiaS. RAMALINGAM. E. MATHAI Department of Microbiology

Correspondence to E. MATHAI

© The National Medical Journal of India 1996

TABLE I. Potential vaccine antigens

AllliKCIISfrom pre-erythrocytic stagesIrradiated sporozoites Circumsporozoite protein (CSP)Liver stage antigen-I (LSA-I)

Antigens [rom merozoites and erythrocytesErythrocyte binding antigen-175 (EBA-175)Merozoite surface antigen-I (MSA-I/MSP-I)Merozoite surface antigen-2 (MSA-2/MSP-2)Ring-infected erythrocyte surface antigen (RESA)Serine repeat antigen (SERA/SERP)Rhoptry-associated proteins (RAP)Apical membrane antigen-I (AMA-I) Histidine-rich proteins (HRP)Transmission blocking antigensPfs-25 4R/45K Pfs-2JOCombined vaccineSPf66

ANTIGENS FROM THE PRE-ER YTHROCYTIC STAGESSporozoites are the first stage of the malarial parasite that ourimmune system encounters: hence effective immunity againstsporozoites can abort infection. The antigens on the sporozoitesurface are highly immunogenic, inducing both cell-mediated aswell as humoral immunity.':" However, anti sporozoite immunityhas its drawbacks in being stage-specific, species-specific andrequiring large immunizing doses ..•.."··-II Further. sporozoitesremain in circulation for very short periods. and their develop-ment cannot be stopped if a few escape. Both fractions and wholesporozoites have been tried for vaccine production.

Irradiated sporozoitesThis is one of the earliest immunizing methods tried. As sporozoi-tes cannot be cultured, irradiated, infected mosquitoes are madeto feed on volunteers. Effective protective immunity is inducedfor a long duration. In a recent study antibodies to sporozoiteswere found 9-12 months after booster bites by irradiated. infectedmosquitoes. One volunteer was protected from challenge 9 monthsafter immunization. I I Since the immunizing dose required is large(around 400-1800 mosquito bites per person), this method is notpractical for routine immunizanon+w-!'

Circumsporozoite proteins (CSP)The circumsporozoite protein of 412 amino acids with a molecu-lar weight of 4~66.kDa is expressed on the surface of the sporo-zoites, 12 with a sequence of amino acid repeats in the middle. andcharged areas on either side. In the charged areas, there are twogenus conserved regions RI and RII.12.B

Monoclonal antibodies to CSP completely protect mice fromchallenge with infected mosquitoes. 14 Also, monoclonal antibod-

RAMAUNGAM, MATHAI: VACCINES AGAINST FALCIPARUM MALARIA

ies were inhibited from binding to CSP of P. falciparum bysynthetic peptides of the repeat region."

The repetitive area of the CSP is immunodominant and has40-46 tetrapeptide repeats, mostly NANP (Asn, Ala, Asn, Pro).There are also 3-4 NVDP (Asn, Val, Asp, Pro) variants. Atripeptide of NANP is considered to be the protecti ve determinantof CSP.13It induces humoral immunity and has been included inmany vaccines with varying success. 15·17

For example, a 12 amino acid synthetic peptide Ac-Cys-(NANP)3, coupled to tetanus toxoid, with aluminium hydroxideas adjuvant seroconverted 53% and 73% of volunteers receiving100 ug and 160 ug respectively. Of three volunteers challenged,two developed parasitaemia on day I I, while the third did noteven till day 29. All controls developed parasitaemia within JOdays.P'Ihis antigen elicited either an antipeptide, anticircumsporo-zoite or an anlisporozoite antibody response. However, the finespecificity varied among volunteers, with some producing mainlyantipeptide antibodies.'? Another recombinant DNA sporozoitevaccine (FSV-I) produced in E. coli induced anticircumsporozoiteantibodies which inhibited invasion of hepatoma cells in vitro. Noside-effects were observed. However, the protection on challengewas only partial in volunteers. IX

Yet another vaccine, R32TOX-A has been tried recently. Itconsists of a recombinant protein R32LR [(NANP) 15(NVDP)2LR], made from the repeat region of CSP, conjugated to detoxi-fied toxin-A of Pseudomonas aeruginosa, and adsorbed to alu-minium hydroxide, with merthiolate as preservative." In thePhases I and II trials on non-immune volunteers, this vaccineproved to be safe, well tolerated and immunogenic." But theprotective immunity was very low. In another Phase II study on199 Thai volunteers (immune and non-immune), its safety andimmunogenicity were proven, with maximum antibody responsein the immune volunteers. However, the response appears to beshort-lived and its protective efficacy has not been proved. ,H

Other epitopes stimulating B and T cells have also beenidentified outside the repeat region." RII is a sporozoite ligand forthe hepatocyte receptor. It has been found to be conserved in otherplasmodium species. Recently it has been shown that peptidesbased on Rll inhibit invasion ofHep-G2 cells by P. berghei sporo-zoites. Also. mice immunized with a peptide P32 from the Rllregion of P.falciparum were protected against a lethal challengewith P. berghei sporozoites." Studies in mice show that T cellstimulation leads to IFN-y production, which induces liver cellsto produce nitrous oxide and kill the sporozoites.tv" The geneencoding CSP has been cloned. '5

Liver stage antigen-I (LSA-l)LSA-I is the only antigen that is expressed only at the liver stage.It is a 200 kDa protein found in infected hepatocytes within theparasitophorous vacuole.2.~·26It has highly conserved repeat andnon-repeat regions. contains T and B cells stimulating epitopes"and the gene encoding for it has been cloned and sequenced."

LSA-I is highly immunogenic inducing both humoral andcell-mediated immunity. There is a naturally occurring HLArestricted cytotoxic T cell response to this antigen which protectsfrom severe malaria. In a study in a less endemic area. antibodiesto LSA-I were detected in more than 70% of the general popula-tion. and in more than 97% of the population in moderately hightransmission areas. Also. in an area of low transmission, morethan 50% of the children below 5 years had raised antibody titresto LSA-l. IFN-y secretion could also be induced by this antigen.LSA-I was highly conserved between the 2 strains tested. 2~

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ANTIGENS FROM MEROZOITES AND ERYTHROCYTESThese are the next line of antigens detected by our immunesystem. They are specific for species and stage. Immunity againstthese antigens will not abort infection, but will prevent Iheinvasion of erythrocytes. hence reducing the severity of infection.The different antigens of this stage are situated in variousorganelles of the merozoites.

Erythrocyte binding antigen- 175 (EBA-175)This is a 175 kDl! protein ligand found in the micronemes ofmerozoites and helps the merozoites bind 10 RBCs in a two-stageprocess. 2K.NAt the time of invasion. EBA-175 is cleaved, and a 65kDa protein remains attached to the RBC. Hyperimmune serumagainst this region reduces invasion of erythrocytes by homolo-gous merozoites by 80%.NThe gene encoding EBA-175 has beencloned. W EBA is also recognized by T cells and induces a specificproliferative response in previously exposed individuals. A studyusing EBA suggests that it could also be a target for T cell-independent antibody response.'

Merozoite surface antigen-I (MSA-I/MSP-/)MSA-I is a precursor protein of molecular weight 195kDa,n seenon developing schizonts, It is processed by proteolysis to smallerfragments of 83 kDa and 42 kDa protein at the time of invasion bymerozoites.Il-J4 MSA 1-19, which is a 19kDa sub-fragment of the42 kDa protein, is retained by the merozoites and is seen on thering forms within the invaded erythrocytes. U ..lh

The gene encoding MSA-I has been cloned and the specificfragments located. J4 Recombinant proteins have been producedfrom yeasts.

Monoclonal antibody to MSA 1-19 reduces the efficacy oferythrocyte invasion. IgG. IgM antibodies occur naturally 10 thisantigen ..l~Also lymphocytes from an endemic population show aproliferative response and IFN-y release in response to thisantigen.":"

There are conflicting reports on the protective efficacy of thisantigen. A recombinant vaccinia virus expressing MSA-I did notprotect or show an immune response in Saimiri monkeys, butthere was an increase in antibody levels following challenge ..l·Immunization using a recombinant hybrid of MSA-I, SERA andHRP-2 in Aorus monkeys led to a high antibody level and verylow level of parasitaemia on challenge." There appears to be somedifference between strains in the repeat regions of MSA_14u

Merozoite surface antigen-2 (MSA-2IMSP-2)This is another protein of molecular weight 45 kDa situated on thesurface of the merozoites of P.falciparum. The Nand C terminalsare conserved while the central repeat region is variable andirnmunodorninant."

Saimiri monkeys immunized with a recombinant vacciniavirus with MSA-2 showed a very low antibody response whichincreased on challenge. but there was no protection." Miceinoculated with MSA-2 of P. falciparum coupled to diphtheriatoxoid elicited an antibody response that recognized MSA-2 fromdifferent strains of P.falciparum and also P. chaubaudii (Rodentmalarial parasite). in immunoblots." Therefore, the conservedregion of MSA-2 could form the basis of a malaria vaccine in asuitably immunogenic form.

Ring-infected erythrocyte surface antigen (RESAlPF 155)RESA is a soluble, heat-stable protein of molecular weight 155kDa found on the surface of ring-infected erythrocytes." It is

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probably produced late in schizogony in the micronemes ofmerozoites, from where it is transferred to the rhoptry organ-elies." At the time of invasion, RESA is released through theapical pore, and is then found on the cytoplasmic surface ofinfected erythrocytes."

RESA consists of tandemly repeated subunits of 8, 4 and 3amino acids and is present in all strains of P.Jalciparum. The geneencoding RES A has been cloned and has small and large exons,connected by an intron."

Aotus monkeys immunized with an E. coli-derived fusionprotein were partially protected. Antibody to an 8 amino acidsequence appears to be more protective." Saimiri monkeys vac-cinated with a recombinant vaccine did not show protection orincreased antibody levels, but the antibody level increased after achallenge." Human antibodies to RESA effectively block RBCinvasion by merozoites ill vitro and also lyse recently infectedcells." A direct relationship seems to exist between the antibodylevels to RESA and immunity. People living in endemic areashave a high titre of anti-RESA antibodies."

Serine repeat antigen (SERA)SERA (also called SERP, P 126, PI 13, P 140) is a protease similarto the cathepsin LBH groups of cystine protease.v-" It has amolecular weight of III kDa and consists of 989 amino acids,with a stretch of serine 35 amino acids long. It is secreted bytrophozoites and schizonts into the parasitophorous vacuole, andis released at the time of merozoite release."

The gene responsible for SERA has been cloned." Whenrecombinant proteins with SERA and HRP-2 or MSA-I were usedfor immunizing Aotus monkeys, they showed very high antibodylevels and low parasitaernia." The SERA sequences in theserecombinant proteins were found to be highly conserved."

Rhoptry-associated proteins (RAP-IlRAP-2)Rhoptries are apical organelles of merozoites that release theircontents into erythrocytes at the time of invasion. The antigens,RAP-I (82 kDa; also called P82) and RAP-2 (42 kDa) form acomplex in the parasite. Monoclonal antibodies against RAP-I,RAP-2 complex inhibit in vitro invasion by the parasite. The geneencoding RAP-l has been cloned. Saimiri monkeys immunizedwith RAP-l were protected from lethal parasitaemia" and lym-phocytes from Ghanaian children responded by proliferation toRAP_].31 In a study on human volunteers, nearly all possessedantibodies to RAP-I following infection."

Apical membrane antigen-I (AMA-l)AMA-JIPF83 is an 83 kDa protein antigen that is seen at the apicalend of merozoites. It distributes across the surface of the freemerozoites before it invades erythrocytes.Y" AMA-l is rela-tively conserved between different strains of P.Jalciparum.

AMA-J of P. knowlesi (PK66) is a 66 kDa protein. Antibodyto PK66 prevents invasion of Rhesus monkey erythrocytes byP. knowlesi and protects against a live challenge." AMA-J ofP.Jragile has been expressed in Bacuolo viruses. Squirrel mon-keys, when infected by these viruses were completely protectedagainst a P.Jalciparum challenge, suggesting inter-species simi-larity in AMA-J. 51Naturally occurring antibodies to this antigenhave been demonstrated in areas of high malaria transmission. 52

Histidine-rich proteins (HRP)Three types of histidine-rich proteins-HRP-l, HRP-2, HRP-3are produced by P. Jalciparum-infected erythrocytes."

THE NATIONALMEDICALJOURNALOF INDIA VOL.9, NO.3, 1996

HRP-2. It is a water-soluble protein, with molecular weightbetween 60-150 kDa, secreted into the plasma by most erythro-cytic stages of the parasite even as early as 6-8 hours after ringdevelopment. The period of maximum release is during schizontrupture.r' It has an extended repeat region of tripeptide units Ala-His-His and Ala-Ala-Asp. The histidine content in HRP-2 is 34%.It is seen in both knob-positive and negative strains. HRP-2 hasbeen demonstrated to occur naturally in the plasma of infectedindividuals and is a potential target for the immune system."

As stated earlier, Aotus monkeys immunized with two recom-binant proteins containing HRP-2 had a high antibody level andlow parasitaernia."

HRP-l and 3. HRP-I is a knob-associated histidine-rich pro-tein (KAHRP), with molecular weight between 80 and 115 kDalocated in the sub-membrane electron-dense materials. HRP-3has a molecular weight of 40-55 kDa, with a histidine content of28%.5) There is a high degree of sequence homology betweenHRP-2 and 3. which points to an evolution from a common gene.

TRANSMISSION BLOCKING ANTIGENSThese antigens are seen in the sexual stages of the malarialparasite. Antibody to these antigens prevent either ferti lization, ormaturation of gametocytes. zygotes or ookinetes. Also. bloodfrom non-exposed individuals has a high frequency of gametereactive T cells which can be stimulated by intracellular gameto-cytes to proliferate and secrete IFN_y.55

Transmission blocking vaccines will prevent the infection ofmosquitoes. Since the antigen is not exposed in the parasite stagesthat affect man. it would not come under selective immunepressure and thus the parasite may not develop resistance to thisvaccine. For the same reason, the immune response would not benaturally boosted. These vaccines will be of use only in endemicareas and are not for travellers.

PJs-25Pfs-25 is a protein antigen of molecular weight 25 kDa, seen onthe surface of mature gametocytes, zygotes and ookinetes.v' It isprobably useful for the penetration of the stomach wall of themosquito by the ookinete. It has 2 epitopes, Region-I, a relativelyconserved region which is the target of transmission blockingantibodies, and Region-II, a well conserved region, not associatedwith protection. 57 Monoclonal antibody to Pfs-25 interferes withthe development of the ookinete in the mosquito."

Mice inoculated with transfected vaccinia virus producepolyclonal antibody capable of inhibiting the sexual stage devel-opment." An analog of Pfs-25 synthesized from yeast reacts withmonoclonal antibodies to Pfs-25 and also elicits transmissionblocking antibodies in monkeys and mice."

The other transmission blocking antigens described are a48/45 kDa doublet, seen on gametocytes and macrogametes, a230 kDa protein seen on freshly emerged macrogametes, alwaysassociated with the 48/45 kDa doublet and a 45/10 kDa antigenseen on gametocytes. Monoclonal antibodies to the 48/45 kDaantigen can also prevent the development of oocysts."

COMBINED VACCINESThese vaccines may ultimately be the solution for malaria control.As different stages of the parasite have specific antigens and as noimmunogenic antigen common to all the stages has been found, avaccine against one stage is not useful against another. Hence theconcept of combined vaccines is based on the incorporation ofantigens from different stages into one vaccine, to produce an

RAMAUNGAM, MATHAI: VACCINES AGAINST FALCIPARUM MALARIA

immune response, blocking all stages of parasite development.The SPf66 vaccine is a promising option at hand.

SPf66 vaccineSPf66, a synthetic molecule, was first described by a Colombiangroup led by Dr Manuel Patarroyo in 1988.lb It has 3 merozoitepeptides linked by a sporozoite peptide, the NANP repeat. Two ofthe merozoite peptides of molecular weight 55 kDa and 35 kDaare not well characterized, while the third peptide of molecularweight 83 kDa is a part of MSP-I."" The NANP repeat facilitatesthe polymerization of these antigens into a p pleated sheet. 16Thisvaccine is available. solubilized in sterile saline. and adsorbed toaluminium hydroxide. It is undergoing field trials. mostly in dosesof I mg for children below 5 years, and 2 mg for children above5 years and adults. on days O. 30 and 180.bl

SPf66 has proved to be a safe and immunogenic vaccine. Ahigh level of previous exposure does not enhance susceptibility toallergic reactions.v-" The reported seroconversion rates rangefrom 33- 100% in different studies.b1.64MChildren living in areaswith perennial transmission in Africa show a substantial antibodyresponse to 3 doses without major side-effects. A booster effectwas seen after the third dose with a clear dose-response effect."Anti-SPf66 antibody levels declined to baseline levels within1year after the third dose in most trials.This is probably related tothe half-life of immunoglobulins and lack of natural boosting. Asignificant protection at 12 months reported in Colombia may bedue to natural boosting."

Natural anti-SPf66 IgG antibody with titres more than 1:100using lFA has been reported from different areas, the prevalenceranging from 3.3% to greater than 80%.b2.116 There is controversyregarding the effect of prevaccination antibody levels on immuneresponse to SPf66 vaccination.v-? A lower immune response toSPf66 has been reported among HLA DR4 antigen carriers."

Controversy exists regarding the protective efficacy of thisvaccine. In a group of soldiers the protective efficacy againstnatural infection was estimated to be 60-80%,67 while in a largerfield trial at La Tola, Colombia. the protective efficacy was only39%.64In another trial at Ecuador, a protective efficacy of 67%with a wide confidence interval (CI) was reported. The 95% CIranges from -3% to 89%."1Recent studies in Tanzania showed aprotective efficacy of 31% in children 1-5 years old, while in astudy held in The Gambia the protective efficacy was only 8% ininfants 6-11 months old against the first episode of clinicalmalaria.68.6~Cross-protection to P. vivax has been reported fromVenezuela." Studies on larger populations are being conducted inTanzania, Thailand. Colombia and The Gambia.

CONCLUSIONThe 'ideal' malaria vaccine would be one that prevents infection,decreases the level of infection if the first step fails, and preventsmalaria transmission. Considerable progress has been made in thepast few decades in the pursuit of such a vaccine. Many antigenshave been characterized and tested on animals and a few onhumans, with varying results. The antigens studied are stage- andspecies-specific and provide immunity for about a year. However,no vaccine has as yet provided total protective immunity. andsome potential vaccine candidates have been disregarded as beingimpractical.

Much work remains to be done. The process of identifyingantigens must continue along with animal and human experi-ments. As one antigen alone is unable to provide good protection.a mixture of antigens synthetically put together may be the

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solution. The addition of transmission blocking antigens will helpstop the spread of the disease. Some of the antigens that we knownow may be more effective if they are presented to the body in adifferent form. by the addition of a suitable adjuvant. or bychanging the route of administration of the antigen.

The World Health Organization (WHO) in its recent publica-tions has given special mention to SPf66. MSA-I. AMA, SERA,EBA, and Pfs-25. as deserving the most urgent development."The antigens listed are all immunogenic. and no single antigen hasproved to be superior thus far.

SPf66 is under Phase III trials and the results should soon beavailable. Plans for Phase I and II trials of MSA-l and Pfs-25 areunderway. Field trials for AMA-l, SERA and EBA-175 may startin a few years."

There is hope that a day will soon come when antimalarialchemoprophylaxis is a thing of the past.

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