Abstract:
The present invention provides an antigenically restricted subset of the highly variant PfEMP1 rosetting antigen which possess epitopes which may be exploited to raise immune responses effective against many diverse strains and isolates of the malaria parasite,  Plasmodium falciparum . In this regard, the invention provides one or more  P. falciparum  Erythrocyte Membrane Protein-1 (PfEMP1) antigen(s) or a fragment or fragments thereof, for use in raising immune responses in humans.

Description:
FIELD OF THE INVENTION 
       [0001]    This invention provides antigens which may be exploited to raise immune responses in humans. In particular, the invention provides antigens which raise immune responses which exhibit cross-reactivity to a variety of  Plasmodium falciparum  strains and which may be exploited in the treatment and/or prevention of malaria and in particular severe malaria. 
       BACKGROUND OF THE INVENTION 
       [0002]    Sequence diversity in pathogen antigens is an obstacle to development of interventions against many infectious diseases. In malaria caused by  Plasmodium falciparum , the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. 
         [0003]    Every  P. falciparum  isolate has 50-60 diverse PfEMP1 variants, and the PfEMP1 repertoires of different isolates are largely non-overlapping [3,4,5,6]. PfEMP1 variants are expressed in a mutually exclusive fashion, and transcriptional switching from one var gene to another results in antigenic variation of  P. falciparum  infected erythrocytes [7]. PfEMP1 variants sampled from broad global parasite populations show essentially unlimited sequence diversity [5,8], making PfEMP1 an extremely challenging therapeutic target [9](see also: Pierce S K, Miller L H. J Immunol 2009; 182:5171-7). 
         [0004]    Surface-reactive antibodies to PfEMP1 on live infected erythrocytes that occur after natural infections [10,11] or after immunization with recombinant PfEMP1 domains [11,12] are predominantly variant-specific, as expected for highly polymorphic parasite antigens. However, children living in endemic areas develop antibodies during the first few years of life that protect against life-threatening malaria [13] suggesting that strain-transcending antibody responses may occur, or that the parasites that cause severe malaria are of restricted antigenic types [14,15]. Antigenically-restricted subsets of parasite surface antigens that induce strain-transcending antibodies have not yet been identified. 
         [0005]    In addition to their role in immunity and immune evasion, PfEMP1 variants are adhesion proteins that mediate interactions with a variety of human cell types and surface receptors [16,17]. Three major PfEMP1 families (A, B and C, based on conserved upstream sequence and genomic location) differ in their adhesive function [16]. Group B and C variants (approximately 40-50 variants per haploid parasite genome) bind to the endothelial protein and scavenger receptor CD36 [18,19]. In contrast, Group A variants (approximately 10 variants per haploid parasite genome) do not bind CD36 [18,19] but do mediate rosetting [11,12,20,21], an adhesion phenotype in which infected erythrocytes bind to uninfected erythrocytes [22]. Transcription of Group A var genes is linked to severe malaria in a variety of geographical settings [23,24,25,26] and laboratory experiments [27], whereas transcription of B and C var genes occurs in less virulent infections causing uncomplicated disease [23,24,25,26]. 
         [0006]    Rosetting is an important parasite virulence factor, associated with life-threatening malaria in African children [28,29,30,31,32] and high parasite burden in a primate malaria model [33]. Rosetting causes pathological obstruction to microvascular blood flow [34] and human erythrocyte polymorphisms that reduce the ability of  P. falciparum  to form rosettes confer substantial protection against severe malaria [35,36].  P. falciparum  rosetting parasites can be divided into two distinct phenotypes: those that bind IgM natural antibodies (“non-immune” IgM) from normal human serum [37,38] and those that do not. Non-immune IgM binding is thought to strengthen the adhesion interactions between infected and uninfected erythrocytes in rosettes [37,39,40] and may also play a role in immune evasion by masking key epitopes [41]. Previous studies on PfEMP1 and rosetting have focussed on parasites with the non-IgM binding phenotype [11,12,20,21,42]. Detailed examination of IgM binding rosetting parasites has been neglected to date, despite the clinical importance of this phenotype, as rosetting parasites from clinically ill children are predominantly of the IgM binding type [38]. 
       SUMMARY OF THE INVENTION 
       [0007]    The present invention is based on the discovery of an antigenically restricted subset of the highly variant PfEMP1 rosetting antigen. Moreover, the inventors have discovered that despite substantial sequence diversity, PfEMP1 variants possess epitopes which can be exploited to raise immune responses effective against many diverse strains and isolates of the malaria parasite,  Plasmodium falciparum.    
         [0008]    In a first aspect, the present invention provides one or more  P. falciparum  Erythrocyte Membrane Protein-1 (PfEMP1) antigen(s) or a fragment or fragments thereof, for use in raising immune responses in humans. 
         [0009]    In a second aspect, the invention provides use of one or more  P. falciparum  Erythrocyte Membrane Protein-1 (PfEMP1) antigen(s) or a fragment or fragments thereof for the manufacture of a medicament for raising immune responses in humans. 
         [0010]    In a further aspect, the invention provides a method of raising an immune response in a human, the method comprising the step of administering an immunogenic amount of one or more  P. falciparum  Erythrocyte Membrane Protein-1 (PfEMP1) antigen(s) or a fragment or fragments thereof to a human subject. 
         [0011]    In one embodiment, the immune response raised in the human subject comprises a systemic antibody response, comprising one or more antibody isotypes. By way of example, the immune response raised in the human may comprise IgM, IgG, IgA, IgD and/or IgE antibody isotypes. 
         [0012]    Advantageously, the immune responses described herein are cross reactive; that is to say, the immune response comprises antibodies which exhibit a degree of affinity, selectivity and/or specificity not only to the specific PfEMP1 antigen(s) exploited by this invention, but also to other PfEMP1 variants. Given the comparable lack of sequence identity/homology between PfEMP1 antigens, the ability to raise cross reactive antibody responses from only a limited selection of PfEMP1 antigens is both advantageous and surprising. 
         [0013]    The immune responses described herein may further assist in alleviating, reducing and/or eliminating the symptoms of malaria/severe malaria. Additionally or alternatively, the immune response raised in the human subject may reduce the parasite ( Plasmodium  sp.) burden and/or clear or eliminate the parasite from the host. One of skill will appreciate that immune responses of this type may be known as “protective immune responses”. 
         [0014]    As such, the antigens, uses and/or methods described herein may be exploited to raise cross-reactive and/or protective antibodies in human hosts, which antibodies exhibit a degree of affinity, selectivity and/or specificity for the PfEMP1 antigen of  P. falciparum.    
         [0015]    In one embodiment, the PfEMP1 antigens to be exploited by this invention are IgM rosetting variants. One of skill in this field will appreciate that IgM rosetting PfEMP1 variants are characterised by their ability to bind IgM antibodies from human serum. As such, in a yet further embodiment, the invention provides PfEMP1 IgM rosetting variants (or a fragment(s) thereof) for use in raising immune responses in humans; use of PfEMP1 IgM rosetting variants (or a fragment(s) thereof) for the manufacture of medicaments for raising immune responses in humans; and methods of raising immune responses in humans, the methods comprising the step of administering an immunogenic amount of one or more PfEMP1 IgM rosetting variants or a fragment or fragments thereof, to a human subject. 
         [0016]    In one embodiment, the PfEMP1 antigens of this invention represent specific PfEMP1 variants. In this regard, the inventors have discovered that the (IgM rosetting) PfEMP1 variants designated HB3var6, TM284var1 and ITvar60 comprise epitopes capable of inducing protective and/or cross reactive immune responses. 
         [0017]    Accordingly, one embodiment of this invention provides one or more PfEMP1 antigen(s) or a fragment(s) thereof, for use in raising an immune response in a human subject, wherein the PfEMP1 antigen is selected from the group consisting of:
       (i) HB3var6   (ii) TM284var1; and   (iii) ITvar60.       
 
         [0021]    In addition, it should be understood that the various medicaments and methods described in this invention may also exploit the specific PfEMP1 variants (or fragment(s) thereof) listed as (i)-(iii) above. 
         [0022]    One of skill will appreciate that the HB3var6 PfEMP1 antigen may be obtained from the  P. falciparum  strain HB3. Similarly the TM284var1 PfEMP1 antigen and the ITvar60 PfEMP1 antigen may be obtained from  P. falciparum  strains TM284 and IT/PAR+ respectively. 
         [0023]    The PfEMP1 variant designated HB3var6 has the following amino acid sequence: 
         [0000]    
       
         
               
             
           
               
                 SEQ ID NO: 1 
               
               
                 MGNTIPKPPDPIYINESYQSTRNVLERYAESIKQQAAADAEKCEKSLKGD 
               
               
                   
               
               
                 LTKAEFRGAHIETVGVQKYSYSNPCGLNHTWNTNLLHDRVKDRDPCHGRN 
               
               
                   
               
               
                 QKRFDEGQVYECGSGIIKGNGNNRNGGSYAPPRRRHICDKNLEALTVQNT 
               
               
                   
               
               
                 KNSNDLLGNILVTAKYEGESIVNSYANSGMFNVCTALARSFADIGDIVRG 
               
               
                   
               
               
                 KDLYSGNKQEKEKRKQLEKNLQKIFRNIYDKLLEYNKTNGEIEARYGSDK 
               
               
                   
               
               
                 ENFFQLREDWWKANRDQVWRAITCKAPQDANYFRKISGDTKVFTSQGQCG 
               
               
                   
               
               
                 HSETNVPTNLDYVPQFLRWFDEWAEEFCRVREHKLKKIKEACRGKNDEKD 
               
               
                   
               
               
                 CSREGYDCNKTNLRLNEIFMDLECPNCEKACTSYKEWIENKQKEFNKQKK 
               
               
                   
               
               
                 KYEKEIENDESNSHSTYDNELYNNLKRNYPSFENFVETLKEGAYCTNGII 
               
               
                   
               
               
                 EGKIDFNKQYDTFSHSQYCKSCPILGAKCKNGQCNSFNDINCTKIPTMTN 
               
               
                   
               
               
                 IRIHSTESPKDIYILVNDKKNREHSLELKDAFNDCDIFKRIRKQKWYCKY 
               
               
                   
               
               
                 KCNLDVCELKNFNRDMDDERLISIEVLIKRWLKYFLNDYNQIKENLNQCI 
               
               
                   
               
               
                 NNGTNTLCINDCHKNCECIEKWIKEKEKEWKVIKDRYVEQYNNNDKDVSS 
               
               
                   
               
               
                 KLKTFLKQDLFTNYVKNALDPDETLDKMKESSVCNVPNKLNGTSCKKKDV 
               
               
                   
               
               
                 INILLNRLNEKIDPCKNQHKATKGKECCDKLPKIADGDTSDDEDDDEEDV 
               
               
                   
               
               
                 SVTSGEKQNVKQDCAGKKSDEVCEMVKKLIGDNNGMSGRIESCNPKTGTY 
               
               
                   
               
               
                 PPWKNHASLVEDNRTWMPPRRQKLCVSALTQEGKIKNKEDIRKHFITCAA 
               
               
                   
               
               
                 IETHFAWHRYKNHNANAESKLKTGKIPDDFLRSMKYTFGDYRDIFFGTDI 
               
               
                   
               
               
                 SSCDKIKNASNTIKSILENKTKKKKGDKLIEDNEKHKEWWNEHGKEIWEG 
               
               
                   
               
               
                 MLCALEKVGGSVNIKSTYNYDTIKNDLEDFASRPQFLRWFTEWSDEFCQE 
               
               
                   
               
               
                 RKKLEAKVKEYCKKDYVGCNKQNTKANNSCVSACEAFQHYMKSKMSEYDT 
               
               
                   
               
               
                 QKKKFEAEKSGKEPEYEGFSTKDASEYLKEKCLHGTCDCMEKVKNIDDYW 
               
               
                   
               
               
                 KNPHKTYDDNKLETKCECPQTPPKPCEIVKTLLEDNNGRHVDACNLKYEG 
               
               
                   
               
               
                 KKEKHTSWNCNPNKFKNGEEGACIPPRRQKLYIYNLEKFTGGTSEIELRK 
               
               
                   
               
               
                 AFIECAAIETFFSWHKFKKDKEREDKEKQDLVGYTSTVDEKHQKDLQSGK 
               
               
                   
               
               
                 IPEEFKRQMFYTFGDYRDICLGNDMGNDNYNKNISTKVRSILNSGETPEE 
               
               
                   
               
               
                 WWQKHGPQIWEGMLCALSYDTEKQKKVQDVHNNLIAPPNNNKYNDVKLVS 
               
               
                   
               
               
                 KSGKLHTSLSDFATVPQFLRWEEENVEEFCRKKKIKIDKIEDECRGEYDN 
               
               
                   
               
               
                 GGKKYCSGDGYDCDKRYLSHNKMFADLNCLGCEKECRNYKKWIEEKVEEF 
               
               
                   
               
               
                 YKQKKKYEKGFENTRTNLDNKYVKEFYETSAGKYKSVDLFLDTLKERSHC 
               
               
                   
               
               
                 SMGMVNRKIDEKNPLETFSPSIYCKTCPLYGVNCNSRECVDITENEFKKK 
               
               
                   
               
               
                 NVLDEIIINDKSHTSIDIEMIDRRGQYMQENLDNPLFKESYLLKSVRDQK 
               
               
                   
               
               
                 WDCNFIHNKIDLCEINKFNENIDTDESITFKVLIERWLQDFLEGYYISKK 
               
               
                   
               
               
                 QIDLFTKKEENKCECVKKWAEKKEGEWEKINEHFNKQKHDDAFDMDFKVK 
               
               
                   
               
               
                 NYFEKNASDLKDWIDNFKRLNNIDDYQVCNVHNNCKSADKKNKIDMVSIL 
               
               
                   
               
               
                 LSELKKEIETCKNQGNEKTKIKCDASPTNDELDEEYELGTTDTSPSAAPD 
               
               
                   
               
               
                 ICKDVIQSKSEETICRDDKRVDCNKVGKDDPIKVPMDPKSGEDHLNEMGD 
               
               
                   
               
               
                 KHNCSGIIIKTNGEWKNTKQLNYPNPCESIYASPRRQKFCVHELDKAKNQ 
               
               
                   
               
               
                 KELRTKLLTVAANQGYNLAIKHHEYKDKYTVNPCNALKYSFYDYQHIILG 
               
               
                   
               
               
                 DDPMEPEKWDTESALKRIFGNRNTEDAKPLSRKRKDFWKENKECVWSAMK 
               
               
                   
               
               
                 CGYNEGIKKGNKSNNIPECKDSIPTQFDGVPQFLMWFTEWSEDFCNHKKT 
               
               
                   
               
               
                 HLKKLEQGCRGCTLRIDGTCEKDGSGCQKCSQACEEYKAWLQNWKDQYKK 
               
               
                   
               
               
                 QSKKYSGDKKKELYKTVPKVKNSTHAYEYLQTQLEKLCEKGKCDYTCMKN 
               
               
                   
               
               
                 PSTENSTENMPESLDVKPDIVKDKCPCPPQKIEKPDSTLNCIDRSAFELY 
               
               
                   
               
               
                 AKAKSDLHGVKDKLKGNNTKNIYEETTNGKNDDNIICKINESISKQNNVC 
               
               
                   
               
               
                 KKNENLFDDIDKWDCKKRTNTVPIENICIPPRRKLMCAYPLKNLGVKKNT 
               
               
                   
               
               
                 SEVLFNKVLRTAAYEGKHIKESWEKAEKSKKKKTQICDAMKYSFADLGDI 
               
               
                   
               
               
                 IRGRDILIFNNGNNEIERDLKAVFQSIYDKWKSDSNNNKDKYPDLTSFRS 
               
               
                   
               
               
                 AWWDANRKDIWKAMTCGAPEDATLFKKLEKWGIPNLILSQHKCGHNDDPP 
               
               
                   
               
               
                 IDDYIPQRLRWMKEWGEYVCKILNENVNDMKNDCDKCTLNDKKCSDEDDG 
               
               
                   
               
               
                 NKCRSCKEKCKEYTKLIYNLKSQFYILEKHYNELYTKAQNNTTYFTNDND 
               
               
                   
               
               
                 KKVIEFFKKVKKDCDVGTPDKYLDKAIHCIHYDFTKNGTKSKPYVFNNQP 
               
               
                   
               
               
                 EKYKNLCSCTITNHPLDKCPLPDKTDDYCKIIRHINPCITINLDNNLDTW 
               
               
                   
               
               
                 TGFVVHNISHKNKGVLVPPRRRHLCTRELTGIRYRKNDKDNLKQNLIDSA 
               
               
                   
               
               
                 FNQGILLGKTFKDYSDQGLEYMKYSFADYGDIIKAKDMIGGSNIDDFNND 
               
               
                   
               
               
                 LKKMFPEHHSENMGKTTISREQWWEANKTHVWHAMLCGYHQGIINPNLSR 
               
               
                   
               
               
                 RRPKPLEEGTQSSIATKTIPSNWCQLPNDYSTDQFLRWFQEWIENFCTRK 
               
               
                   
               
               
                 KVLEKEAQEQCKNITCNNDTGKTNTKCTEACKNYSNFILIKKKEYESLNS 
               
               
                   
               
               
                 QYDMNYKKIVEHKNALEYFKDKCKNNCECLSKHIDNGKNWKEPYETIDDS 
               
               
                   
               
               
                 ELIGKCKCKKVKPKTPDVIPAGATETKEKDTPHAPEKPQQPPQPLPPSDE 
               
               
                   
               
               
                 PFDPTILQTTIPFGIALALGSIAFLFMKKKPKSPVDLLRVLNIPKRDYEM 
               
               
                   
               
               
                 PTLKSKNRYIPYASDRYKGKTYIYMEGDSSGDEKYAFMSDTTDVTSSESE 
               
               
                   
               
               
                 YEELDINDIYVPGSPKYKTLIEVVLEPSKSNGNTLGDDMVPTTNTFTDEE 
               
               
                   
               
               
                 WNELKHDFISQYVQREPLDVPQYDESTQLPMNIVGNVLDDGMDEKPFITS 
               
               
                   
               
               
                 IHDRDLYTGEEISYNINMSTNSMDDPKYVSNNVYSGIDLINDTLSGDRID 
               
               
                   
               
               
                 IYDELLKRKENELFGTNHVKQTSIHSVAKLTNSDPIHNQLDLFHTWLDRH 
               
               
                   
               
               
                 RDMCNTWNTKEELLDKLNEQWNKDNDGGDIPNDNKKLNTDVSFEIDMDET 
               
               
                   
               
               
                 KGKKEFSNMDTILDDMEDDIYYDVNDENPSVDNIPMDHNKVDVPKKVHVE 
               
               
                   
               
               
                 MKILNNTSNGSLEQEFPISDVWNI 
               
             
          
         
       
     
         [0024]    Moreover, the HB3var6 PfEMP1 variant is encoded by the following nucleic acid sequence: 
         [0000]    
       
         
               
             
           
               
                 SEQ ID NO: 2 
               
               
                 ATGGGGAATACAATACCAAAGCCTCCGGATCCAATTTATATAAATGAAAG 
               
               
                   
               
               
                 TTATCAAAGTACCAGAAATGTTTTGGAACGTTATGCCGAAAGTATAAAGC 
               
               
                   
               
               
                 AACAGGCAGCTGCTGATGCAGAAAAATGTGAGAAATCGTTGAAAGGAGAT 
               
               
                   
               
               
                 TTGACAAAAGCAGAATTTCGTGGTGCTCATATAGAGACAGTTGGTGTCCA 
               
               
                   
               
               
                 GAAATATAGTTATTCTAATCCATGTGGTTTGAATCATACATGGAATACTA 
               
               
                   
               
               
                 ATTTATTGCATGATAGAGTGAAGGATAGAGATCCGTGCCATGGTAGAAAT 
               
               
                   
               
               
                 CAAAAACGTTTTGATGAAGGTCAAGTATATGAATGTGGTAGTGGTATAAT 
               
               
                   
               
               
                 CAAAGGTAATGGAAATAACAGAAATGGGGGATCCTATGCGCCACCCAGAA 
               
               
                   
               
               
                 GAAGACATATATGTGATAAAAACTTGGAAGCTCTAACTGTGCAAAATACA 
               
               
                   
               
               
                 AAAAATTCTAACGACTTGTTAGGAAATATCTTGGTCACAGCAAAATATGA 
               
               
                   
               
               
                 AGGCGAATCTATTGTGAATAGTTATGCAAATAGCGGAATGTTCAATGTAT 
               
               
                   
               
               
                 GTACTGCACTTGCACGAAGTTTTGCAGATATAGGTGATATCGTACGCGGC 
               
               
                   
               
               
                 AAAGATCTATATAGTGGTAATAAACAAGAAAAAGAAAAAAGAAAACAATT 
               
               
                   
               
               
                 AGAGAAGAATTTACAAAAAATTTTTAGGAATATATATGACAAATTATTAG 
               
               
                   
               
               
                 AGTATAACAAGACGAATGGGGAGATAGAAGCTCGCTACGGAAGTGATAAA 
               
               
                   
               
               
                 GAAAATTTTTTTCAATTAAGAGAAGATTGGTGGAAAGCGAACAGAGATCA 
               
               
                   
               
               
                 AGTATGGAGAGCTATAACATGTAAAGCTCCACAAGACGCTAATTATTTTA 
               
               
                   
               
               
                 GAAAAATTTCAGGAGATACTAAGGTGTTTACAAGTCAAGGACAATGTGGG 
               
               
                   
               
               
                 CATAGTGAAACAAACGTTCCTACGAATCTAGATTATGTCCCTCAATTTTT 
               
               
                   
               
               
                 ACGATGGTTTGATGAATGGGCAGAAGAGTTTTGCAGAGTAAGAGAACATA 
               
               
                   
               
               
                 AGTTGAAAAAGATTAAAGAAGCGTGTCGTGGGAAAAATGATGAAAAAGAT 
               
               
                   
               
               
                 TGTAGTCGTGAGGGTTACGATTGTAATAAAACAAATCTAAGACTTAATGA 
               
               
                   
               
               
                 AATTTTTATGGATTTAGAATGTCCAAATTGTGAAAAAGCGTGTACAAGTT 
               
               
                   
               
               
                 ATAAGGAATGGATAGAAAATAAACAAAAGGAATTTAATAAACAAAAGAAA 
               
               
                   
               
               
                 AAATACGAAAAGGAAATAGAAAATGATGAAAGTAATTCTCATAGTACATA 
               
               
                   
               
               
                 TGATAACGAATTATATAATAATCTGAAAAGAAATTATCCTTCCTTTGAAA 
               
               
                   
               
               
                 ATTTTGTAGAAACATTAAAAGAAGGAGCGTATTGTACTAATGGCATTATA 
               
               
                   
               
               
                 GAAGGTAAAATAGATTTTAATAAACAATATGACACTTTTTCCCATTCACA 
               
               
                   
               
               
                 GTACTGTAAATCATGTCCTATATTAGGTGCCAAATGCAAGAATGGACAAT 
               
               
                   
               
               
                 GCAATTCATTTAATGATATAAATTGTACAAAAATACCGACTATGACTAAT 
               
               
                   
               
               
                 ATAAGAATTCACAGCACTGAAAGTCCTAAGGATATTTATATTCTGGTGAA 
               
               
                   
               
               
                 TGATAAAAAAAATAGAGAACATTCTCTTGAATTAAAGGATGCTTTTAATG 
               
               
                   
               
               
                 ATTGTGATATTTTTAAAAGAATAAGAAAACAAAAATGGTATTGTAAATAT 
               
               
                   
               
               
                 AAATGTAACTTAGATGTATGTGAACTAAAAAATTTTAATAGGGATATGGA 
               
               
                   
               
               
                 TGATGAAAGATTAATTTCAATCGAAGTATTGATTAAACGTTGGTTAAAGT 
               
               
                   
               
               
                 ATTTTTTAAATGATTATAATCAAATAAAAGAAAATTTAAATCAATGTATA 
               
               
                   
               
               
                 AATAATGGAACAAATACATTATGTATAAACGATTGTCATAAAAATTGTGA 
               
               
                   
               
               
                 ATGCATAGAGAAATGGATAAAAGAAAAAGAGAAAGAGTGGAAAGTTATAA 
               
               
                   
               
               
                 AAGATCGTTATGTAGAACAATATAATAATAACGATAAAGACGTTTCTTCT 
               
               
                   
               
               
                 AAACTTAAAACTTTTTTGAAACAGGATTTGTTTACTAACTATGTTAAAAA 
               
               
                   
               
               
                 TGCCTTGGACCCCGATGAAACGTTAGATAAGATGAAAGAATCTAGTGTAT 
               
               
                   
               
               
                 GCAATGTACCTAATAAACTCAATGGAACATCATGTAAAAAAAAGGACGTG 
               
               
                   
               
               
                 ATAAATATTTTACTTAATAGACTTAATGAAAAAATAGATCCTTGCAAAAA 
               
               
                   
               
               
                 TCAACATAAAGCAACCAAAGGTAAAGAATGTTGTGATAAATTACCTAAAA 
               
               
                   
               
               
                 TTGCAGATGGTGATACATCAGACGATGAAGATGACGATGAAGAGGATGTT 
               
               
                   
               
               
                 TCTGTGACTTCTGGAGAAAAACAAAATGTAAAACAAGATTGTGCTGGTAA 
               
               
                   
               
               
                 GAAATCGGATGAGGTGTGCGAAATGGTGAAGAAACTTATTGGAGATAACA 
               
               
                   
               
               
                 ATGGAATGAGTGGTCGAATAGAGAGTTGTAATCCAAAAACTGGAACTTAT 
               
               
                   
               
               
                 CCTCCATGGAAAAACCACGCAAGTTTAGTGGAAGACAACAGAACGTGGAT 
               
               
                   
               
               
                 GCCTCCAAGAAGACAGAAATTATGCGTAAGTGCTTTAACACAAGAAGGTA 
               
               
                   
               
               
                 AAATAAAAAATAAAGAAGATATAAGAAAACATTTTATTACATGTGCGGCT 
               
               
                   
               
               
                 ATAGAAACACATTTTGCGTGGCATAGATATAAAAACCATAATGCGAATGC 
               
               
                   
               
               
                 TGAAAGCAAATTAAAAACTGGAAAAATTCCTGATGATTTTTTAAGATCCA 
               
               
                   
               
               
                 TGAAATATACTTTTGGTGATTATAGAGATATATTTTTTGGAACAGATATT 
               
               
                   
               
               
                 TCATCATGTGATAAAATTAAAAATGCCTCAAATACTATAAAATCTATATT 
               
               
                   
               
               
                 AGAAAATAAAACAAAGAAGAAAAAAGGAGATAAACTCATTGAAGATAATG 
               
               
                   
               
               
                 AAAAACACAAAGAGTGGTGGAATGAACATGGGAAGGAGATATGGGAAGGA 
               
               
                   
               
               
                 ATGTTATGTGCACTAGAAAAAGTTGGAGGAAGCGTCAATATCAAATCCAC 
               
               
                   
               
               
                 GTACAACTACGATACTATAAAAAATGATCTAGAAGACTTTGCATCTAGGC 
               
               
                   
               
               
                 CACAATTTTTACGGTGGTTCACCGAATGGAGTGATGAATTTTGTCAGGAA 
               
               
                   
               
               
                 CGGAAGAAATTGGAGGCAAAGGTTAAAGAATATTGTAAGAAGGATTATGT 
               
               
                   
               
               
                 CGGATGTAATAAACAAAACACGAAGGCTAATAATAGTTGTGTTAGCGCTT 
               
               
                   
               
               
                 GTGAAGCATTTCAACATTACATGAAGTCCAAAATGTCAGAATACGATACA 
               
               
                   
               
               
                 CAAAAAAAAAAATTTGAGGCTGAAAAAAGCGGGAAGGAACCAGAATATGA 
               
               
                   
               
               
                 AGGTTTTTCAACTAAAGACGCTTCTGAATACTTAAAAGAAAAATGTTTGC 
               
               
                   
               
               
                 ATGGTACATGTGATTGTATGGAGAAAGTAAAAAACATTGATGATTATTGG 
               
               
                   
               
               
                 AAAAATCCTCATAAAACGTATGACGATAACAAACTTGAAACTAAATGTGA 
               
               
                   
               
               
                 GTGTCCTCAAACACCACCAAAACCATGTGAAATAGTAAAAACACTTTTGG 
               
               
                   
               
               
                 AAGATAACAATGGAAGACATGTAGATGCTTGCAATCTCAAATATGAGGGT 
               
               
                   
               
               
                 AAAAAAGAAAAACATACTTCATGGAATTGTAATCCAAATAAGTTTAAAAA 
               
               
                   
               
               
                 TGGAGAAGAAGGTGCCTGTATACCTCCGAGAAGACAAAAATTATACATAT 
               
               
                   
               
               
                 ATAATTTAGAGAAATTCACTGGTGGAACATCAGAAATTGAATTGAGAAAA 
               
               
                   
               
               
                 GCTTTTATTGAATGTGCTGCAATAGAAACGTTTTTTTCTTGGCATAAATT 
               
               
                   
               
               
                 TAAAAAGGATAAAGAAAGAGAGGATAAAGAAAAACAAGATCTAGTAGGAT 
               
               
                   
               
               
                 ATACATCAACCGTCGATGAAAAACATCAAAAGGATTTACAAAGTGGAAAA 
               
               
                   
               
               
                 ATTCCTGAAGAATTTAAACGTCAAATGTTCTATACATTTGGTGATTATAG 
               
               
                   
               
               
                 AGATATATGTTTAGGAAACGATATGGGTAATGATAACTATAATAAAAATA 
               
               
                   
               
               
                 TATCTACAAAAGTTAGGAGTATTTTAAATAGTGGGGAAACACCTGAAGAA 
               
               
                   
               
               
                 TGGTGGCAAAAACATGGACCTCAGATATGGGAAGGTATGTTATGTGCTTT 
               
               
                   
               
               
                 AAGTTACGATACCGAAAAACAAAAAAAGGTTCAAGATGTGCACAATAATC 
               
               
                   
               
               
                 TTATAGCCCCCCCAAACAACAACAAGTACAACGACGTAAAATTGGTTTCG 
               
               
                   
               
               
                 AAAAGTGGGAAACTTCATACCTCTTTATCCGATTTTGCAACTGTTCCGCA 
               
               
                   
               
               
                 GTTTTTAAGATGGTTCGAAGAATGGGTCGAAGAGTTTTGTAGAAAAAAAA 
               
               
                   
               
               
                 AAATTAAAATTGATAAAATTGAAGACGAATGTCGTGGAGAATATGATAAT 
               
               
                   
               
               
                 GGTGGTAAAAAATATTGTAGTGGTGATGGTTATGATTGTGACAAAAGATA 
               
               
                   
               
               
                 TTTATCCCATAATAAAATGTTTGCAGATTTAAATTGTCTAGGTTGTGAGA 
               
               
                   
               
               
                 AAGAATGTAGAAATTATAAAAAATGGATAGAAGAAAAAGTAGAAGAATTT 
               
               
                   
               
               
                 TATAAACAAAAAAAGAAATACGAAAAGGGGTTTGAGAACACACGAACTAA 
               
               
                   
               
               
                 CCTTGATAATAAATATGTTAAAGAATTTTATGAAACATCTGCTGGAAAAT 
               
               
                   
               
               
                 ATAAGTCTGTTGACTTATTTTTAGATACATTGAAAGAAAGATCTCATTGT 
               
               
                   
               
               
                 AGTATGGGTATGGTAAATAGAAAAATAGATTTTAAGAATCCGCTCGAAAC 
               
               
                   
               
               
                 ATTTTCCCCTTCAATATATTGTAAAACGTGCCCTTTATATGGAGTTAACT 
               
               
                   
               
               
                 GTAATTCGAGAGAATGTGTAGACATTACTGAAAATGAGTTTAAGAAAAAA 
               
               
                   
               
               
                 AATGTTTTAGATGAGATTATTATAAACGATAAGTCACATACAAGTATTGA 
               
               
                   
               
               
                 TATCGAAATGATTGATCGTAGGGGACAGTATATGCAAGAGAATTTAGACA 
               
               
                   
               
               
                 ATCCTTTGTTTAAAGAATCATATCTTTTAAAAAGTGTCAGAGATCAAAAA 
               
               
                   
               
               
                 TGGGATTGTAACTTTATTCATAATAAGATCGATTTATGTGAAATAAATAA 
               
               
                   
               
               
                 GTTTAATGAAAACATAGACACTGATGAAAGCATTACATTTAAGGTTTTGA 
               
               
                   
               
               
                 TAGAACGGTGGTTACAAGATTTCTTAGAAGGATATTATATATCAAAAAAA 
               
               
                   
               
               
                 CAAATCGATCTATTTACAAAAAAAGAAGAAAATAAATGTGAATGCGTGAA 
               
               
                   
               
               
                 GAAATGGGCAGAAAAAAAGGAAGGAGAATGGGAAAAAATAAACGAACATT 
               
               
                   
               
               
                 TTAATAAACAAAAACATGATGATGCATTTGATATGGATTTTAAAGTCAAA 
               
               
                   
               
               
                 AATTATTTTGAGAAAAATGCAAGTGATTTAAAGGATTGGATAGATAATTT 
               
               
                   
               
               
                 TAAACGTCTAAACAATATAGATGATTATCAGGTTTGCAATGTTCATAACA 
               
               
                   
               
               
                 ATTGTAAAAGTGCGGATAAAAAAAATAAAATAGATATGGTATCTATTTTA 
               
               
                   
               
               
                 CTTTCTGAGCTTAAAAAAGAAATAGAAACTTGTAAAAATCAAGGTAACGA 
               
               
                   
               
               
                 AAAAACAAAAATAAAATGTGATGCATCACCTACAAACGACGAACTAGATG 
               
               
                   
               
               
                 AAGAATACGAACTAGGCACAACGGACACATCTCCATCCGCAGCTCCGGAT 
               
               
                   
               
               
                 ATTTGTAAGGATGTGATCCAAAGTAAATCTGAAGAAACAATATGTAGAGA 
               
               
                   
               
               
                 TGATAAAAGGGTAGATTGTAACAAGGTGGGTAAAGATGATCCAATAAAGG 
               
               
                   
               
               
                 TCCCTATGGATCCAAAATCTGGTGAAGATCATCTCAACGAGATGGGAGAT 
               
               
                   
               
               
                 AAACATAATTGTAGTGGAATTATCATTAAAACAAATGGTGAATGGAAAAA 
               
               
                   
               
               
                 TACAAAACAATTAAATTACCCGAACCCATGTGAAAGCATATATGCTTCAC 
               
               
                   
               
               
                 CTCGAAGACAAAAATTCTGTGTACATGAACTTGACAAAGCAAAAAACCAA 
               
               
                   
               
               
                 AAGGAATTAAGAACTAAATTATTGACTGTTGCTGCAAATCAAGGATATAA 
               
               
                   
               
               
                 TCTAGCTATTAAACATCATGAATATAAAGACAAATATACTGTTAATCCTT 
               
               
                   
               
               
                 GTAATGCATTGAAATATAGTTTTTACGATTATCAGCATATAATTCTAGGA 
               
               
                   
               
               
                 GATGACCCGATGGAACCTGAAAAATGGGATACGGAAAGTGCATTGAAAAG 
               
               
                   
               
               
                 AATATTTGGAAATAGAAATACAGAAGATGCCAAACCTCTTAGTAGAAAAC 
               
               
                   
               
               
                 GTAAAGATTTTTGGAAAGAAAACAAAGAATGCGTTTGGTCAGCAATGAAA 
               
               
                   
               
               
                 TGTGGTTACAACGAAGGAATAAAAAAGGGTAATAAGAGTAATAATATTCC 
               
               
                   
               
               
                 AGAATGTAAGGACAGTATACCTACCCAATTTGATGGTGTTCCTCAATTTT 
               
               
                   
               
               
                 TGATGTGGTTTACTGAATGGAGTGAAGATTTTTGTAATCATAAGAAGACA 
               
               
                   
               
               
                 CATTTGAAAAAATTGGAGCAGGGGTGTAGGGGATGTACTCTTCGTATTGA 
               
               
                   
               
               
                 TGGCACATGTGAGAAAGATGGCTCAGGATGCCAAAAATGTTCACAAGCGT 
               
               
                   
               
               
                 GTGAAGAATATAAAGCATGGCTTCAAAATTGGAAAGACCAATATAAGAAA 
               
               
                   
               
               
                 CAAAGCAAAAAATATAGTGGTGATAAAAAAAAAGAGCTATATAAAACTGT 
               
               
                   
               
               
                 TCCTAAAGTAAAAAATTCAACACATGCCTATGAATATTTACAAACACAAT 
               
               
                   
               
               
                 TAGAAAAACTTTGTGAAAAAGGTAAATGTGATTATACTTGTATGAAAAAC 
               
               
                   
               
               
                 CCATCGACAGAAAATAGTACTGAAAATATGCCCGAATCATTGGACGTAAA 
               
               
                   
               
               
                 ACCCGATATAGTTAAGGATAAATGCCCTTGTCCACCACAGAAAATAGAAA 
               
               
                   
               
               
                 AACCCGATTCCACATTAAATTGCATAGATAGAAGTGCATTTGAATTATAT 
               
               
                   
               
               
                 GCAAAAGCAAAAAGTGATTTACATGGTGTAAAAGATAAATTAAAGGGTAA 
               
               
                   
               
               
                 TAATACAAAAAATATATACGAAGAAACAACTAATGGTAAAAATGATGATA 
               
               
                   
               
               
                 ATATTATCTGTAAAATTAATGAGAGTATTTCTAAACAAAACAATGTATGT 
               
               
                   
               
               
                 AAAAAAAATGAAAATCTTTTTGATGATATAGACAAATGGGACTGTAAAAA 
               
               
                   
               
               
                 ACGAACAAATACAGTGCCCATTGAAAATATATGTATTCCTCCAAGAAGGA 
               
               
                   
               
               
                 AACTTATGTGTGCATATCCATTAAAAAATTTAGGAGTAAAAAAAAATACT 
               
               
                   
               
               
                 TCAGAAGTATTGTTCAACAAAGTATTGCGTACAGCAGCATATGAAGGAAA 
               
               
                   
               
               
                 ACATATAAAGGAATCATGGGAAAAAGCAGAAAAATCCAAGAAAAAAAAAA 
               
               
                   
               
               
                 CCCAAATATGTGATGCTATGAAATACAGTTTTGCAGATTTAGGAGATATA 
               
               
                   
               
               
                 ATTAGAGGAAGAGATATATTGATATTTAATAATGGTAATAATGAAATTGA 
               
               
                   
               
               
                 GAGAGACTTAAAAGCTGTTTTTCAGTCAATATACGATAAATGGAAATCTG 
               
               
                   
               
               
                 ACAGTAATAATAATAAAGATAAATACCCCGACTTAACCTCTTTTCGTTCT 
               
               
                   
               
               
                 GCCTGGTGGGATGCTAATAGAAAAGATATTTGGAAAGCTATGACATGTGG 
               
               
                   
               
               
                 TGCACCGGAAGATGCTACGCTTTTTAAAAAACTAGAAAAATGGGGAATTC 
               
               
                   
               
               
                 CTAATTTAATTTTGTCACAACATAAATGTGGGCATAATGACGATCCTCCT 
               
               
                   
               
               
                 ATTGATGATTACATACCTCAACGGTTAAGATGGATGAAGGAATGGGGAGA 
               
               
                   
               
               
                 ATATGTTTGCAAAATATTAAATGAAAACGTGAATGATATGAAGAACGATT 
               
               
                   
               
               
                 GTGATAAATGTACACTAAATGATAAAAAATGTTCAGATGAAGATGATGGT 
               
               
                   
               
               
                 AATAAATGTAGAAGTTGTAAAGAAAAATGTAAAGAATATACTAAACTTAT 
               
               
                   
               
               
                 ATACAATCTGAAATCACAATTTTATATACTAGAAAAACATTATAACGAAT 
               
               
                   
               
               
                 TATATACAAAAGCACAAAATAATACAACATATTTTACAAATGATAACGAT 
               
               
                   
               
               
                 AAAAAGGTTATTGAATTTTTTAAAAAAGTTAAAAAGGATTGTGATGTGGG 
               
               
                   
               
               
                 AACTCCTGATAAATATCTCGATAAAGCTATTCATTGTATCCATTATGATT 
               
               
                   
               
               
                 TTACTAAAAATGGAACCAAATCTAAGCCATATGTCTTCAACAATCAACCA 
               
               
                   
               
               
                 GAAAAGTATAAAAATCTTTGTAGTTGTACTATTACTAATCATCCGTTAGA 
               
               
                   
               
               
                 CAAATGTCCTTTACCTGATAAAACAGATGATTATTGCAAAATCATTAGAC 
               
               
                   
               
               
                 ATATTAATCCGTGTATAACAATAAATTTGGATAATAATTTGGATACGTGG 
               
               
                   
               
               
                 ACTGGATTTGTTGTGCATAATATAAGTCACAAAAATAAAGGTGTGCTTGT 
               
               
                   
               
               
                 ACCTCCAAGAAGAAGACATTTATGTACAAGAGAATTAACTGGAATTAGAT 
               
               
                   
               
               
                 ATCGTAAAAATGATAAAGATAATTTGAAACAAAATCTTATTGATTCTGCT 
               
               
                   
               
               
                 TTTAATCAAGGAATACTTTTAGGAAAAACATTTAAAGATTACAGCGATCA 
               
               
                   
               
               
                 AGGTTTGGAATATATGAAATATAGTTTTGCTGATTATGGAGATATAATTA 
               
               
                   
               
               
                 AAGCTAAAGATATGATAGGAGGTTCAAATATTGATGATTTCAATAATGAT 
               
               
                   
               
               
                 TTAAAAAAAATGTTTCCAGAACATCATAGTGAGAATATGGGAAAAACTAC 
               
               
                   
               
               
                 TATTAGTCGTGAACAGTGGTGGGAAGCAAATAAAACACACGTATGGCACG 
               
               
                   
               
               
                 CTATGTTATGCGGGTATCATCAAGGAATAATTAATCCAAACTTATCAAGA 
               
               
                   
               
               
                 AGAAGACCAAAACCATTAGAAGAAGGAACACAATCGTCGATAGCAACTAA 
               
               
                   
               
               
                 AACTATTCCTTCAAATTGGTGTCAATTACCTAATGATTATAGCACTGATC 
               
               
                   
               
               
                 AGTTTCTTCGTTGGTTTCAGGAATGGATTGAAAATTTTTGTACAAGGAAA 
               
               
                   
               
               
                 AAAGTATTAGAGAAAGAAGCACAAGAACAATGTAAGAATATTACATGTAA 
               
               
                   
               
               
                 TAACGATACTGGAAAAACGAACACTAAATGTACTGAAGCATGTAAAAATT 
               
               
                   
               
               
                 ATAGTAATTTTATTTTAATAAAAAAAAAGGAGTATGAGTCACTAAATAGT 
               
               
                   
               
               
                 CAATACGATATGAATTATAAAAAAATAGTAGAACATAAAAATGCCCTAGA 
               
               
                   
               
               
                 ATATTTCAAAGATAAATGTAAAAATAATTGTGAATGTCTCTCTAAACATA 
               
               
                   
               
               
                 TTGATAATGGAAAAAATTGGAAAGAACCATATGAAACTATCGATGACTCA 
               
               
                   
               
               
                 GAACTCATAGGTAAATGTAAATGCAAAAAAGTTAAACCCAAAACTCCTGA 
               
               
                   
               
               
                 CGTAATTCCTGCAGGGGCAACTGAAACAAAAGAAAAAGATACACCTCATG 
               
               
                   
               
               
                 CACCTGAAAAACCTCAACAACCCCCACAACCCTTACCACCATCCGACGAA 
               
               
                   
               
               
                 CCCTTTGACCCGACCATCCTACAAACGACCATTCCTTTTGGAATCGCTTT 
               
               
                   
               
               
                 GGCATTAGGATCGATAGCGTTTCTTTTCATGAAAaaaaaaccgaaatctc 
               
               
                   
               
               
                 cagttgacctcttacgtgtactgaatatcccgaaacgagattatgaaatg 
               
               
                   
               
               
                 cctacgttgaaatcaaaaaatcgatatataccctatgctagtgatcgata 
               
               
                   
               
               
                 taaaggtaaaacatacatttatatggaaggagatagcagtggagatgaaa 
               
               
                   
               
               
                 aatatgcatttatgtctgatactactgatgtaacttcctcagaaagtgaa 
               
               
                   
               
               
                 tatgaagaattggatattaatgatatatatgtaccaggtagtcctaaata 
               
               
                   
               
               
                 taaaacattgatagaagtagtattggaaccatcaaaaagtaatggtaaca 
               
               
                   
               
               
                 cactaggtgatgatatggtacctaccactaatacatttacagatgaggaa 
               
               
                   
               
               
                 tggaatgaattgaaacatgattttatatcacaatatgtacaacgtgaacc 
               
               
                   
               
               
                 actggatgtaccacaatatgatgaatcaacacagttaccaatgaatatag 
               
               
                   
               
               
                 taggtaacgttttagatgatggtatggatgaaaaaccttttattacttct 
               
               
                   
               
               
                 attcatgatagagatttatatactggagaagaaattagttataatattaa 
               
               
                   
               
               
                 tatgagtactaatagtatggatgatccaaaatatgtatcaaataatgtat 
               
               
                   
               
               
                 attctggtatagatttaattaatgatacattaagtggtgatcgtattgat 
               
               
                   
               
               
                 atatatgatgaattattgaaacgaaaagaaaatgaattatttggtacaaa 
               
               
                   
               
               
                 tcatgtgaaacaaacaagtatacatagtgttgccaaactaacaaatagtg 
               
               
                   
               
               
                 accccatccacaaccaattagatttgttccatacatggttagatagacat 
               
               
                   
               
               
                 agagatatgtgcaatacgtggaataccaaggaagaattattagataaatt 
               
               
                   
               
               
                 gaatgaacaatggaataaagataatgatggtggtgatataccaaatgata 
               
               
                   
               
               
                 acaaaaagttgaatacggatgtttcgtttgaaatagatatggatgaaact 
               
               
                   
               
               
                 aaaggaaagaaggaatttagtaatatggatactatcttggatgatatgga 
               
               
                   
               
               
                 agatgatatatattatgatgtaaatgatgaaaacccatctgtggataata 
               
               
                   
               
               
                 tacctatggatcataataaagtagatgtaccaaagaaagtacatgttgaa 
               
               
                   
               
               
                 atgaaaatccttaataatacatccaatggatcgttggaacaagaatttcc 
               
               
                   
               
               
                 catatcggatgtatggaatatataa 
               
             
          
         
       
     
         [0025]    PfEMP1 variant designated TM284var1 has the following amino acid sequence: 
         [0000]    
       
         
               
             
           
               
                 SEQ ID NO: 3 
               
               
                 MTSKRGNRTVINLSVTDVLEKIALQIYKEENEKKIPHESELIGTLWKAQF 
               
               
                   
               
               
                 SDGLSGSFGDVRSGPSNSCNLHHKYYTNIKNGYPPARNPCDGRNEKRFSN 
               
               
                   
               
               
                 EGEAECGSDKIRVIGKGDGTACVPFRRQNMCDKNLEYLINKNTKTTHDLL 
               
               
                   
               
               
                 GNVLVTAKYEGASIVAKHPHKDTSEVCTALARSFADIGDIVRGRDMFLPN 
               
               
                   
               
               
                 KDDKVQKGLREVEKKIHDNLSSSVKPHYKDDGSGNYVKLREDWWAINRKE 
               
               
                   
               
               
                 VWNALTCEAPQSVHYFIKTSHGTRGFTSQGKCGRNETNVPTNLDYVPQYL 
               
               
                   
               
               
                 RWFDEWAEEFCRLRNHKLQNVKKECRGENIGDKYCSGDGEDCEKIVRQDY 
               
               
                   
               
               
                 NIRSDFLCPSCKKECTNYKKWIDTKQGEFNKQKKKYEKEIKKVESNSDTT 
               
               
                   
               
               
                 YDKKVYKILKEMYPLNSEFVATLKEAPYCNNNNVDGTIDENKPDDTFSRS 
               
               
                   
               
               
                 DYCKSCPVFGVICTRGECTEVKEDTCSKMNVKVPKKITNKEDPINTGILV 
               
               
                   
               
               
                 SDDRVSVIPNELENVCKDTGLFKGIRKDQRSCNYLCNLDVCDLSHNKNNT 
               
               
                   
               
               
                 HIDKRISIRVLFKRWLEYFFKDYSKLKKKLNSCTNNGEKSICINKCKKKC 
               
               
                   
               
               
                 ECVGKWVEEKRTEWEKVRKRYFSQYNVDDSQKSYTVKSIVNGNVDRSDIN 
               
               
                   
               
               
                 NSLDESEDIETLKESDTCYNSDSAKKQKCEKNDVITILTDRLKKKIDDCE 
               
               
                   
               
               
                 KQHDNRTNQICCDELPESKEDDEDEEEEGKKKKNAKQLEVTNEKKEQEDK 
               
               
                   
               
               
                 NLFQVCQKMKKVITDNNGERIRNQRCNEKTDRKWDCSTNEIPTNHTGACM 
               
               
                   
               
               
                 PPRRISLCIRPLRYLVDNGGKKSIDDYKNAFTECASIETYLLWQKYKRTN 
               
               
                   
               
               
                 GAEDKLKDGEIPNDFLRIMYYTYGDYRDIFLGTDISKNPNIKNISNKVKN 
               
               
                   
               
               
                 ILKFKKSMDESGKNQDENAKVQSSWDEHKRDIWKGMLCGLTYDIQNEKKD 
               
               
                   
               
               
                 ILKILNNKYNYPCDLEVFASKPQFFRWFIEWAEDYCRKYNDEYEKLQTAC 
               
               
                   
               
               
                 STVDCSKDPTDSEKQKCKNACDNFKTFVEGWKKQYDSQKNKFNKIKIEAN 
               
               
                   
               
               
                 IKNTYKGIENKEAYVFLSEECKGKCDCIKYKTDYDTNANDPKGFDTPPKE 
               
               
                   
               
               
                 QKDNCECVLRKKSACENNEVPKGRTQSQMTCADLKNESPSKGNNNTGNNH 
               
               
                   
               
               
                 KETITFSCNKSNLIGLGAQWKKITDDGLYASPRTRQLCLKHVIDIGRNNT 
               
               
                   
               
               
                 KKNNITEEEFINVLQKDAYAEGKLLYMYYNSNGKISIFQNGEKLKLDDIE 
               
               
                   
               
               
                 KHTHEAMKRSYADYGDLIKGTTKYTQYNDYNKISDIINVVTKKKNSASIN 
               
               
                   
               
               
                 DIYEREEFWEKYRADVWNAMLCGYKDVSNKTFDGNDDMCNLPNTDKEEEF 
               
               
                   
               
               
                 LRWEKEWNENFCITQIKRAEKLKNECNNFNCSSIKSKKDDIKSKCVKACI 
               
               
                   
               
               
                 NYKKFVKESKTQYEDQKRTYNERHNKTNKDIPTFLKDNCIHKNCDCISIK 
               
               
                   
               
               
                 FNHKDNWEKSFFESLDSSDIKNKCECLKLEEESNTTERYISKEDPQYHPE 
               
               
                   
               
               
                 YKGDGKVNYKYEKGKPKALPSIYPLNCAEKVADELRMYAENSLDTNTKLK 
               
               
                   
               
               
                 AKISKSIDTNEQNATNDEIDCNIYNNISNGQKNTCEHNGNTFHDKDEWDC 
               
               
                   
               
               
                 NKGTNKLYENDICLPPRRKHMCTKQLENISTASITTTDDLLKEVLITAVN 
               
               
                   
               
               
                 EGKRLKQQWEKTENEAQKKKHFLCDAMKYSFADLADIIRGTDIWKGNREQ 
               
               
                   
               
               
                 QKIQERLVKIFRNIYDNLEKDEYEKYKYGTKYQNLRSAWWDAHRKKIWNA 
               
               
                   
               
               
                 MTCSAPGDFLFVKRGKGDGSDIEFLTFSEHKKCGHDKEPPVYDYVPQILR 
               
               
                   
               
               
                 WITEWSEHFCELQEKNYYLLKEKCADYIQKDSKPIDDSHNIKCNTCKTKC 
               
               
                   
               
               
                 EEYSKFIKKWNSQYINLEKKFKELYDEANNTKSYEELYRIGKPSHRNHYE 
               
               
                   
               
               
                 DENLIQFLQNVKSECNEPNTVDKYLMYTSDCRRVKFSNTIDTNVNKPTAD 
               
               
                   
               
               
                 VTHNTINGPSSNLPVVTETNIKNELREYAFLETPEGYGNACKCKGPEPLD 
               
               
                   
               
               
                 RCPENDNISNYCNDFVSVPECTAKIYKDEIDHWNNANVKFKTSINNGVLV 
               
               
                   
               
               
                 PPRRSHICLKNMITKNYDKKKNGNEKEKTDLLQVAYNEGYFLCQKYDKQP 
               
               
                   
               
               
                 RDVLEAMKYTFADIADIVKGRDMINKDISAKLRKLLDIKVEPKAPRKWWK 
               
               
                   
               
               
                 YNKAHVWHAMLCGYRKGGGTITNDECNVPDEEYTYQFLRWFQEWIKKFCT 
               
               
                   
               
               
                 GQQKLYDDVQTKCSSANCNRDDGTISLPECESSCVQYKNYITRKRQEYRS 
               
               
                   
               
               
                 LNHQYNMNFKEQKAQGMKATQYIDDKCNSKCDCLIKYIDREKEWTNIYDS 
               
               
                   
               
               
                 LENNDLKNKCDCKQIKPKRHPKEVNPEEEPANSEPDYIVPLVPQKPSTPE 
               
               
                   
               
               
                 VPPPPPPPLPTPSDEPFNRDILEKTIPFGIALALCSIAFLFIKKKPKSSV 
               
               
                   
               
               
                 DLLRVIDIHKGDYDIPTLKSKNRYIPYKSAQYKGKTYIYMEGDSDSGHYY 
               
               
                   
               
               
                 EDTTDITSSESEYEEMDINDIYVPGSPKYKTLIEVVLEPSKRDTQNDIPS 
               
               
                   
               
               
                 DNTPSYKLTDEEWNQLKHDFISQYLPNTEPNNNYRSGNSPTNTNNTTTSH 
               
               
                   
               
               
                 DNMGEKPFITSIHDRDLYTGEEISYNINMSTNTNNDIPKYVSNNVYSGID 
               
               
                   
               
               
                 LINDTLSGNKHIDIYDEVLKRKENELFGTNHPKNTSNNSVAKLTNSDPIM 
               
               
                   
               
               
                 NQLDLLHKWLDRHRDMCDKWNTKEELLDKLNEQWNKDNDVGGDISTSNGN 
               
               
                   
               
               
                 KTLNTNVSIEIDMDETKGKKEFSNMDTILDNIEDDIYYDVNDENPSMDDI 
               
               
                   
               
               
                 PMDHNKVDVPKKVHVEMKILNNTFNGSLEPEFPISDVWNI 
               
             
          
         
       
     
         [0026]    Moreover, the TM284var1 PfEMP1 variant is encoded by the following nucleic acid sequence: 
         [0000]    
       
         
               
             
           
               
                 SEQ ID NO: 4 
               
               
                 ATGACGTCAAAACGTGGAAATCGAACTGTAATTAATCTTAGTGTAACGGA 
               
               
                   
               
               
                 TGTTCTAGAAAAAATTGCATTACAAATATATAAAGAGGAAAATGAAAAAA 
               
               
                   
               
               
                 AGATTCCACATGAAAGTGAATTGATAGGCACATTATGGAAAGCACAATTT 
               
               
                   
               
               
                 TCTGATGGCTTGAGTGGTTCATTTGGTGATGTAAGGTCTGGTCCTTCAAA 
               
               
                   
               
               
                 TTCATGCAATCTTCATCACAAATACTATACTAATATAAAGAATGGATATC 
               
               
                   
               
               
                 CACCCGCAAGGAATCCTTGCGATGGTAGAAATGAAAAACGTTTTTCAAAC 
               
               
                   
               
               
                 GAAGGTGAAGCAGAATGTGGTAGTGATAAAATAAGGGTTATTGGAAAAGG 
               
               
                   
               
               
                 TGATGGTACAGCATGTGTACCATTTAGAAGGCAAAATATGTGTGATAAAA 
               
               
                   
               
               
                 ATTTAGAATATTTGATTAATAAAAACACGAAAACTACTCATGATTTATTG 
               
               
                   
               
               
                 GGAAATGTATTAGTTACAGCAAAATATGAAGGTGCCTCTATTGTTGCAAA 
               
               
                   
               
               
                 GCATCCACATAAAGATACTTCAGAAGTATGTACTGCACTTGCACGAAGTT 
               
               
                   
               
               
                 TTGCAGATATAGGTGATATTGTAAGAGGAAGAGATATGTTTTTACCTAAT 
               
               
                   
               
               
                 AAGGATGATAAAGTACAAAAAGGTCTAAGAGAAGTTTTCAAGAAAATACA 
               
               
                   
               
               
                 TGATAATTTGTCATCTTCCGTAAAACCACATTACAAAGATGATGGATCTG 
               
               
                   
               
               
                 GAAATTACGTCAAATTAAGAGAAGATTGGTGGGCAATTAATAGAAAGGAG 
               
               
                   
               
               
                 GTATGGAATGCATTAACATGTGAAGCTCCACAAAGTGTTCATTATTTTAT 
               
               
                   
               
               
                 AAAAACGTCACATGGAACAAGAGGTTTTACAAGTCAAGGAAAATGTGGCC 
               
               
                   
               
               
                 GTAATGAAACAAACGTTCCTACAAATCTTGACTATGTTCCTCAATATTTA 
               
               
                   
               
               
                 CGCTGGTTCGATGAATGGGCAGAAGAGTTTTGTCGATTAAGGAATCATAA 
               
               
                   
               
               
                 GTTACAAAACGTTAAGAAAGAGTGTCGTGGAGAAAATATAGGTGACAAAT 
               
               
                   
               
               
                 ATTGTAGTGGTGATGGTGAGGATTGTGAAAAGATTGTTCGTCAGGATTAT 
               
               
                   
               
               
                 AATATTCGTTCGGATTTTTTATGTCCGAGCTGTAAAAAGGAATGTACAAA 
               
               
                   
               
               
                 TTATAAAAAATGGATAGACACAAAACAGGGAGAATTTAATAAACAGAAAA 
               
               
                   
               
               
                 AAAAATACGAAAAAGAAATTAAAAAAGTTGAAAGTAATTCTGATACCACA 
               
               
                   
               
               
                 TATGATAAAAAAGTTTATAAAATTCTAAAAGAAATGTACCCTTTAAATTC 
               
               
                   
               
               
                 AGAATTTGTAGCAACATTAAAAGAAGCTCCCTATTGTAATAACAATAATG 
               
               
                   
               
               
                 TAGACGGTACAATAGATTTTAATAAACCAGATGATACATTTTCTCGTTCA 
               
               
                   
               
               
                 GACTATTGTAAATCATGTCCTGTATTTGGTGTTATTTGTACAAGAGGTGA 
               
               
                   
               
               
                 GTGTACTGAAGTTAAGGAAGATACATGTAGTAAAATGAATGTTAAGGTTC 
               
               
                   
               
               
                 CGAAAAAAATTACAAATAAGGAAGATCCTATTAATATAGGTATTCTTGTT 
               
               
                   
               
               
                 AGTGATGACAGAGTAAGTGTAATTCCAAATGAATTAGAGAATGTTTGCAA 
               
               
                   
               
               
                 AGATACAGGTCTCTTTAAAGGTATTAGAAAAGATCAACGGTCATGTAATT 
               
               
                   
               
               
                 ACTTATGTAATTTAGATGTATGTGACCTGAGTCATAATAAAAACAATACA 
               
               
                   
               
               
                 CATATAGATAAACGTATTTCTATTAGAGTATTGTTTAAACGTTGGTTAGA 
               
               
                   
               
               
                 ATATTTTTTTAAAGATTATAGTAAATTAAAAAAAAAACTGAATTCATGTA 
               
               
                   
               
               
                 CAAATAATGGAGAAAAATCCATATGTATAAATAAATGTAAAAAAAAATGT 
               
               
                   
               
               
                 GAATGTGTGGGAAAATGGGTAGAAGAAAAAAGGACAGAATGGGAAAAAGT 
               
               
                   
               
               
                 AAGAAAGCGTTACTTCAGTCAATATAATGTTGATGATTCACAAAAATCGT 
               
               
                   
               
               
                 ATACAGTGAAAAGTATTGTAAATGGAAATGTAGATCGTAGTGATATTAAT 
               
               
                   
               
               
                 AATTCATTAGATGAGAGCGAAGATATAGAAACGTTGAAAGAATCAGATAC 
               
               
                   
               
               
                 ATGTTATAATTCTGATAGCGCAAAAAAACAAAAATGTGAAAAAAACGACG 
               
               
                   
               
               
                 TCATAACTATTTTAATTGATAGACTTAAAAAAAAAATTGATGATTGTGAA 
               
               
                   
               
               
                 AAGCAACATGATAATAGAACTAATCAAATTTGTTGTGATGAGTTACCTGA 
               
               
                   
               
               
                 AAGTAAAGAAGATGATGAAGATGAAGAGGAAGAAGGGAAAAAGAAAAAAA 
               
               
                   
               
               
                 ATGCAAAGCAATTGGAAGTAACTAATGAGAAAAAAGAACAAGAAGACAAA 
               
               
                   
               
               
                 AACTTGTTTCAAGTGTGCCAAAAAATGAAGAAGGTAATTACGGATAATAA 
               
               
                   
               
               
                 TGGAGAAAGAATCAGAAACCAGCGTTGCAATGAAAAAACTGATAGAAAAT 
               
               
                   
               
               
                 GGGATTGTAGTACTAATGAAATTCCTACAAATCATACTGGAGCTTGTATG 
               
               
                   
               
               
                 CCACCAAGAAGAATATCATTATGTATTCGGCCTTTACGATATTTGGTAGA 
               
               
                   
               
               
                 TAACGGAGGAAAAAAAAGCATAGATGATTATAAAAATGCGTTTACTGAAT 
               
               
                   
               
               
                 GTGCATCAATAGAAACGTATTTGTTATGGCAAAAATACAAAAGAACTAAT 
               
               
                   
               
               
                 GGAGCAGAAGATAAATTAAAAGATGGAGAGATTCCAAATGATTTTCTAAG 
               
               
                   
               
               
                 AATAATGTATTATACATATGGAGATTATAGAGATATATTTTTGGGAACAG 
               
               
                   
               
               
                 ATATTTCTAAAAATCCTAATATTAAAAATATATCAAATAAGGTTAAAAAT 
               
               
                   
               
               
                 ATATTGAAATTCAAAAAGAGCATGGACGAATCAGGTAAAAATCAGGATGA 
               
               
                   
               
               
                 AAATGCGAAAGTTCAATCTTCGTGGGATGAACATAAAAGGGACATATGGA 
               
               
                   
               
               
                 AAGGAATGTTATGTGGATTAACCTATGATATCCAAAATGAAAAGAAAGAT 
               
               
                   
               
               
                 ATTCTCAAAATTCTCAATAACAAGTACAATTACCCATGCGATCTTGAAGT 
               
               
                   
               
               
                 GTTTGCATCTAAACCACAATTTTTTCGTTGGTTTATTGAATGGGCAGAAG 
               
               
                   
               
               
                 ATTATTGTAGAAAATACAATGATGAGTATGAAAAATTACAGACGGCGTGT 
               
               
                   
               
               
                 AGTACGGTAGATTGTAGTAAAGACCCTACTGATTCTGAAAAACAAAAATG 
               
               
                   
               
               
                 TAAAAACGCTTGTGATAATTTCAAAACATTCGTTGAAGGTTGGAAAAAAC 
               
               
                   
               
               
                 AATATGATAGTCAAAAAAATAAATTTAATAAGATAAAAATTGAAGCTAAT 
               
               
                   
               
               
                 ATAAAGAATACATATAAAGGTATAGAAAATAAAGAAGCTTATGTATTTTT 
               
               
                   
               
               
                 AAGTGAAGAATGTAAAGGAAAATGTGACTGTATAAAATATAAAACAGACT 
               
               
                   
               
               
                 ATGATACAAATGCAAATGATCCTAAAGGTTTCGATACACCACCGAAAGAA 
               
               
                   
               
               
                 CAAAAAGATAATTGTGAATGTGTGTTGAGAAAAAAATCGGCATGTGAAAA 
               
               
                   
               
               
                 TAATGAAGTACCTAAAGGTCGAACACAATCTCAAATGACATGTGCTGATC 
               
               
                   
               
               
                 TAAAAAATGAATCTCCTAGTAAAGGAAATAATAATACTGGGAACAATCAT 
               
               
                   
               
               
                 AAAGAAACCATTACATTCTCGTGCAATAAAAGCAATTTAATTGGCTTAGG 
               
               
                   
               
               
                 AGCACAATGGAAAAAAATAACTGATGATGGTTTATATGCTTCTCCAAGAA 
               
               
                   
               
               
                 CTCGACAATTATGTTTGAAACACGTAATAGACATAGGAAGGAATAATACT 
               
               
                   
               
               
                 AAAAAAAACAATATAACAGAAGAAGAGTTCATTAATGTATTACAAAAAGA 
               
               
                   
               
               
                 TGCATATGCTGAAGGTAAATTACTTTATATGTACTACAACAGTAATGGTA 
               
               
                   
               
               
                 AAATATCTATATTTCAAAATGGCGAAAAGTTAAAATTGGATGACATAGAA 
               
               
                   
               
               
                 AAACATACACATGAAGCCATGAAAAGATCATATGCTGATTATGGTGATTT 
               
               
                   
               
               
                 AATTAAAGGAACAACAAAATATACACAATACAATGATTATAACAAAATTA 
               
               
                   
               
               
                 GCGATATTATAAACGTTGTGACTAAAAAGAAAAATTCCGCTTCAATTAAT 
               
               
                   
               
               
                 GATATTTATGAGCGTGAAGAATTTTGGGAAAAATATAGAGCTGATGTATG 
               
               
                   
               
               
                 GAATGCTATGTTATGTGGTTACAAAGATGTATCAAATAAAACATTTGATG 
               
               
                   
               
               
                 GAAACGATGATATGTGTAACTTACCAAATACTGATAAGGAGGAAGAATTT 
               
               
                   
               
               
                 CTCAGATGGTTTAAGGAATGGAATGAAAATTTTTGTATTACACAAATAAA 
               
               
                   
               
               
                 ACGCGCAGAGAAATTAAAAAATGAATGCAATAATTTTAACTGTTCTTCCA 
               
               
                   
               
               
                 TTAAGAGTAAAAAGGACGATATTAAATCTAAATGTGTAAAAGCATGTATA 
               
               
                   
               
               
                 AATTATAAAAAGTTTGTAAAGGAATCAAAAACGCAATATGAAGATCAAAA 
               
               
                   
               
               
                 GAGAACATACAATGAAAGACATAATAAGACAAATAAGGATATTCCTACTT 
               
               
                   
               
               
                 TTTTGAAAGATAATTGTATTCATAAAAACTGTGATTGTATTTCTATAAAA 
               
               
                   
               
               
                 TTTAATCATAAAGATAATTGGGAAAAATCTTTTTTTGAGAGTTTAGATAG 
               
               
                   
               
               
                 TTCCGATATTAAAAATAAGTGTGAATGTTTAAAACTTGAAGAAGAGTCAA 
               
               
                   
               
               
                 ATACTACAGAACGATATATTTCTAAAGAAGACCCACAATATCATCCAGAA 
               
               
                   
               
               
                 TATAAAGGTGATGGAAAGGTTAATTATAAATATGAGAAAGGAAAACCAAA 
               
               
                   
               
               
                 AGCTCTTCCTTCTATATACCCTTTGAACTGTGCTGAAAAGGTTGCTGACG 
               
               
                   
               
               
                 AGTTACGAATGTATGCTGAAAATTCTTTGGATACTAATACTAAATTGAAG 
               
               
                   
               
               
                 GCAAAAATATCAAAAAGTATAGATACAAATGAACAAAATGCTACGAATGA 
               
               
                   
               
               
                 TGAGATTGATTGCAATATTTACAATAATATATCTAATGGACAGAAAAATA 
               
               
                   
               
               
                 CTTGTGAACATAATGGAAACACTTTTCATGATAAGGATGAATGGGATTGT 
               
               
                   
               
               
                 AACAAAGGAACAAATAAATTATATGAAAATGATATTTGTTTACCTCCAAG 
               
               
                   
               
               
                 AAGAAAACATATGTGTACAAAACAACTAGAAAATATCAGCACGGCATCAA 
               
               
                   
               
               
                 TTACAACTACGGATGATTTACTGAAAGAAGTGTTAATTACAGCTGTAAAT 
               
               
                   
               
               
                 GAAGGAAAGCGTTTAAAACAGCAATGGGAGAAAACAGAAAATGAAGCACA 
               
               
                   
               
               
                 AAAAAAGAAACACTTTTTATGTGATGCTATGAAATATAGTTTTGCTGATT 
               
               
                   
               
               
                 TAGCTGATATTATAAGAGGAACAGACATATGGAAAGGAAATAGAGAGCAA 
               
               
                   
               
               
                 CAAAAAATACAAGAAAGATTAGTAAAAATCTTCAGAAATATATATGATAA 
               
               
                   
               
               
                 CTTAGAGAAGGATGAATATGAGAAATATAAATATGGTACAAAATATCAAA 
               
               
                   
               
               
                 ATTTAAGATCGGCTTGGTGGGATGCACATAGAAAGAAAATATGGAATGCT 
               
               
                   
               
               
                 ATGACATGTTCAGCACCAGGTGATTTCCTTTTTGTAAAAAGAGGAAAAGG 
               
               
                   
               
               
                 AGATGGAAGTGACATCGAATTTTTAACTTTTTCAGAACATAAAAAATGTG 
               
               
                   
               
               
                 GACATGATAAAGAACCACCTGTTTATGATTATGTGCCTCAAATACTTAGA 
               
               
                   
               
               
                 TGGATTACAGAATGGTCTGAACATTTTTGTGAATTGCAAGAAAAAAATTA 
               
               
                   
               
               
                 TTATCTTCTAAAAGAAAAATGTGCTGATTATATACAAAAGGATTCCAAAC 
               
               
                   
               
               
                 CTATTGATGATTCACATAATATAAAATGTAATACTTGTAAGACGAAATGT 
               
               
                   
               
               
                 GAAGAATATAGTAAATTTATTAAGAAATGGAACTCTCAGTATATAAATCT 
               
               
                   
               
               
                 GGAAAAAAAATTTAAAGAATTATATGACGAGGCAAATAATACTAAAAGTT 
               
               
                   
               
               
                 ATGAAGAACTTTACAGAATTGGGAAGCCTTCACACAGAAACCACTATGAA 
               
               
                   
               
               
                 GATGAAAACCTGATTCAGTTCTTACAAAATGTAAAATCTGAGTGTAACGA 
               
               
                   
               
               
                 ACCTAACACTGTTGATAAATATCTTATGTATACAAGTGATTGTAGAAGAG 
               
               
                   
               
               
                 TTAAATTTTCTAATACTATCGATACAAATGTTAACAAACCTACTGCGGAT 
               
               
                   
               
               
                 GTTACTCATAATACTATTAATGGTCCTAGTAGTAACCTCCCAGTTGTTAC 
               
               
                   
               
               
                 TGAAACAAATATTAAAAATGAACTAAGAGAATATGCTTTCTTAGAAACAC 
               
               
                   
               
               
                 CAGAAGGATATGGTAATGCTTGTAAATGTAAGGGTCCTGAACCATTAGAT 
               
               
                   
               
               
                 CGTTGCCCTGAAAATGATAATATTAGTAATTACTGTAACGATTTTGTTAG 
               
               
                   
               
               
                 TGTTCCTGAATGCACAGCAAAAATATATAAAGATGAAATTGATCATTGGA 
               
               
                   
               
               
                 ATAATGCAAATGTAAAATTTAAGACATCAATAAATAACGGTGTGTTAGTT 
               
               
                   
               
               
                 CCTCCAAGAAGAAGTCATATATGTCTTAAGAATATGATAACAAAAAACTA 
               
               
                   
               
               
                 TGATAAAAAGAAAAATGGGATGGAAAAATTTAAAACTGATCTTCTACAGG 
               
               
                   
               
               
                 TTGCATACAATGAAGGTTATTTCCTATGTCAAAAATATGATAAGCAACCT 
               
               
                   
               
               
                 AGAGACGTATTGGAAGCGATGAAATACACATTTGCAGATATTGCTGATAT 
               
               
                   
               
               
                 AGTAAAAGGTAGAGATATGATTAACAAAGATATATCCGCAAAACTACGAA 
               
               
                   
               
               
                 AATTATTGGATATTAAGGTTGAACCCAAAGCTCCTAGAAAATGGTGGAAA 
               
               
                   
               
               
                 TACAATAAAGCACATGTATGGCACGCTATGTTATGTGGATATAGAAAAGG 
               
               
                   
               
               
                 TGGAGGAACAATTACGAATGATGAGTGTAATGTTCCAGATGAAGAGTACA 
               
               
                   
               
               
                 CTTATCAATTTCTTCGATGGTTTCAAGAATGGATTAAAAAATTTTGTACT 
               
               
                   
               
               
                 GGACAACAAAAATTATATGACGACGTACAAACGAAATGTTCATCTGCCAA 
               
               
                   
               
               
                 TTGTAATAGAGATGATGGGACGATTAGCCTACCTGAATGTGAAAGTTCTT 
               
               
                   
               
               
                 GTGTTCAATATAAGAATTACATTACAAGGAAGAGACAAGAGTATCGGTCA 
               
               
                   
               
               
                 CTAAACCATCAATATAACATGAATTTTAAAGAACAAAAGGCACAAGGTAT 
               
               
                   
               
               
                 GAAAGCCACACAGTACATAGATGATAAATGTAATAGTAAATGTGATTGTC 
               
               
                   
               
               
                 TCATTAAATATATTGATAGAGAAAAAGAATGGACAAACATATATGACTCA 
               
               
                   
               
               
                 TTGGAAAATAATGATCTGAAAAATAAATGTGATTGTAAGCAAATTAAACC 
               
               
                   
               
               
                 CAAACGTCATCCAAAAGAAGTAAATCCTGAGGAAGAACCTGCTAATTCTG 
               
               
                   
               
               
                 AACCCGATTACATTGTTCCCCTTGTACCACAAAAACCTTCAACACCAGAG 
               
               
                   
               
               
                 GTACCCCCACCTCCTCCTCCACCTTTACCAACCCCTTCGGACGAACCATT 
               
               
                   
               
               
                 CAATCGTGACATTCTGGAAAAAACCATTCCTTTTGGAATTGCATTGGCAT 
               
               
                   
               
               
                 TATGTTCGATAGCTTTTCTCTTCATAAAGaaaaaacctaaatcatctgtt 
               
               
                   
               
               
                 gacctcttgcgagtaattgacatccacaaaggagattatgatatacctac 
               
               
                   
               
               
                 attgaaatccaaaaataggtacataccatataaaagtgctcaatataaag 
               
               
                   
               
               
                 gtaaaacatacatttatatggaaggagatagtgatagtggacactactac 
               
               
                   
               
               
                 gaagatacaactgatattacttcctccgaaagtgaatatgaagagatgga 
               
               
                   
               
               
                 tattaatgatatatatgttcctggtagtccaaaatacaaaacgttgatag 
               
               
                   
               
               
                 aagttgttctggagccatcaaaaagagatacacaaaatgatatacctagt 
               
               
                   
               
               
                 gataatacacctagttataaacttacagatgaggaatggaatcaattgaa 
               
               
                   
               
               
                 acatgattttatatcacaatatttaccaaatacagaaccaaataataatt 
               
               
                   
               
               
                 atagaagtggaaatagtccaacaaataccaataatactaccacgtcacat 
               
               
                   
               
               
                 gataatatgggagaaaaaccttttattacttctattcatgatagggattt 
               
               
                   
               
               
                 atatactggagaagaaattagttataatattaatatgagtactaacacta 
               
               
                   
               
               
                 ataatgatattccaaaatatgtatcaaataatgtatattctggtatagat 
               
               
                   
               
               
                 ttaattaatgacacattaagtggtaacaaacatattgatatatatgatga 
               
               
                   
               
               
                 agtgctaaaaagaaaagaaaatgaattatttggaacaaatcatccgaaaa 
               
               
                   
               
               
                 atacatcaaacaatagtgtagctaaattaacaaatagtgatccaattatg 
               
               
                   
               
               
                 aaccaattagatttgttacataaatggttagatagacatagagatatgtg 
               
               
                   
               
               
                 cgataaatggaataccaaggaagaattgttagataaattaaatgaacaat 
               
               
                   
               
               
                 ggaataaagataatgatgttggtggtgatatttccactagtaatggtaat 
               
               
                   
               
               
                 aaaacgttgaatactaatgtttcgattgaaatagatatggatgaaactaa 
               
               
                   
               
               
                 aggaaagaaggaatttagtaatatggatactatcttggataatatagaag 
               
               
                   
               
               
                 atgatatatattatgatgtaaatgatgaaaacccatctatggatgatata 
               
               
                   
               
               
                 cctatggatcataataaagtagatgtacctaagaaagtacatgttgaaat 
               
               
                   
               
               
                 gaaaatccttaataatacattcaatggatccttggaaccagaatttccca 
               
               
                   
               
               
                 tatcggatgtatggaatatataa 
               
             
          
         
       
     
         [0027]    The PfEMP1 variant designated ITvar60 has the following amino acid sequence: 
         [0000]    
       
         
               
             
           
               
                 SEQ ID NO: 5 
               
               
                 MAPKGRSTNEIELSARDVLENIGIGIYNQEKIKKNPYEQQLKGTLSNARF 
               
               
                   
               
               
                 HDGLHKAADLGVIPGPSHFSQLYYKKHTNNTKYYKDDRHPCHGRQGKRFD 
               
               
                   
               
               
                 EGQKFECGNDKIIGNSDKYGSCAPPRRRHICDQNLEFLDNNHTDTIHDVL 
               
               
                   
               
               
                 GNVLVTAKYEGESIVNDHPDKKNNGNKSGICTSLARSFADIGDIVRGRDM 
               
               
                   
               
               
                 FKPNDKDAVRHGLKVVFKKIYDKLSPKVQEHYKDVDGSGNYYKLREDWWT 
               
               
                   
               
               
                 ANRDQVWKAITYKAPQDANYFRNVSGTTMAFTSAGKCRHNDNSVPTNLDY 
               
               
                   
               
               
                 VPQFLRWYDEWADDFCRIRNHKLQKVKDTCQGYNNSGYRIYCSGDGEDCT 
               
               
                   
               
               
                 NILKQNFNIVSDFFCPSCKTECTNYKKWINKKQGEFNKQKKKYEKEINNI 
               
               
                   
               
               
                 ASNSDNTYDKKVYKTLKSMYPLDTKFVATLKEAPFCNNNNVDGIIDFNKP 
               
               
                   
               
               
                 DDTFSSSTYCDSCPAFGVICENGTCTKVNEDTCSKMNVQVPKIITNKEDP 
               
               
                   
               
               
                 TNIGILVSDDRVNVIPNELENVCKNTGIFKGIRKDEWSCKYLCNLDVCDL 
               
               
                   
               
               
                 SHNKNNTHIDKRISIRVLFKRWLEYFLKDYSKLKKKLNSCTNNGKESICI 
               
               
                   
               
               
                 NECKKKCECVGKWAEEKRKEWEKVRKRFFNQYNVDDSLKSYEVKTFVNGN 
               
               
                   
               
               
                 VDRSDIKNALNEGENLEALQDSDECIKPHNSKKDTCVKNDVVNILINRLK 
               
               
                   
               
               
                 KKIDDCKIQHDNRTNQICCDELPESKEDNEDEEEEGEKKKNSKHLEETKE 
               
               
                   
               
               
                 KKELDDNNFLDLCNNVKKYIEDNNKQISIQHKCNTKGDGNWNDSTKKIDI 
               
               
                   
               
               
                 QHTGAHMPPRRKSLCIRELRYLVEIGGDKNIDDYKNAFTKCASIETYLLW 
               
               
                   
               
               
                 QKYKKSNRSEEDKLKGGEIPEDFRGIMYYTFGDYRDIFLGTDISSDGNIK 
               
               
                   
               
               
                 NISNKIKDLMKEKYSKATGHKGENHNSNLQSSWDEHKRTIWKGMLCGLTY 
               
               
                   
               
               
                 GISNEQQKKNIRKMLNNKYKYPCDLETESKKPQFLRWFNEWSEDFCKNYK 
               
               
                   
               
               
                 NAIDILKKDCTEADCMNKLVNNREKNKKCKEACEHFKEWIKGWKNQYEQQ 
               
               
                   
               
               
                 RKKFNIDKNVEQKETAYINVNGLEPYEFFQNQYFVGTCECMKNKSESSAN 
               
               
                   
               
               
                 NDENIPEAFDEKPKEFKDKCPCTYDIPEPSETMSCIEKAAFKLRYASEDK 
               
               
                   
               
               
                 IHSKISSKLKGNGSAFSCTNSASDNIFDETSCYKNEFNKTENINSVKASN 
               
               
                   
               
               
                 MNRFDTNIIWDCDGKTKYEQINLCVPPRRENMCIKGLEHLNETKHSDNKT 
               
               
                   
               
               
                 LLKELQEIASTEGKGISKNFKQMDRENDDGICDAMKYSFADLADIVRGTD 
               
               
                   
               
               
                 NYKNSNGNNNKVEENLKKIFEKIHNINSLKKEYSKDKPDYQRLRSDWWDT 
               
               
                   
               
               
                 NRKEIWKALTCSARDNNKIYKKGQKNTNNGKNKCGNEEDPPDDDYIPQPF 
               
               
                   
               
               
                 RWLQEWSEHFCRVQYDNLNKLKEECGECNENKNGLACMMNSNIKDTKCMN 
               
               
                   
               
               
                 CKDACKDYRNMINTWNSQWKKQQEDYKELYNTKNKININKCKVIEFLDKT 
               
               
                   
               
               
                 NDTCHYKPGSAEKFLKESSHCTDLTFDKTKNSNNIPYAFENPPDGYKVLC 
               
               
                   
               
               
                 GTTYRKSCKKLKKLGMNYTSENKIDLSGENAKWEKLNDLIYVPPRTQQLC 
               
               
                   
               
               
                 LQPLQTLISRTNKTTKVTEYDFSRALQICAYNEANSLHNYYSKYGKDFVF 
               
               
                   
               
               
                 SAGKSQDTKDEIKTHILENMKRSFADYGNLIKGKTQYEYNGLNKKLQDYI 
               
               
                   
               
               
                 KTNLKYNGTDRKTGEDLWNKHKSDIWNSMLCGYNEENPSEPLHDKDIRCK 
               
               
                   
               
               
                 LPDNDSEDEFLRWFQEWREDFCVIKGILIQNVKDACNFNNCEDANNKSIR 
               
               
                   
               
               
                 SCQKPCVKYKTWVEQRKIEYENQIQKYKNLNNNSNEGKESLLFLNDKCKG 
               
               
                   
               
               
                 KCECIVQKKSTDNIDKIFEEYPEEYKTQCECQPDPCSDLSITDSGFPDAS 
               
               
                   
               
               
                 PFGGGQPRSACPTRRGNHNNCPTEEICKKYDSYINGCRPKTYHDNTNNWD 
               
               
                   
               
               
                 SRGMLNSSSENEGVLIPPRRRHLCTRNIIKNLSRIKNKDHFKDYLMKSAY 
               
               
                   
               
               
                 EEGKLLREKYRNNSRDGLNAMMFTFADYADIVKGTDIFGSILSQKLGEIT 
               
               
                   
               
               
                 GISNDINERKKWWSEIKNNIWEVMLCSYNRTKNNNNFEGNIVRENCNVPN 
               
               
                   
               
               
                 TDEKDQFLRWLLEWGIQACKEKKIRKQALQTKCYCSNPNEISGSDIIKHY 
               
               
                   
               
               
                 PCKSELTKYIQWNLMIKELLDQLNIKYQNIKASNNPKNPSEINAEEYIET 
               
               
                   
               
               
                 ELKEGECNLVDIERDYNKIKQEHNPLKEILMYLCPNLEFPDDTFEYIGKT 
               
               
                   
               
               
                 ETEDTTIEPETPTSDNPEDSIPSISPEDVHPTTGEDTNIFNSNILSSTIP 
               
               
                   
               
               
                 FGIALALSSTAFLFLKKKTLSPVDLLRVLDIHKGDYGIPTLKSKNRYIPY 
               
               
                   
               
               
                 RSGTYKGKTYLYVEGDSDSGHYYEDTTDITSSESEYEEMDINDIYVPGSP 
               
               
                   
               
               
                 KYKTLIEVVLEPSKSDGHIPHSAGEPLDDMVGTTIFTDEEWNELKHDFIS 
               
               
                   
               
               
                 QYVQRESMGVPQYDVSTELPMNIGGNVLDDGMEEKPFITSIHDRDLYTGE 
               
               
                   
               
               
                 EFSYNINMGTNSMDDPKYVSNNVYSGIDLINDTLSGNQHIDIYDEVLKRK 
               
               
                   
               
               
                 ENELFGTNYKKNISNNRVAKLTNNDPIMNQLDLLHKWLDRHRDMCNTWNT 
               
               
                   
               
               
                 KEELLDKLNEQWNKDNDAGDIPSDSNKKLNTDVSIQIDMDDPKGKKEFSN 
               
               
                   
               
               
                 MDTILDDIEDDIYYDVNDENPFVDDIPMDHNKVDVPKKVHVEMKILNNTS 
               
               
                   
               
               
                 NGSLEPEFPISDVWNI 
               
             
          
         
       
     
         [0028]    Moreover, the ITvar60 PfEMP1 variant is encoded by the following nucleic acid sequence: 
         [0000]    
       
         
               
             
           
               
                 SEQ ID NO: 6 
               
               
                 ATGGCACCAAAGGGTAGAAGTACAAATGAAATTGAACTTAGCGCAAGAGA 
               
               
                   
               
               
                 TGTTTTGGAAAATATTGGAATAGGAATATATAATCAGGAAAAAATAAAAA 
               
               
                   
               
               
                 AGAATCCATATGAACAACAATTGAAAGGCACATTATCAAACGCCCGATTT 
               
               
                   
               
               
                 CATGATGGCTTGCACAAGGCAGCTGATTTGGGGGTAATACCTGGTCCTTC 
               
               
                   
               
               
                 ACATTTTTCTCAGCTTTATTACAAAAAGCATACTAATAACACAAAATATT 
               
               
                   
               
               
                 ATAAGGATGATAGGCATCCTTGTCATGGTAGACAAGGAAAACGTTTTGAT 
               
               
                   
               
               
                 GAAGGTCAAAAATTTGAATGTGGTAATGATAAAATAATTGGTAATAGCGA 
               
               
                   
               
               
                 TAAATATGGATCCTGTGCTCCACCTAGAAGAAGACATATATGTGATCAAA 
               
               
                   
               
               
                 ATTTAGAATTCTTAGATAACAATCATACTGATACTATTCATGATGTATTG 
               
               
                   
               
               
                 GGAAATGTGTTGGTCACAGCAAAATATGAAGGTGAATCTATTGTTAATGA 
               
               
                   
               
               
                 TCATCCAGATAAAAAGAACAATGGTAATAAATCAGGTATATGTACTTCTC 
               
               
                   
               
               
                 TTGCACGAAGTTTTGCCGATATAGGTGATATTGTAAGAGGAAGAGATATG 
               
               
                   
               
               
                 TTTAAACCTAATGACAAAGATGCAGTGCGGCATGGTTTAAAGGTAGTTTT 
               
               
                   
               
               
                 TAAGAAAATATATGATAAATTGTCACCTAAAGTACAAGAACATTACAAAG 
               
               
                   
               
               
                 ATGTTGATGGATCTGGAAATTACTATAAATTAAGGGAAGATTGGTGGACA 
               
               
                   
               
               
                 GCGAACAGAGATCAAGTATGGAAAGCCATAACATATAAAGCTCCACAAGA 
               
               
                   
               
               
                 CGCTAATTATTTTAGAAATGTTTCAGGAACAACTATGGCGTTTACAAGTG 
               
               
                   
               
               
                 CAGGAAAATGTAGACACAATGACAATAGCGTCCCAACGAATCTAGATTAT 
               
               
                   
               
               
                 GTCCCTCAATTTTTACGTTGGTACGATGAATGGGCAGATGATTTTTGTCG 
               
               
                   
               
               
                 AATAAGAAATCATAAGTTGCAAAAGGTTAAAGACACATGTCAGGGATATA 
               
               
                   
               
               
                 ATAATAGTGGTTATAGAATATATTGTAGTGGTGATGGTGAGGATTGTACA 
               
               
                   
               
               
                 AATATTCTTAAACAGAATTTTAATATTGTTTCGGATTTTTTTTGTCCGAG 
               
               
                   
               
               
                 CTGTAAAACCGAATGTACAAATTATAAAAAATGGATAAATAAAAAACAAG 
               
               
                   
               
               
                 GTGAATTTAATAAACAAAAAAAAAAATACGAAAAGGAAATTAACAATATT 
               
               
                   
               
               
                 GCAAGTAATTCTGATAACACATATGATAAAAAAGTTTATAAAACTCTAAA 
               
               
                   
               
               
                 AAGCATGTACCCTTTAGATACAAAATTTGTAGCAACACTAAAAGAGGCTC 
               
               
                   
               
               
                 CCTTTTGCAATAACAATAATGTAGATGGTATAATAGATTTTAATAAACCA 
               
               
                   
               
               
                 GATGATACATTTTCTAGTTCAACATATTGTGATTCATGTCCTGCATTTGG 
               
               
                   
               
               
                 TGTTATTTGTGAAAATGGTACGTGTACTAAGGTTAATGAAGATACATGTA 
               
               
                   
               
               
                 GTAAAATGAATGTTCAGGTTCCAAAAATAATTACAAATAAGGAAGATCCT 
               
               
                   
               
               
                 ACTAATATAGGTATTCTTGTTAGTGATGACAGAGTAAATGTAATTCCAAA 
               
               
                   
               
               
                 TGAATTAGAGAATGTTTGCAAAAATACAGGTATCTTTAAAGGTATTAGAA 
               
               
                   
               
               
                 AAGATGAATGGTCATGTAAATATTTATGTAATTTAGATGTCTGTGACCTG 
               
               
                   
               
               
                 AGTCATAATAAAAACAATACACATATAGATAAACGTATTTCAATTAGAGT 
               
               
                   
               
               
                 ACTGTTTAAACGTTGGTTAGAATATTTTTTAAAAGATTATAGTAAATTAA 
               
               
                   
               
               
                 AAAAAAAACTGAATTCATGTACAAATAATGGAAAAGAATCCATATGTATA 
               
               
                   
               
               
                 AATGAATGTAAAAAAAAATGTGAATGTGTGGGAAAATGGGCAGAAGAGAA 
               
               
                   
               
               
                 AAGAAAAGAATGGGAAAAAGTAAGAAAGCGTTTCTTTAATCAATATAATG 
               
               
                   
               
               
                 TTGATGATTCACTAAAATCTTACGAAGTGAAAACATTTGTAAATGGAAAT 
               
               
                   
               
               
                 GTAGATCGTAGTGATATTAAGAATGCATTAAATGAGGGTGAAAATTTAGA 
               
               
                   
               
               
                 AGCGTTGCAAGATTCTGATGAATGTATTAAACCTCATAATTCCAAGAAAG 
               
               
                   
               
               
                 ACACATGTGTAAAAAATGACGTCGTAAATATTTTAATTAATAGACTTAAA 
               
               
                   
               
               
                 AAAAAAATTGATGATTGTAAAATCCAACATGATAATAGAACTAATCAAAT 
               
               
                   
               
               
                 TTGTTGTGATGAGTTACCTGAAAGTAAAGAAGATAATGAAGATGAAGAGG 
               
               
                   
               
               
                 AAGAAGGGGAAAAGAAAAAAAATTCAAAACATTTGGAGGAGACTAAAGAG 
               
               
                   
               
               
                 AAAAAAGAACTGGATGACAACAATTTTTTGGATTTGTGCAACAATGTGAA 
               
               
                   
               
               
                 GAAATATATTGAGGATAATAATAAGCAAATAAGTATACAACATAAATGCA 
               
               
                   
               
               
                 ATACGAAAGGAGATGGAAATTGGAATGATAGTACAAAAAAGATCGATATT 
               
               
                   
               
               
                 CAACATACTGGAGCTCATATGCCACCAAGAAGAAAATCATTATGTATTCG 
               
               
                   
               
               
                 TGAGTTACGATATTTGGTAGAAATTGGAGGAGATAAAAACATAGATGATT 
               
               
                   
               
               
                 ATAAAAATGCGTTTACTAAATGTGCATCAATAGAAACATATTTGTTATGG 
               
               
                   
               
               
                 CAAAAATACAAAAAATCTAATAGATCAGAAGAAGATAAATTAAAAGGTGG 
               
               
                   
               
               
                 AGAGATTCCAGAAGATTTTAGAGGAATAATGTATTATACATTTGGTGATT 
               
               
                   
               
               
                 ACAGAGATATATTTTTGGGAACAGATATTTCTTCAGATGGTAATATTAAA 
               
               
                   
               
               
                 AATATATCAAATAAAATAAAAGATTTAATGAAAGAAAAGTATAGTAAAGC 
               
               
                   
               
               
                 AACAGGTCATAAAGGAGAAAACCATAATTCAAATCTTCAATCTTCGTGGG 
               
               
                   
               
               
                 ATGAACATAAAAGGACCATATGGAAAGGAATGTTATGTGGATTAACCTAT 
               
               
                   
               
               
                 GGTATATCAAATGAACAACAAAAAAAAAATATTCGCAAAATGCTCAACAA 
               
               
                   
               
               
                 CAAGTACAAATACCCATGCGATCTAGAAACATTTTCAAAGAAACCACAAT 
               
               
                   
               
               
                 TTCTTCGTTGGTTTAACGAATGGAGTGAAGATTTTTGTAAAAATTACAAA 
               
               
                   
               
               
                 AATGCTATTGATATATTAAAAAAGGATTGTACAGAAGCAGATTGTATGAA 
               
               
                   
               
               
                 TAAATTAGTAAATAATCGTGAAAAAAACAAGAAATGTAAAGAAGCGTGTG 
               
               
                   
               
               
                 AACATTTTAAAGAGTGGATAAAAGGATGGAAAAATCAATATGAACAACAA 
               
               
                   
               
               
                 AGAAAAAAATTTAATATTGATAAAAATGTTGAACAAAAGGAGACAGCATA 
               
               
                   
               
               
                 TATAAACGTAAATGGTCTGGAACCCTATGAATTTTTTCAAAACCAATATT 
               
               
                   
               
               
                 TTGTGGGAACATGTGAATGCATGAAAAATAAATCAGAGTCATCTGCAAAT 
               
               
                   
               
               
                 AATGATGAAAATATACCAGAAGCATTCGATGAAAAACCAAAAGAGTTCAA 
               
               
                   
               
               
                 GGACAAATGTCCATGTACTTATGATATACCTGAACCTAGCGAAACTATGA 
               
               
                   
               
               
                 GTTGTATAGAGAAAGCTGCCTTCAAATTACGTTATGCTTCTGAAGATAAA 
               
               
                   
               
               
                 ATTCATAGTAAAATTAGTAGTAAATTGAAAGGAAACGGTTCAGCGTTTTC 
               
               
                   
               
               
                 ATGTACGAACAGCGCAAGTGACAATATTTTTGATGAGACAAGTTGTTATA 
               
               
                   
               
               
                 AGAATGAATTTAACAAAACAGAAAATATTAATTCAGTGAAAGCTTCAAAC 
               
               
                   
               
               
                 ATGAATCGTTTTGATACAAATATTATATGGGATTGTGATGGAAAAACAAA 
               
               
                   
               
               
                 ATATGAGCAAATTAATTTATGTGTTCCGCCTAGAAGAGAAAATATGTGTA 
               
               
                   
               
               
                 TAAAGGGGCTAGAACACTTGAACGAAACTAAACATTCTGATAATAAAACG 
               
               
                   
               
               
                 CTATTAAAAGAGCTTCAAGAAATTGCAAGTACTGAAGGGAAAGGCATATC 
               
               
                   
               
               
                 AAAAAATTTTAAACAAATGGATAGAGAAAATGATGACGGAATATGTGATG 
               
               
                   
               
               
                 CCATGAAGTACAGTTTTGCCGATTTGGCAGATATAGTAAGAGGTACAGAT 
               
               
                   
               
               
                 AATTACAAAAATTCTAATGGTAATAATAATAAAGTAGAGGAAAACCTTAA 
               
               
                   
               
               
                 AAAAATTTTCGAAAAAATACACAATATTAATAGTCTTAAAAAAGAATATA 
               
               
                   
               
               
                 GTAAGGACAAACCGGATTATCAAAGATTACGATCTGACTGGTGGGATACG 
               
               
                   
               
               
                 AATAGAAAAGAAATATGGAAAGCATTAACGTGTTCAGCAAGGGATAATAA 
               
               
                   
               
               
                 TAAAATATACAAGAAAGGGCAAAAAAATACTAATAATGGAAAGAATAAAT 
               
               
                   
               
               
                 GTGGAAATGAAGAGGATCCTCCTGATGATGATTATATTCCACAACCTTTT 
               
               
                   
               
               
                 CGTTGGTTACAAGAATGGAGTGAACATTTTTGTAGAGTTCAATATGATAA 
               
               
                   
               
               
                 TCTGAATAAACTGAAAGAAGAATGTGGAGAATGTAATGAAAATAAAAATG 
               
               
                   
               
               
                 GTTTAGCTTGTATGATGAATTCAAATATTAAAGATACAAAATGTATGAAT 
               
               
                   
               
               
                 TGCAAAGATGCATGCAAGGACTACAGAAATATGATTAACACATGGAATAG 
               
               
                   
               
               
                 TCAATGGAAAAAACAACAAGAAATATATAAAGAATTATATAATACTAAAA 
               
               
                   
               
               
                 ATAAAATAAATATtAATAAATGTAAGGTGATAGAATTTTTAGATAAAACA 
               
               
                   
               
               
                 AATGATACGTGTCACTATAAACCAGGAAGTGCAGAAAAGTTTCTTAAAGA 
               
               
                   
               
               
                 ATCTAGTCATTGTACTGACCTTACATTCGACAAAACAAAGAATTCAAATA 
               
               
                   
               
               
                 ATATTCCTTATGCCTTTGAAAATCCACCTGATGGATATAAAGTTTTATGT 
               
               
                   
               
               
                 GGTACAACATATAGAAAATCATGTAAAAAGCTAAAAAAATTAGGGATGAA 
               
               
                   
               
               
                 TTATACGTCAGAAAACAAAATTGATTTAAGTGGAGAAAACGCTAAGTGGG 
               
               
                   
               
               
                 AAAAACTTAATGATTTGATATATGTCCCTCCACGAACACAACAATTATGT 
               
               
                   
               
               
                 TTACAACCTTTGCAAACGTTGATATCACGTACCAATAAAACCACGAAAGT 
               
               
                   
               
               
                 AACAGAATATGATTTCTCCAGAGCATTACAAATATGTGCATATAATGAAG 
               
               
                   
               
               
                 CAAATTCTCTTCACAATTATTATAGTAAGTATGGCAAAGATTTTGTATTT 
               
               
                   
               
               
                 AGTGCTGGTAAGTCTCAAGATACTAAAGATGAAATAAAAACACACATTCT 
               
               
                   
               
               
                 TGAAAATATGAAAAGAAGCTTTGCTGATTATGGTAATTTAATTAAAGGAA 
               
               
                   
               
               
                 AGACCCAATACGAATATAATGGTTTAAACAAAAAGCTTCAGGATTACATA 
               
               
                   
               
               
                 AAGACTAATTTAAAATATAATGGAACTGACAGAAAAACAGGTGAAGATCT 
               
               
                   
               
               
                 TTGGAATAAACATAAATCCGATATTTGGAATTCTATGCTATGTGGATATA 
               
               
                   
               
               
                 ATGAAGAAAACCCAAGTGAACCACTTCATGATAAAGACATAAGATGTAAA 
               
               
                   
               
               
                 TTACCTGATAATGATAGTGAAGATGAATTTTTACGTTGGTTTCAAGAATG 
               
               
                   
               
               
                 GAGAGAAGATTTTTGTGTTATCAAAGGTATACTGATACAAAATGTGAAAG 
               
               
                   
               
               
                 ATGCATGTAATTTTAATAATTGTGAAGATGCAAATAATAAATCCATAAGA 
               
               
                   
               
               
                 TCTTGTCAAAAACCATGTGTCAAATACAAAACATGGGTTGAACAAAGAAA 
               
               
                   
               
               
                 AATTGAATACGAAAATCAAATCCAGAAATATAAAAATCTCAACAATAATT 
               
               
                   
               
               
                 CAAATGAAGGAAAAGAATCACTTTTATTTTTAAACGATAAATGTAAAGGG 
               
               
                   
               
               
                 AAATGTGAATGTATAGTTCAAAAAAAAAGTACTGATAATATCGATAAGAT 
               
               
                   
               
               
                 TTTTGAGGAATATCCTGAAGAATACAAAACGCAATGTGAATGTCAGCCTG 
               
               
                   
               
               
                 ATCCATGTAGTGACCTAAGTATTACAGATAGTGGATTTCCTGATGCAAGT 
               
               
                   
               
               
                 CCTTTTGGTGGAGGTCAACCACGTTCCGCGTGTCCTACAAGGCGAGGAAA 
               
               
                   
               
               
                 TCATAATAATTGCCCTACTGAAGAAATATGTAAAAAATATGATAGCTATA 
               
               
                   
               
               
                 TTAATGGTTGTCGCCCTAAAACATATCACGACAATACAAATAATTGGGAT 
               
               
                   
               
               
                 AGCAGAGGCATGCTAAACAGTTCATCTGAAAATGAAGGTGTATTAATTCC 
               
               
                   
               
               
                 TCCTAGAAGGCGACATTTATGCACAAGAAATATAATTAAAAATTTGTCAC 
               
               
                   
               
               
                 GTATTAAAAATAAAGATCATTTTAAAGATTACCTAATGAAATCTGCTTAT 
               
               
                   
               
               
                 GAAGAAGGAAAACTTTTAAGGGAAAAATATAGAAATAATAGTAGAGATGG 
               
               
                   
               
               
                 ATTAAATGCAATGATGTTCACTTTTGCTGATTATGCAGATATAGTTAAAG 
               
               
                   
               
               
                 GTACTGATATATTTGGCAGTATATTATCTCAAAAATTAGGTGAAATAACT 
               
               
                   
               
               
                 GGAATAAGCAATGATATAAATGAGCGTAAAAAATGGTGGAGTGAAATTAA 
               
               
                   
               
               
                 AAATAATATATGGGAAGTTATGTTGTGTTCTTATAATAGAACAAAGAATA 
               
               
                   
               
               
                 ATAATAATTTTTTCGGAAACATTGTGAGAGAAAACTGTAATGTTCCTAAC 
               
               
                   
               
               
                 ACAGATGAAAAGGACCAATTTTTGAGATGGTTACTTGAATGGGGTATACA 
               
               
                   
               
               
                 AGCATGTAAAGAAAAAAAAATCAGAAAACAAGCCCTTCAAACGAAGTGTT 
               
               
                   
               
               
                 ACTGTTCAAACCCAAACGAAATATCGGGTTCCGATATTATAAAACATTAC 
               
               
                   
               
               
                 CCTTGTAAAAGTGAACTCACCAAATATATACAATGGAATTTAATGATTAA 
               
               
                   
               
               
                 AGAATTATTAGATCAATTAAATATAAAATATCAAAATATAAAAGCATCTA 
               
               
                   
               
               
                 ATAATCCGAAAAATCCCTCGGAAATAAATGCAGAGGAATACATAGAAACA 
               
               
                   
               
               
                 GAACTTAAGGAAGGTGAATGTAATTTGGTTGATATAGAACGAATATATAA 
               
               
                   
               
               
                 TAAAATTAAACAAGAACATAATCCATTAAAAGAAATATTAATGTATTTAT 
               
               
                   
               
               
                 GTCCTAATTTAGAATTTCCTGATGATACATTCGAATACATTGGGAAAACC 
               
               
                   
               
               
                 GAAACTGAAGATACAACTATTGAACCAGAAACCCCTACATCCGATAACCC 
               
               
                   
               
               
                 TGAGGATAGTATTCCTTCCATTTCACCTGAAGACGTTCACCCAACAACAG 
               
               
                   
               
               
                 GAGAAGACACAAACATTTTCAACAGTAACATATTATCATCTACTATCCCT 
               
               
                   
               
               
                 TTTGGAATTGCTTTGGCCTTAAGTTCGATCGCTTTCCTGTTCCTGAAGaa 
               
               
                   
               
               
                 aaaaaccctatcccctgtggacctcttacgtgtacttgatatccataaag 
               
               
                   
               
               
                 gcgattatggaatacctacactgaaatccaaaaataggtatataccatat 
               
               
                   
               
               
                 agaagtggtacatataaaggcaaaacatatttatatgttgaaggagatag 
               
               
                   
               
               
                 tgatagtggacactactacgaagatacaactgatattacttcatccgaaa 
               
               
                   
               
               
                 gtgaatatgaagaaatggatatcaatgatatatatgtaccaggtagtcct 
               
               
                   
               
               
                 aaatataaaacattgatagaagtagtactggaaccatccaaaagtgatgg 
               
               
                   
               
               
                 tcatataccacatagtgctggtgaaccactagatgatatggtaggtacca 
               
               
                   
               
               
                 ctatatttacagatgaggaatggaatgaactgaaacacgattttatatca 
               
               
                   
               
               
                 caatatgtgcaacgtgaatcaatgggtgtaccacaatatgatgtatcaac 
               
               
                   
               
               
                 cgagttaccaatgaatataggaggtaatgttttagatgatggcatggaag 
               
               
                   
               
               
                 aaaaaccttttattacttctattcatgatagggatttatatactggagaa 
               
               
                   
               
               
                 gaatttagttataatattaatatgggtactaatagtatggatgatccaaa 
               
               
                   
               
               
                 atatgtatcaaataatgtatattctggtatcgatttaattaatgacacat 
               
               
                   
               
               
                 taagtggtaatcaacatattgatatatatgatgaagtattgaaaagaaaa 
               
               
                   
               
               
                 gaaaatgaattatttgggacaaattataagaaaaatatatcgaataaccg 
               
               
                   
               
               
                 tgtagctaaattaacaaataatgatccaattatgaaccaattagatttgt 
               
               
                   
               
               
                 tacataaatggttagatagacatagagatatgtgcaatacgtggaatacc 
               
               
                   
               
               
                 aaggaagaattattagataaattgaatgaacaatggaataaagataatga 
               
               
                   
               
               
                 tgctggtgatataccaagtgatagtaacaaaaagttgaatacggatgttt 
               
               
                   
               
               
                 cgatacaaatagatatggatgatcctaaaggaaagaaggaatttagtaat 
               
               
                   
               
               
                 atggatactatcttggatgatatagaagatgatatatattatgatgtaaa 
               
               
                   
               
               
                 tgatgaaaacccatttgtggatgatatacctatggatcataataaagtag 
               
               
                   
               
               
                 atgtaccaaagaaagtacatgttgaaatgaaaatccttaataatacatcc 
               
               
                   
               
               
                 aatggatccttggaaccagaatttcctatatcggatgtatggaatatat 
               
               
                   
               
               
                 aa 
               
             
          
         
       
     
         [0029]    In view of the above, the invention relates to PfEMP1 antigens encoded by or having the sequences given as SEQ ID NOS 1, 2, 3, 4, 5 or 6 above. One of skill will appreciate that PfEMP1 antigens encoded by sequences exhibiting a degree of homology or identity with the sequences provided herein, are also within the scope of this invention. In particular, PfEMP1 antigens encoded by sequences exhibiting at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 095%, 96%, 97%, 98% or 99% homology/identity with any of SEQ ID NOS; 1, 2, 3, 4, 5 or 6 are to be regarded as PfEMP1 antigens having potential utility in this invention. The invention further relates to recombinantly or synthetically generated PfEMP1 antigens encoded by or having a sequence having homology and/or identity to those given as SEQ ID NOS: 1, 2, 3, 4, 5 or 6. 
         [0030]    In all cases, the invention encompasses PfEMP1 antigens which, relative to those encoded by or having the sequences given as SEQ ID NOS: 1, 2, 3, 4, 5 or 6, comprise (i) one or more mutations—a mutation being characterised as an amino acid/nucleic acid deletion, addition, substitution and/or inversion and (ii) with respect to SEQ ID NOS: 2, 4 and/or 6, one or more conservative amino acid substitutions—the term “conservative substitution” embracing the act of replacing one or more amino acids of a protein or peptide with an alternate amino acid with similar properties and which does not substantially alter the physico-chemical properties and/or structure or function of the native (or wild type) protein. Of course, one of skill will appreciate that the degeneracy of the genetic code permits substitution of one or more bases in a codon without changing the primary amino acid sequence. Consequently, although SEQ ID NOS: 1, 3 and 5 encode specific PfEMP1 antigens, the degeneracy of the code may be exploited to yield variant nucleic acid sequences which encode the same primary amino acid sequences. 
         [0031]    One of skill will appreciate that the PfEMP1 antigens described herein may be produced recombinantly using expression vector and/or systems. In this regard, the invention provides vectors, for example bacterial expression vectors (pET or pGEX system vectors for example) comprising nucleic acid sequences encoding PfEMP1 antigens of this invention. One of skill will appreciate that the nucleic acid sequences encoding PfEMP1 antigens of this invention may be codon optimised to ensure maximum expression in a particular expression system. The invention further provides host cells (for example bacterial cells ( E. coli )) transformed with the vectors of this invention. 
         [0032]    In other embodiments, the invention provides medicaments for use in raising immune responses in humans—the medicaments comprising PfEMP1 antigen(s) (or fragments thereof) selected from the group consisting of HB3var6; TM284var1; and ITvar60. Additionally, the invention provides methods of raising immune responses in humans, comprising administering to a human subject, an immunogenic amount of one or more PfEMP1 antigen(s) (or a fragment thereof), the PfEMP1 antigens being selected from the group consisting of HB3var6; TM284var1; and ITvar60. 
         [0033]    One of skill will appreciate that the term “fragment” encompasses immunogenic and/or antigenic fragments of the PfEMP1 antigens described herein—including those encoded by SEQ ID NOS 1, 2, 3, 4, 5 or 6. In one embodiment, a fragment may comprise a complete domain or region of the PfEMP1 antigen—including for example, one or more of the Duffy Binding Like (DBL) domains. In other embodiments, suitable fragments may comprise between about 10 and n−1 amino acids—where “n” is the total number of amino acids of the complete PfEMP1 antigen (HB3var6: n=3424; TM284var1: n=2790; ITvar60: n=2716). By way of example, a PfEMP1 antigen fragment may comprise or consist of about 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2700, 2800, 2900, 3000, 3100, 3200, 3300 or 3400 (contiguous) PfEMP1 amino acids. In other embodiments, a PfEMP1 antigen fragment suitable for use in this invention may comprise at least one epitope capable of raising a cross-reactive/protective immune response. 
         [0034]    In addition to identifying PfEMP1 antigens suitable for raising cross reactive and/or protective immune responses in humans, the inventors have further identified specific fragments, regions and/or domains of the PfEMP1 antigen(s) which are also suitable for raising cross reactive and/or protective immune responses in humans. 
         [0035]    As such, in a fourth aspect, the invention provides one or more Duffy Binding Like (DBL) domains of  P. falciparum  Erythrocyte Membrane Protein-1 (PfEMP1) for use in raising immune responses in humans. 
         [0036]    In a fifth aspect, the invention provides use of one or more Duffy Binding Like (DBL) domains of  P. falciparum  Erythrocyte Membrane Protein-1 (PfEMP1) for the manufacture of a medicament for raising immune responses in humans. 
         [0037]    In a sixth aspect, the invention provides a method of raising an immune response in a human, the method comprising the step of administering an amount of one or more Duffy Binding Like (DBL) domains of  P. falciparum  Erythrocyte Membrane Protein-1 (PfEMP1) to a human subject. 
         [0038]    As with the PfEMP1 antigens, exploited in the first, second and/or third aspects of this invention, the DBL domains exploited in the fourth, fifth and sixth aspects are suitable for raising cross reactive and/or protective immune responses/antibodies. That is to say, an immune response raised against a PfEMP1 DBL domain provided by this invention may comprise antibodies which exhibit a degree of specificity, selectivity and/or affinity for the PfEMP1 antigens of many different  P. falciparum  strains. 
         [0039]    In one embodiment, the DBL domains for use in the fourth, fifth and/or sixth aspects of this invention are one or more selected from the group consisting of:
       (i) the NTS-DBL1α domain;   (ii) the DBL4ε domain; and   (iii) a(n immunogenic/antigenic) fragment of (i) and/or (ii).       
 
         [0043]    In one embodiment, the NTS-DBL1α domains for use in the fourth, fifth and/or sixth aspects of this invention are derived from the PfEMP1 variant HB3var6, TM284var1 and/or ITvar60. In one embodiment, the NTS-DBL1α domains for use in the fourth, fifth and/or sixth aspects of this invention are derived from HB3var6, and/or TM284var1. 
         [0044]    In other embodiments, the DBL4ε domain is obtained from the PfEMP1 variant ITvar60. 
         [0045]    In view of the above, the invention provides antigens for use, uses and methods comprising one or more PfEMP1 DBL domains selected from the group consisting of:
       (i) the NTS-DBL1α domain of PfEMP1 variant HB3var6;   (ii) the NTS-DBL1α domain of PfEMP1 variant TM284var1;   (iii) the DBL4ε domain of PfEMP1 variant ITvar60; and   (iv) a (n immunogenic/antigenic) fragment of any of (i)-(iii) above.       
 
         [0050]    It should be understood that all references to DBL domains encompass DBL domain fragments. As defined above, a DBL fragment for use in this invention may be immunogenic and/or antigenic and thus capable of generating protective/cross-reactive immune responses in humans. In other words, a DBL fragment may be capable of raising (or generating) a host immune response which is substantially similar or identical to an immune response raised when a host is administered a complete DBL antigen. DBL domain fragments suitable for use may include those comprising or consisting of 10, 20, 30, 40 or 50 (preferable contiguous) amino acids of the complete DBL sequence. In one embodiment the fragments may comprise or consist of any number of amino acids of a DBL domain from about 10 amino acids to about n−1 amino acids, where n is the total number of amino acids of the relevant DBL domain. 
         [0051]    For convenience, the various PfEMP1 antigens, mutants/variants, fragments and domains thereof described in this specification shall be collectively referred to as “PfEMP1 antigens”. Specifically, the following are to be regarded as PfEMP1 antigens according to this invention:
       (i) PfEMP1 (including mutants or sequence variants thereof as defined above);   (ii) an IgM binding rosetting PfEMP1;   (iii) PfEMP1 variant HB3var6;   (iv) PfEMP1 variant TM284var1;   (v) PfEMP1 variant ITvar60;   (vi) a PfEMP1 DBL domain;   (vii) the NTS-DBL1α domain;   (viii) the DBL4ε domain;   (ix) the NTS-DBL1α domain of PfEMP1 variant HB3var6;   (x) the NTS-DBL1α domain of PfEMP1 variant TM284var1;   (xi) the DBL4ε domain of PfEMP1 variant ITvar60; and   (x) a(n immunogenic/antigenic) fragment of any of (i)-(ix).       
 
         [0064]    One of skill will appreciate that the PfEMP1 antigens, fragments and/or domains may find particular application as vaccines for raising immune responses. While immunity to malaria can develop naturally, repeated exposure to the malarial parasite (for example,  P. falciparum ) is required. Indeed, without wishing to be bound by theory, this invention is based, in part, upon the discovery that antibodies to PfEMP1 antigens are detected in children recovering from severe (cerebral) malaria—moreover the inventors have noted that these antibodies exhibit a degree of specificity for the specific PfEMP1 antigens described herein (namely PfEMP1 variants, HB3var6; TM284var1 and ITvar60). As such, PfEMP1 variants HB3var6; TM284var1 and ITvar60 may represent exemplary vaccine candidates. 
         [0065]    Of course, in the young and people from areas of the world where malaria is not a problem (for example Europe), levels of natural immunity are low as there has been insufficient exposure to the malaria parasite. Moreover in a person with low/no immunity to a  Plasmodium  sp. (for example  P. falciparum ), infection can bring about severe malaria. One of skill in this field will appreciate that instances of severe malaria can result in secondary complications including death, coma (cerebral malaria), respiratory difficulties, hypoglycaemia and severe anaemia. As such, children and/or travelers not regularly exposed to malarial parasites are susceptible to developing malaria and/or severe malaria. 
         [0066]    The vaccines provided by this invention may be used to raise immune responses in children, wherein the immune response is protective against malaria and the development of severe malaria. In other embodiments, the invention provides vaccines which may be used to raise immune responses in humans who are infrequently exposed to malaria or who have not been exposed to malaria—this may include, for example travelers from those areas of the world where malaria does not exist. As above, immune responses raised through use of the vaccines provided by this invention may offer protection against the development of malaria and severe malaria. 
         [0067]    In a seventh aspect, the invention provides the PfEMP1 antigens of this invention as compositions comprising a pharmaceutically acceptable diluent, carrier and/or excipient. In one embodiment, the compositions of this invention may be sterile. 
         [0068]    The compositions may be formulated or prepared for parenteral administration—including, for example injection by subcutaneous, intradermal, intramuscular and/or intravenous injection. In one embodiment compositions of this invention may comprise aqueous or oleaginous suspensions formulated according to the known art using suitable dispersing, wetting and/or suspending agents. In this regard, one of skill will appreciate that an acceptable carrier, diluents and/or excipient may comprise 1,3-butanediol, water, Ringer&#39;s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable compositions according to this invention. 
         [0069]    In one embodiment, the compositions and vaccines provided by this invention may further comprise one or more adjuvants. One of skill will appreciate that the term “adjuvant” may encompass any agent which enhances or promotes the immune response of an immunised host to the antigens of a vaccine or immunogenic composition. Suitable adjuvants may include alum and/or freund&#39;s adjuvant. 
         [0070]    The compositions and/or vaccines provided by this invention may be administered by either single or multiple dosages of an effective amount. Preferably two or more doses are administered over a predetermined period of time. 
         [0071]    By way of example, an initial vaccine dose (primary immunisation) may be administered followed by one or more booster doses (secondary immunisation(s)) given at 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 week intervals after the primary immunisation. 
         [0072]    Other modes of administration are contemplated by the present invention and include intranasal, intraperitoneal, intrathecal, rectal, infusion and intrapulmonary administration. Administration may also be by nasal drip, aerosol, infusion through the skin or membrane surfaces or ingestion. 
         [0073]    In an eight aspect, the invention provides a vaccine comprising a PfEMP1 antigen, for use in treating or preventing malaria or severe malaria. In one embodiment, the vaccines of this invention may prevent, limit or inhibit the development of severe malaria. One of skill will appreciate that a vaccine may be administered prophylactically to prevent the development of malaria or severe malaria in susceptible populations. A susceptible population may comprise children or juveniles and/or those not routinely, regularly exposed to the malarial parasite (for example European travelers). 
         [0074]    In a ninth aspect, the invention provides the use of a vaccine comprising a PfEMP1 antigen in the manufacture of a medicament for treating or preventing malaria and/or severe malaria. 
         [0075]    The PfEMP1 antigens provided by this invention may be combined with other antigens, for example other  Plasmodium  ( falciparum ) antigens and/or other bacterial or viral antigens to yield a combined vaccine for use. In a tenth aspect, the invention provides a method of treating or preventing malaria/severe malaria, the method comprising administering a vaccine to a human subject, the vaccine comprising an immunogenic amount of a PfEMP1 antigen. 
         [0076]    In an eleventh aspect, the invention provides one or more of the PfEMP1 antigens described herein, for use in diagnosing or detecting malaria and/or severe malaria, in human subjects—particularly children (i.e. young or juvenile human subjects). The diagnostic procedures provided by the eleventh aspect of this invention may comprises the step of detecting a level of PfEMP1 antigen in a sample provided by a subject suspected of suffering from malaria/severe malaria, or being infected with a malaria parasite (for example  P. falciparum ). One of skill will appreciate that levels of PfEMP1 antigen may be detected by way of immunological techniques such as ELISA (exploiting antibodies exhibiting affinity, specificity and/or selectivity for a predetermined PfEMP1 antigen), immunoblot, SDS PAGE and/or PCR based techniques which probe samples for levels of  P. falciparum  nucleic acid—in particular, those sequences which encode PfEMP1 antigens. 
         [0077]    In a twelfth aspect, the invention provides antibodies exhibiting a degree of affinity, specificity and/or selectivity for one or more of the PfEMP1 antigens described herein. Without wishing to be bound by theory, interventions that disrupt rosettes could be valuable adjunctive therapies for severe malaria. By way of example, antibodies which exhibit an affinity, specificity and/or selectivity for one or more of the PfEMP1 antigens described herein, may be exploited to unblocking the congested blood flow in the microvasculature that is the primary pathological event caused by rosetting parasites. “Rosette-busting” interventions of this type may be analogous to clot-busting drugs used after stokes and heart attacks, to remove the obstruction to microvascular blood flow caused by rosetting cells, and restore normal tissue oxygenation. 
         [0078]    The term “antibodies” includes polyclonal antibodies as well as monoclonal antibodies and embraces antigen and/or epitope binding antibody fragments such as, for example, Fab, Fab 2 , scFv, V H  domain and/or V L Domain fragments. The production and isolation of polyclonal/monoclonal antibodies specific for protein/peptide sequences is routine in the art, and further information can be found in, for example “Basic methods in Antibody production and characterisation” Howard &amp; Bethell, 2000, Taylor &amp; Francis Ltd. The antibodies described herein may be conjugated to detectable moieties such as, for example, chemiluminescent and/or fluorescent moieties. The antibodies described herein may be used in diagnostic procedures, to, for example, detect  P. falciparum  in samples, or to diagnose malaria or severe malaria (as caused by  P. falciparum ) in humans. Any diagnostic procedure may involve contacting a sample provided by a human subject, with an antibody of this invention—the detection of antibody bound to a PfEMP1 antigen indicating that the sample has been provided by a human infected with the  P. falciparum  parasite. The term “sample” may relate to any biological material and/or fluid provided by a human subject and may include, for example, samples of blood, serum, plasma, sweat, saliva and/or tissue biopsies or cell samples. 
         [0079]    The antibodies provided by this invention may further be used in passive immunisation protocols to treat or prevent malaria and/or severe malaria. As such, one embodiment of this invention provides one or more antibodies according to the twelfth aspect of this invention for use in treating and/or preventing malaria/severe malaria. 
         [0080]    As such, a thirteenth aspect of this invention provides an antibody exhibiting a specificity, selectivity and/or affinity for a PfEMP1 antigen described herein, for use in treating or preventing malaria and/or severe malaria. The invention further provides use of an antibody exhibiting a specificity, selectivity and/or affinity for a PfEMP1 antigen in the manufacture of a medicament for treating or preventing malaria. In a yet further aspect, the invention provides a method of treating or preventing malaria, the method comprising administering a therapeutically effective amount of an antibody exhibiting a specificity, selectivity and/or affinity for a PfEMP1 antigen, to a human subject in need thereof. 
         [0081]    In a fourteenth aspect, the invention provides one or more compounds capable of recognising the interaction between PfEMP1 and uninfected erythrocytes, for use in treating and/or preventing malaria/severe malaria. By way of example, the compound may comprise a small molecule (for example a protein/peptide, nucleic acid carbohydrate, organic/inorganic molecule) or an antibody exhibiting affinity, selectivity and/or specificity for one or more of the PfEMP1 antigens, fragments, epitopes and/or domains provided by this invention. In addition, this invention may extend to the provision of small molecules which may be used in methods and medicaments for treating or preventing rosette formation. 
     
    
     
       DETAILED DESCRIPTION 
         [0082]    The present invention will now be described in detail with reference to the following figures which show: 
           [0083]      FIG. 1 : Identification of key surface antigens (Group A PfEMP1 variants) of  P. falciparum  rosetting parasites and production of recombinant proteins for immunization. a) PfEMP1 domain architecture of the predominantly expressed variants from  P. falciparum  rosetting laboratory strains. The previously described rosetting variant ITvar9 [12,20] is shown for comparison. Domain types are based on conserved motifs [6,45]. NTS: N-terminal segment; DBL: Duffy Binding Like; CIDR: Cysteine-rich InterDomain Region; ATS: Acidic terminal Segment; TM: TransMembrane region. *The IT isolate was originally from Brazil, however following cross-contamination in the early 1980s, current IT/FCR3 strains are thought to be of South-East Asian origin [65]. b) Northern blots of RNA from isogenic rosetting (R+) and non-rosetting (R−) parasites probed with a PfEMP1 domain from the rosette-specific variant for each strain (R+DBL probe, high stringency) and with an Exon II probe (moderate stringency), which detects all var genes [44]. Arrows indicate the major rosette-specific variants. Equal loading of R+ and R− RNA was confirmed by staining with ethidium bromide (Et Br). c) Production of recombinant NTS-DBLα domains in  E. coli  to immunize rabbits. 1: TM180var1, 2: Muz12var1, 3:TM284var1, 4: ITvar60, 5:HB3var6. M: molecular weight marker; R: reduced; NR: non-reduced. 
           [0084]      FIG. 2 : Antibodies to PfEMP1 recognize the surface of live infected erythrocytes, and show cross reactivity between  P. falciparum  rosetting laboratory strains. a) Immunofluorescence assay (IFA) with antibodies to ITvar60 (25 μg/ml) tested on the homologous parasite (IT/PAR+). Left panel: DAPI staining to show position of infected erythrocytes. Right panel: punctate fluorescence over the surface of live infected erythrocytes. b) Flow cytometry to show the titration of antibodies to ITvar60 against IT/PAR+ parasites, compared to a non-immunized rabbit IgG control. The end titre (defined here as the lowest concentration at which more than 50% of infected erythrocytes were positive by IFA/flow cytometry) was 0.1 g/ml. c) PfEMP1 antibodies (four-fold dilutions of total IgG starting at 400 μg/ml) were tested against  P. falciparum  laboratory strains with various different adhesion phenotypes as indicated. The end titre for each antibody:parasite combination (defined as above) is shown inside each rectangle. Homologous antibody: parasite combinations are outlined in bold. Negative controls are non-immunised rabbit IgG, and antibodies against NTS-DBLα from a non-rosetting group A PfEMP1 variant (HB3var3, expressed by HB3-HBEC which are non-rosetting parasites selected for binding to human brain endothelial cells). *The HB3R+ parasites contain a subpopulation of HB3var3-expressing infected erythrocytes that are distinct from the HB3var6-expressing cells, Table S1). 
           [0085]      FIG. 3 : Antibodies to PfEMP1 show cross-reactivity in rosette inhibition and induction of phagocytosis in  P. falciparum  rosetting laboratory strains. a) Rosette inhibition assays to determine the dose-dependent effects of PfEMP1 antibodies on homologous and heterologous rosetting laboratory strains. Data are compared to a control with no added antibody, which contained at least 40% of infected erythrocytes in rosettes. Mean and standard deviation of triplicate values are shown. IC50: concentration of antibody giving 50% rosette inhibition. b) Rosette inhibition assay as above with 1 mg/ml of antibody, except for the Anti-Ros pool which consisted of a mixture of 0.1 mg/ml of each antibody (to HB3var6, TM284var1, ITvar60, Muz12var1, TM180var1 and ITvar9). Controls are as for  FIG. 2   c . c) Phagocytosis assay of opsonised IT/PAR+ infected erythrocytes co-incubated with the monocytic cell line Thp-1 [12]. Data are shown as percentage of the positive control opsonised with a rabbit anti-human erythrocyte antibody. 
           [0086]      FIG. 4 : Antibodies to PfEMP1 show cross-reactivity in surface recognition and rosette inhibition of  P. falciparum  clinical isolates. a) Flow cytometry of clinical isolate MAL43 with 0.4 mg/ml of total IgG from a non-immunised rabbit (negative control, left panel) and antibodies to TM284var1 (middle panel). Infected erythrocytes stained with Hoescht are in the right half, and antibody-positive infected erythrocytes stained with Alexa Fluor 488 are in the upper right quadrant. An overlay of histograms (right panel) shows a clear population of stained infected erythrocytes. b) Fresh clinical isolates tested with PfEMP1 antibodies for surface reactivity (IFA and flow cytometry at 0.4 mg/ml) and rosette inhibition (1 mg/ml). The percentage rosette inhibition is shown inside each rectangle for all isolate:antibody combinations with &gt;25% rosette inhibition. The controls are as for  FIG. 2   c , and the Anti-Ros Pool as for  FIG. 3   b . The Anti-Ros pool was tested for rosette inhibition only. c) Flow cytometry histograms to show negative controls, anti-PfEMP1 positive and IgM-positive infected erythrocytes for five clinical isolates. Other isolates were tested by IFA only. The “negative PfEMP1 Ab” was antibody to TM180var1 and the IgM negative control was a mouse IgG1 isotype control. 
           [0087]      FIG. 5 : Cross-reactive PfEMP1 antibodies do not recognise human IgM. a) ELISA for recognition of human IgM. The positive control is an anti-human IgM antibody. The mean and SD of Optical Density (OD) values from triplicate wells are shown. b) Flow cytometry of IT/PAR+ parasites grown with and without human IgM and stained with anti-TM284var1 antibodies. 
           [0088]      FIG. 6 : Selection for IgM yields rosetting infected erythrocytes that cross-react with PfEMP1 antibodies. a) The culture-adapted Kenyan isolate 9197 was selected three times with anti-human IgM coated Dynabeads. Comparison of the unselected and selected lines by flow cytometry showed that the IgM-selected parasites were recognised by cross-reactive PfEMP1 antibodies to HB3var6. b) An IFA with dual staining (AlexaFluor 488 anti-rabbit IgG to detect PfEMP1 antibody and AlexaFluor 594 anti-mouse IgG to detect anti-human IgM) shows that the same subpopulation of infected erythrocytes bound both IgM and HB3var6 antibodies. 
           [0089]      FIG. 7 . Opsonisation and induction of phagocytosis by PfEMP1 antibodies. Infected erythrocytes were stained with ethidium bromide and opsonised with PfEMP1 antibodies before incubation with the monocytic cell line Thp-1. Thp-1 cells that phagocytosed infected erythrocytes were detected by flow cytometry. a) parasite strain TM284 b) parasite strain HB3R+c) parasite strain IT/R29 d) parasite strain TM180. Data are shown as percentage of the positive control opsonised with a rabbit anti-human erythrocyte antibody. The “Non Ros Group A” negative control consists of antibodies to HB3var3, a PfEMP1 variant that is not involved in rosetting. HB3R+ parasite culture contains a subpopulation of infected erythrocytes expressing HB3var3 (see Table S1) which explains why phagocytosis was induced in this case. The “Control Rabbit IgG” is a negative control consisting of purified IgG from a non-immunized rabbit. 
           [0090]      FIG. 8 : DBL domains from the ITvar60 variant. The underlined regions indicate the recombinant proteins generated. Abbreviations as for  FIG. 1   a.    
           [0091]      FIG. 9 : Inhibition of rosetting by anti-DBL antibodies. Rosette inhibition assays to determine the dose-dependent effects of various ITvar60 PfEMP1 antibodies on IT/PAR+ parasites. Data are compared to a control with no added antibody, which contained at least 40% of infected erythrocytes in rosettes. Mean and standard deviation of triplicate values are shown. 
           [0092]      FIG. 10 : Opsonisation and phagocytosis of infected erythrocytes by ITvar60 antibodies. Infected erythrocytes were stained with ethidium bromide and opsonised with PfEMP1 antibodies before incubation with the monocytic cell line Thp-1. Thp-1 cells that phagocytosed infected erythrocytes were detected by flow cytometry. 
           [0093]      FIG. 11 : Antibodies to various domains of ITvar60 tested against IgM binding rosetting parasites (strain HB3R+ 1st column, strain TM284 2nd column, strain 9197IgM+ 3rd column). Surface reactivity was assessed by flow cytometry, with positive infected cells detected in the upper right quadrant of each dot plot. Positive controls are variant-specific antibodies to each parasite strain (top row). Negative controls are non-immunized rabbit IgG (bottom row) and antibodies to domains from a non-IgM binding variant (ITvar9). Anti-DBL4ε of ITvar60 recognises all three strains, while anti-DBL5ε and anti-DBL2γ of ITvar60 recognise strain TM284 only. 
           [0094]      FIG. 12 : Antibodies to various domains of ITvar60 tested against non-IgM binding rosetting parasites (strain TM180 1st column, strain SA075 2nd column, strain IT/R29 3rd column, strain Muz12 4th column). Surface reactivity was assessed by flow cytometry, with positive infected cells detected in the upper right quadrant of each dot plot. Positive controls are variant-specific antibodies to each parasite strain (top row). Negative controls are non-immunized rabbit IgG (bottom row). Antibodies to ITvar60 domains do not recognise any of these parasite strains. 
           [0095]      FIG. 13 : Case-control study of recognition of live IT/PAR+ infected erythrocytes (expressing the ITvar60 antigen) by plasma samples from young Kenyan children with severe (cerebral) malaria (CM-cases) compared to age- and time of admission-matched controls (CM-controls). Data from flow cytometry; MFI: mean fluorescence intensity. 
           [0096]      FIG. 14 : Case-control study of recognition of live IT/R29 infected erythrocytes by plasma samples from young Kenyan children with severe (cerebral) malaria (CM-cases) compared to age- and time of admission-matched controls (CM-controls). Data from flow cytometry; MFI: mean fluorescence intensity. 
           [0097]      FIG. 15 : Case-control study of recognition of live SA075R+ infected erythrocytes by plasma samples from young Kenyan children with severe (cerebral) malaria (CM-cases) compared to age- and time of admission-matched controls (CM-controls). Data from flow cytometry; MFI: mean fluorescence intensity. 
           [0098]      FIG. 16 : A: Plasma from severe malaria cases tested on parasites expressing HB3var6; B: plasma from uncomplicated malaria controls tested on parasites expressing HB3var6. 
           [0099]      FIG. 17 : A: Plasma from severe malaria cases tested on parasites expressing SA075var1; B: plasma from uncomplicated malaria controls tested on parasites expressing SA075var1. 
           [0100]      FIG. 18 : ELISA showing polyclonal antibody response in mice immunised with TM284var1. 
       
    
    
     EXAMPLE 1 
     Materials and Methods 
     Parasites and Parasite Culture. 
       [0101]    The  P. falciparum  laboratory strains (HB3, Muz12, IT/R29, IT/PAR+, TM180 and TM284) were cultured in supplemented RPMI [59] and selected for rosetting as described [60]. All cultures were checked regularly to exclude mycoplasma contamination [61]. The parasites were genotyped with primers to MSP1, MSP-2 and GLURP [62] and were genetically distinct apart from IT/PAR+ and IT/R29 which share the same genotype but transcribe different PfEMP1 variants. Other parasite strains used were unselected HB3 and 3D7 (CD36-binding), IT/A4 (CD36 and ICAM-1 binding) and three strains selected for binding to human brain endothelial cells (HB3-HBEC, 3D7-HBEC and IT-HBEC, Claessens et al, in press). Clinical isolates were from Cameroon (CAM1), Kenya (KEN7, KEN14, KEN17, 9197, SA075), Mali (MAL27, MAL34, MAL43, MAL81, MAL103) and The Gambia (GAM627). The Malian isolates and KEN7, KEN14 and KEN17 were cryopreserved from previous studies [30,46]. All clinical isolates were examined in the first cycle of in vitro growth except for KEN7, KEN14 and KEN17 (third cycle) and 9197 and SA075 which had been adapted to culture, cloned and selected for rosetting over 3-4 months of in vitro growth. The IgM binding phenotype of the rosetting strains was determined by immunofluorescence assay (IFA) with an anti-human IgM monoclonal antibody (Serotec MCA1662) as described [38,44]. 
       Ethics Statement. 
       [0102]    Collection of clinical isolates (blood samples) from malaria patients was carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from the patients&#39; parents or guardians and was approved by the Lothian Regional Ethical Review Committee (LREC/2002/4/34), the KEMRI Ethical Review Committee, the Gambia Government/MRC Laboratories Joint Ethics Committee, the Cameroon Ministry of Public Health Regional Ethics committee and the University of Bamako Institutional Review Board. Animal immunisations were carried out commercially by BioGenes GmbH (Berlin, Germany) according to European Union guidelines 86/609/EWG of 24.11.1986 and the European Agreement of 18.3.1996 for protection of animals used for scientific purposes. 
       Var Gene Expression Profiling and Var Gene Sequencing. 
       [0103]    RNA extraction and var gene expression profiling were carried out as described previously [24] and in Table S1. The full-length sequence of each predominant rosette-specific var gene was derived from the sequence tag by: a) extraction from parasite genome databases (HB3 at http://www.broadinstitute.org and IT at www.sanger.ac.uk) b) PCR-walking, cloning and sequencing using degenerate primers to upstream and downstream PfEMP1 regions [63] for Muz12var1. c) PCR-walking, cloning and sequencing using vectorette libraries [20] for TM284var1 and TM180var1. 
       Northern Blotting. 
       [0104]    RNA extraction and Northern blotting of isogenic rosetting and non-rosetting pairs of parasites was carried out with Digoxigenin-labelled RNA probes as described [44]. RNA from each parasite strain was hybridised with a specific probe representing one DBL domain from the homologous rosette-specific var gene, as well as an exon II probe to detect all var genes. 
       Recombinant Proteins and Polyclonal Antibodies. 
       [0105]    Recombinant proteins were produced as described previously [12]. The domain boundaries for the NTS-DBL1 recombinant proteins for each rosette-specific variant were as follows: HB3var6 Met1-Pro473; TM284var1 Met1-Pro457; ITvar60 Met1-Pro464; Muz12var1 Met1-Pro458; TM180var1 Met1-Pro485. The non-rosetting group A PfEMP1 variant HB3var3 (Met1-Pro468) was used as a control. Each protein was used to immunize two rabbits which had been pre-screened to avoid animals with pre-existing natural antibodies to human erythrocytes or malaria parasites. Immunization, serum collection and total IgG purification were carried out by BioGenes GmBH (Berlin, Germany). 
       Immunofluorescence Assays (IFA). 
       [0106]    Immune and pre-immune sera were tested in IFA with live infected erythrocytes as described [12,44]. Out of each pair of immunized rabbits, the serum giving the brightest fluorescent signal with the lowest background was chosen for purification of total IgG. In all cases, both rabbit sera gave positive PfEMP1-staining, with only minor differences in intensity of staining. 
       Flow Cytometry. 
       [0107]    Staining for flow cytometry was carried out as for IFA [12,44], except that Hoescht (1.25 μg/ml) was used instead of DAPI to stain infected erythrocytes and 50 μg/ml fucoidan was added after the secondary incubation washes to disrupt rosettes. Cells were fixed with 0.5% paraformaldehyde, with 50 μg/ml fucoidan added to prevent rosettes from re-forming, and 500,000 events per sample were analysed on a Becton-Dickinson LSRII flow cytometer. 
       Rosette Inhibition Experiments. 
       [0108]      P. falciparum  cultures at ring stage were incubated overnight with antibodies and controls at various dilutions, and rosetting assessed the next day by microscopy as described [12]. 
       Phagocytosis Assays. 
       [0109]    Phagocytosis experiments with Thp-1 cells were as described previously [12] except that fucoidan (200 μg/ml) was used for parasite purification and rosette disruption. The positive control was parasite culture opsonized with a rabbit anti-human erythrocyte antibody (ab34858, ABCAM, Cambridge, UK). Muz12var1 antibodies were not included in the phagocytosis assays because they show some background binding to uninfected erythrocytes. 
       IgM ELISA. 
       [0110]    The ability of PfEMP1 antibodies to cross react with human IgM was tested using purified human IgM (5 μg/ml, Rockland) coated onto an ELISA plate at 4° C. overnight. After blocking for 1 hour in PBS containing 0.05% Tween 20 (PBST) and 5% milk, wells were incubated with 10, 1 and 0.1 μg/ml of rabbit anti-NTS DBL1α antibodies in PBST containing 1% milk (PBS™). After 1 hour incubation at room temperature, wells were washed with PBST and incubated with 1:10,000 anti-rabbit IgG-HRP (Sigma) in PBS™ for a further hour. After washing as above, reactions were developed by incubating the wells with substrate 3,3′,5,5′-tetramethylbenzidinedihydrochloride (Sigma) according to the manufacturer&#39;s instructions and absorbance was measured at a wavelength of 450 nm. As a positive control, a rabbit anti-human IgM F(ab′)2-HRP (DAKO) was used at 1:100 (10 μg/ml), 1:1000 (1 g/ml) and 1:10000 (0.1 μg/ml). 
       Surface Cross-Reactivity of PfEMP1 Antibodies in the Absence of IgM. 
       [0111]    Pooled human serum was depleted of IgM by three successive rounds of incubation for 45 mins at room temperature on a rotating wheel (15 rpm) with an equal volume of anti-human IgM (μ-chain specific)-agarose (Sigma A9935). The absence of IgM in the absorbed serum was confirmed by western blotting with an anti-human IgM monoclonal antibody. IT/PAR+ parasites were grown from ring stage overnight in supplemented RPMI with 10% IgM-depleted serum, and an aliquot (positive control culture) was incubated with 1 mg/ml of human IgM (Calbiochem) for 1 hour at 37° C. The IgM-negative and IgM-positive cultures were then washed and testing for surface reactivity with cross-reactive PfEMP1 antibodies to TM284var1 NTS-DBL1α by flow cytometry as described above. 
       Selection for IgM-Positive Infected Erythrocytes. 
       [0112]    Parasites were selected for IgM-positive infected erythrocytes using M-450 Epoxy Dynabeads (Dynal) coated with mouse anti-human IgM antibodies (Serotec MCA1662) as described [64]. 
       Software. 
       [0113]    Flow cytometry data were analysed using FlowJo software (Tree Star Inc.), DNA sequence analysis was done using DNAstar Lasergene (DNAstar Inc.) and graphing and statistical analysis using Prism (GraphPad Software). 
         [0000]    Therapeutic mAbs. 
         [0114]    Monoclonal antibodies with specificity for epitopes of the PfEMP1 variant, TM284var1 have been produced. These antibodies will be used to investigate possibility of developing a therapeutic monoclonal antibody cocktail to reverse rosetting. 
       Results 
     Identification of PfEMP1 Variants Transcribed by Rosetting Parasites 
       [0115]    To identify the key surface antigens of rosetting parasites, five  P. falciparum  laboratory strains (three IgM binding, two non-IgM binding) originating from different countries were grown in vitro and selected for the rosetting phenotype. For each strain, isogenic rosette positive (R+) and rosette negative (R−) populations were selected in parallel [20], and their var gene transcription profiles examined by analysis of short PfEMP1 sequence tags [24]. The rosette-specific variant in each strain was identified as the predominant var gene transcribed by the rosetting population (comprising between one third to one half of all the var gene sequences detected) that was absent/rare in the non-rosetting population (an example is shown in Table S1). The full-length sequence of each predominant rosette-specific var gene was obtained from the sequence tag as described in the methods. The rosetting variants were mostly group A ( FIG. 1   a ), defined by the presence of a conserved upstream sequence (UpsA) and a characteristic N-terminal domain type (called DBLα1 or “Cys2”) that is associated with severe malaria [18,24,26]. The variants from the IgM binding rosetting parasites form a distinct subset that share an unusual PfEMP1 architecture, containing a triplet of domains that occur rarely in PfEMP1 (DBLE and DBLZ) [6]. The binding site for non-immune IgM lies within these DBLε/ζ domains [43,44](AG and JAR, unpublished data). The IgM binding domain triplet is linked via at least one other domain (DBLγ) to a typical group A PfEMP1 head-structure [16,18,45]( FIG. 1   a ). DBLα domains from group A PfEMP1 variants fall into eight subclasses (DBLα1.1 to DBLα1.8) based on sequence homology [6]. The rosetting variants described previously (ITvar9, Palo Alto varO and PF13 — 0003) [11,20,21] are all of the DBLα1.6 subclass. The rosette-specific variants identified here are either DBLα1.5 (HB3var6 and Muz12var1), DBLα1.8 (TM284 and ITvar60) or DBLα2 (a non-group A type, TM180var1) [6]. 
         [0116]    Despite the observed similarities in PfEMP1 architecture, there was considerable sequence diversity amongst the rosette-specific variants from different parasite strains, with the rosette-mediating domain (NTS-DBLα) [11,20,21] showing pair-wise amino acid identities of between 38.9% (ITvar60:TM180var1) and 62.6% (ITvar60:TM284var1) (Table S2). The other extracellular domains from the rosetting variants do not show high levels of amino acid identity apart from the first CIDR domain of TM284var1 and ITvar60 (82.2%) and the first CIDR domain of HB3var6 and Muz12var1 (81.1%; see Tables S2-S7 for pair-wise amino acid identities for all domain types). 
         [0117]    Correct identification of rosette-specific variants was confirmed by Northern blotting ( FIG. 1   b ; shown previously for TM284 [44]). For each parasite strain, the rosette-specific PfEMP1 probe detected a transcript in rosetting parasites (arrowed) that was absent/weak in isogenic non-rosetting parasites. The presence of other transcribed var genes in the non-rosetting parasites was shown using an Exon II probe that identifies all var genes ( FIG. 1   b ). 
         [0118]    In order to raise antibodies against the rosetting PfEMP1 variants, the N-terminal NTS-DBLα region of each rosette-specific variant was expressed as a recombinant protein in  E. coli [ 12], with a shift in mobility of the recombinant proteins upon reduction showing the presence of disulfide bonds in these cysteine-rich proteins ( FIG. 1   c ). NTS-DBLα was chosen because it is the domain that binds erythrocytes to bring about rosetting [20,21], and variant-specific antibodies to this region were the most effective in inhibiting rosetting in previous studies [12,21]. 
         [0000]    Functional Activity and Cross-Reactivity of PfEMP1 Antibodies Against Laboratory-Adapted  P. falciparum  Strains 
         [0119]    The recombinant proteins were used to immunize rabbits [12], to elicit antibodies to determine whether the rosette-specific PfEMP1 variants from different  P. falciparum  strains share common epitopes. Immunofluorescence assays (IFA) showed that antisera to each of the five variants gave punctate fluorescence that is characteristic of PfEMP1 antibodies over the surface of live infected erythrocytes with the homologous parasite strain (shown for IT/PAR+ parasites with ITvar60 antibodies in  FIG. 2   a ). Rabbit pre-immune sera and non-immunised rabbit control sera were negative by IFA. Titration of purified total IgG from each antiserum showed specific surface reactivity against homologous parasites down to low concentrations (end titres of 0.02-1.56 μg/ml of total IgG,  FIG. 2   b  and  FIG. 2   c , rectangles in bold). 
         [0120]    Importantly, the PfEMP1 antibodies also showed surface reactivity with heterologous rosetting strains. This was especially marked between the IgM binding rosetting strains (HB3R+, TM284 and IT/PAR+) and their antibodies (to variants HB3var6, TM284var1 and ITvar60 respectively), with surface reactivity at &lt;10 μg/ml for heterologous antibody:parasite combinations ( FIG. 2   c ). The non-IgM binding rosetting strains (Muz12, TM180 and IT/R29) were also recognised by antibodies to IgM binding rosetting variants, although higher concentrations were required (100-400 μg/ml of total IgG,  FIG. 2   c ). Although these concentrations are high, they still represent a considerable dilution of whole serum (equivalent to 1/100 to 1/25 dilution) therefore they are potentially achievable in vivo. 
         [0121]    The antibodies against rosetting PfEMP1 variants did not recognise parasites selected for other adhesion phenotypes ( FIG. 2   c ), including binding to CD36 or ICAM-1 (parasites expressing Group B and C var genes) or binding to brain endothelial cells (parasites expressing an alternative sub-set of group A and B/A var genes). Thus, only parasites with a shared adhesion phenotype share epitopes that are recognised by cross-reactive antibodies to PfEMP1. 
         [0122]    Surface recognition of live infected erythrocytes by antibodies in vivo is likely to lead to parasite clearance via effector mechanisms such as phagocytosis or complement-mediated lysis [13]. Rosette-inhibition may also be desirable in vivo to prevent pathological microvascular obstruction. We therefore examined whether the cross-reactive surface recognition by PfEMP1 antibodies shown in  FIG. 2 , translated into cross-reactivity in effector functions. The PfEMP1 antibodies showed potent rosette-inhibition against homologous parasite strains with 50% inhibitory concentrations (IC50) for rosetting between 0.8-8 μg/ml of total IgG ( FIG. 3   a , red curves), except for TM180, which was not inhibited ( FIG. 3   a , brown curve) despite good surface reactivity ( FIG. 2   c ). Parasite strains TM284, IT/PAR+ and TM180 all showed rosette inhibition by heterologous antibodies ( FIG. 3   a , blue curves). At a higher concentration (1 mg/ml of total IgG) the cross-reactivity in rosette inhibition was even more marked, with all strains being inhibited by antibodies to at least one of the IgM-binding rosetting PfEMP1 variants ( FIG. 3   b ). 
         [0123]    The antibodies to PfEMP1 variants from IgM binding rosetting parasites were also shown to have cross-reactive opsonising effects, by inducing the phagocytosis of homologous and heterologous infected erythrocytes ( FIG. 3   c  and  FIG. 7   a  and  b ). In contrast, antibodies to PfEMP1 variants from non-IgM binding rosetting parasites only effectively opsonised homologous parasites ( FIG. 7   c  and  d ). 
         [0000]    Functional Activity and Cross-Reactivity of Anti-PfEMP1 Antibodies Against  P. falciparum  Clinical Isolates 
         [0124]    Having shown surface recognition and biological effector functions of cross-reactive PfEMP1 antibodies in  P. falciparum  laboratory strains, we explored the geographical extent of the cross-reactivity using fresh clinical isolates from Cameroon, Kenya, Mali and The Gambia. The proportion of rosetting infected erythrocytes in the clinical isolates varies amongst isolates (15-40%), and was not as high as in the laboratory strains that undergo repeated selection for rosetting. The clinical isolates were selected because they contained at least 15% of infected erythrocytes in rosettes and all isolates tested are shown. Ten fresh clinical isolates were thawed, and all but one were found to be of the IgM binding rosetting phenotype (tested by detection of IgM on the surface of infected erythrocytes by IFA). This is consistent with previous data showing a strong positive correlation between rosette frequency and IgM binding in clinical isolates from Kenyan children [38]. 
         [0125]    Surface reactivity with the panel of PfEMP1 antibodies was detected by punctate fluorescence of live infected erythrocytes by IFA (similar to  FIG. 2   a ) and by flow cytometry ( FIG. 4   a ). Remarkably, antibodies to two PfEMP1 variants (HB3var6 and TM284var1) were sufficient to provide surface reactivity against all of the geographically diverse IgM binding rosetting isolates ( FIGS. 4   b  and  c ). The proportions of anti-PfEMP1 positive and IgM positive cells were closely matched in each isolate (Pearson correlation r=0.934, P=0.006;  FIG. 4   c , compare IgM positive cells with the positive PfEMP1 antibody stained cells). Rosette inhibition was also observed in four isolates, increasing to six isolates when a pool of anti-PfEMP1 antibodies was used ( FIG. 4   b ). The non-IgM binding clinical isolate (MAL103) and two recently culture-adapted rosette-selected non-IgM binding Kenyan strains (9197 and SA075) were not recognized by the PfEMP1 antibodies ( FIG. 4   b ). Therefore, in clinical isolates the cross-reactivity of PfEMP1 antibodies was only seen amongst parasites showing the IgM binding rosetting phenotype. 
         [0126]    We considered the possibility that the above results could be explained by PfEMP1 antibodies cross-reacting with human IgM (which is bound to the surface of the infected erythrocytes from the culture medium) rather than due to shared epitopes within PfEMP1 itself. However, the PfEMP1 antibodies did not recognise human IgM in an ELISA ( FIG. 5   a ), and the surface reactivity with heterologous parasite strains was maintained when the parasites were grown in the absence of IgM (for example, IT/PAR+ parasites show surface reactivity with TM284var1 antibodies in the absence of IgM as shown in  FIG. 5   b ). 
         [0127]    The presence of IgM binding rosetting variants in diverse parasite isolates was shown further by taking the two recently culture-adapted Kenyan strains 9197 and SA075 which showed non-IgM binding rosetting ( FIG. 4   b ), and selecting them for IgM binding using magnetic beads coated with anti-human IgM antibodies. After three rounds of selection, a population of IgM binding rosetting parasites was obtained, which were recognised by antibodies to HB3var6 (9197 IgM-selected,  FIG. 6 ) or by antibodies to TM284var1 (SA075 IgM-selected, data not shown). Dual staining showed that the same subpopulation of infected erythrocytes bound both IgM and HB3var6 antibodies ( FIG. 6   b ). 
       Discussion 
       [0128]    In this work the PfEMP1 variants expressed by  P. falciparum  strains representing two major rosetting phenotypes were examined. IgM binding rosetting parasites were found to express a distinct subset of group A PfEMP1 variants characterised by a DBLα1.5 or DBLα1.8 N-terminal domain and a triplet of DBLε/DBLζ domains adjacent to the transmembrane region. Antibodies raised against the N-terminal region of the IgM binding rosetting variants were potent inhibitors of rosetting when tested against homologous parasites (IC50 for rosette inhibition ≦1 μg/ml of total IgG,  FIG. 3   a ), confirming the role of these variants in rosette formation. Furthermore, the antibodies against IgM binding rosetting variants were cross-reactive, showing surface recognition of live infected erythrocytes and rosette inhibition with globally diverse parasite isolates sharing the same adhesion phenotype. In contrast, antibodies raised against group A PfEMP1 variants from non-IgM binding rosetting parasites were variant specific, as described previously [11,20,21]. These data indicate that not all group A PfEMP1 variants induce cross-reactive antibodies, and that certain subsets of group A variants may be particularly cross-reactive. To our knowledge, this is the first report to describe the successful induction of broadly cross-reactive surface-recognising antibodies to PfEMP1 variants implicated in severe childhood malaria. Broadly cross-reactive antibodies against var2CSA implicated in malaria in pregnancy have been described [47,48], however, var2CSA is a unique case of a strain-transcendent var gene with much more limited sequence diversity than that seen in group A PfEMP1 variants that are non-strain-transcendent [49]. 
         [0129]    Three PfEMP1 variants from IgM binding rosetting parasites were characterised in detail here: HB3var6 from strain HB3, TM284var1 from strain TM284 and ITvar60 from strain IT/PAR+. ITvar60 has previously been linked to rosetting in two other IT/FCR3-derived parasite lines [50,51], and is confirmed here as an IgM binding rosetting variant. As expected for PfEMP1, the three distinct IgM binding rosetting variants show considerable diversity in amino acid sequence (Tables S2-S7), despite their similarities in PfEMP1 architecture ( FIG. 1 ). Other variants with the same “rosetting IgM” type of domain architecture can be seen in the genome of a recently sequenced  P. falciparum  strain IGH (IGHvar12, IGHvar 22 and IGHvar 24 [6]). Furthermore, an ITvar60-like variant occurs in the sequenced  P. falciparum  strain D10 from Papua New Guinea (http://www.broadinstitute.org). Taken together, these data suggest that variants with the rosetting IgM-type of PfEMP1 architecture occur commonly in geographically diverse  P. falciparum  isolates. One limitation of the current study was that there was insufficient material from the clinical isolates to identify and sequence the PfEMP1 variants transcribed by the rosetting parasites to allow comparison with the laboratory strains. The selection of IgM binding rosetting parasites from two culture-adapted clinical isolates ( FIG. 6 ) will allow us to examine their var genes in further detail. The correct identification of rosette-specific variants (Table S1) and sequencing of full-length var genes remains a laborious and time-consuming process for isolates that do not have a full genome sequence available. However, wider studies of PfEMP1 architecture and sequence from rosetting clinical isolates will be essential for a full understanding of how the antibody cross-reactivity documented here relates to sequence diversity and PfEMP1 type. 
         [0130]    Previous studies of non-IgM binding rosetting parasites identified the parasite rosetting ligands as PfEMP1 variants (ITvar9, Palo Alto varO and PF13 — 0003) that show one out of eight possible subclasses of DBLα domain, DBLα1.6 [11,20,21]. In contrast, the group A rosetting variants described here have either DBLα1.5 (HB3var6 and Muz12var1) or DBLα1.8 (ITvar60 and TM284var1). The clinical isolates showed surface reactivity with either HB3var6 antibodies (DBLα1.5 type) or TM284var1 antibodies (DBLα1.8), but rarely with both ( FIG. 4   b ). These data are suggestive that these two main DBLα1 types may underlie the IgM binding rosetting phenotype in diverse field isolates, although clearly further sequence information is needed to substantiate this idea. TM180var1 differs from the other rosette-associated variants as it has an UpsB sequence and a DBLα2 subtype, and this may represent a distinct type of rosetting phenotype that requires further investigation. Taking together the findings from this study and previously published work, we hypothesize that all group A PfEMP1 variants with DBLα1.5, DBLα1.6 or DBLα1.8 domains may be rosette-mediating variants. If true, this would indicate that a substantial proportion (approximately one third to one half) of the group A var gene repertoire from every  P. falciparum  isolate may encode rosetting variants [6]. This would represent a substantial investment by the parasite in an adhesion phenotype whose benefit to parasite fitness remains unknown. 
         [0131]    Rask et al [6] recently presented an alternative way of assessing PfEMP1 types by looking at “domain cassettes” (sets of PfEMP1 domains that usually occur together). They identified seven domain cassettes commonly found in group A var genes [6]. Our data suggest that two of these domain cassettes are linked to the rosetting phenotype: domain cassette 16, characterised by DBLα1.5/6 linked to CIDRδ delta as seen in HB3var6, and domain cassette 11 characterised by DBLα1.8 linked to CIDRβ2 and DBLγ7 as seen in ITvar60 and TM284var1. Although much more work is needed to generalize these results and determine whether particular DBLα subclasses and domain cassettes are always linked to rosetting, our data represent an important step in relating PfEMP1 sequence to parasite adhesion phenotype. 
         [0132]    One unexplained feature of the current data is why the IgM binding rosetting variants show cross-reactivity in recognition by PfEMP1 antibodies indicating shared epitopes, whereas the non-IgM binding variants do not, despite apparently equivalent amino acid diversity in the two sets of variants. We considered the possibility that the IgM itself could be the cause of the cross-reactivity, however we showed that the PfEMP1 antibodies did not recognise human IgM in an ELISA, and the PfEMP1 antibodies still recognized heterologous strains when the parasites were grown in the absence of human IgM ( FIG. 5 ). It may be that a small sequence motif such as one of the homology blocks described by Rask et al [6] present only in the IgM binding variants explains the cross-reactivity. Additional examples of IgM binding rosetting variants will be needed to investigate this possibility. Alternatively, it is possible that the binding of IgM to PfEMP1 affects its tertiary or quaternary structure, making it more accessible to antibodies directed against the N-terminus of the molecule. However, recent data suggest that IgM-binding makes PfEMP1 less accessible to specific antibodies [41]. 
         [0133]    Previous work on PfEMP1 suggests that antibody responses are predominantly variant-specific [10,11,12] with little cross-reactivity between domains [52]. However, other reports suggest that cross-reactive antibodies can occur [53,54,55] and could play a role in structuring PfEMP1 expression during antigenic variation [56]. Whether the gradual acquisition of immunity to clinical malaria is linked to acquisition of a broad repertoire of antibodies to numerous distinct variant types, or due to development of cross-reactive responses remains unresolved. In the case of life-threatening malaria in particular, the role of antibodies to PfEMP1 is unclear. It is known that children become immune to severe malaria after a small number of infections [13,57], and that severe malaria is associated with the acquisition of antibodies to commonly recognised variants [14,15,58]. Current thinking suggests that severe malaria is caused by parasites expressing an antigenically-restricted subset of variant surface antigens [2], probably encoded by Group A var genes [26,27]. Such an “antigenically-restricted” subset of parasites would be expected to have variant surface antigens (probably PfEMP1) showing conserved sequence and/or conserved epitopes that would be recognised by antibodies that show surface reactivity with diverse parasite strains. The findings reported here of a subset of variants with shared epitopes underlying a virulence-associated phenotype may represent the first example of such an “antigenically-restricted” subset of parasites. 
         [0134]    In summary, these data show that antibodies raised against a subset of Group A PfEMP1 variants from IgM binding rosetting laboratory strains are broadly cross-reactive against global parasite isolates that share the same adhesion phenotype. This discovery of shared epitopes amongst  P. falciparum  isolates with a shared virulence-associated phenotype may underlie the epidemiological observations that children acquire immunity to life-threatening malaria after a small number of infections [13,57]. Most importantly, the ability to elicit broadly cross-reactive antibodies by immunizing with key PfEMP1 variants underlying a virulence phenotype, suggests that designing interventions to prevent severe malaria is a realistic goal. 
       EXAMPLE 2 
       [0135]    The data presented in Example 1 is focussed on antibodies to the N-terminal region (NTS-DBL1a) from the IgM-binding rosetting PfEMP1 variants. The experiments presented in Example 2, investigate whether antibodies to other DBL domains from these PfEMP1 variants would also show cross-reactivity. This would not be predicted from examination of the amino acid similarities between the domains (which are low, mostly between 20-40% amino acid identity). 
       Materials &amp; Methods 
       [0136]    Recombinant proteins were made in  E. coli  and antibodies generated in rabbits as described in the main manuscript. Surface reactivity assessed by flow cytometry, rosette inhibition and phagocytosis induction were as described in Example 1. 
       Results: 
       [0137]    Antibodies were raised to all DBL domains from the ITvar60 variant ( FIG. 1A  and  FIG. 8  below regions shown in red) and also to DBL4ε and DBL5e from the HB3var6 variant ( FIG. 1A ). 
       Tests of ITvar60 Antibodies Against the Homologous Parasite Strain (IT/PAR+) 
       [0138]    The ITvar60 antibodies were tested for surface reactivity against live infected erythrocytes of the homologous parasite strain (IT/PAR+). All antibodies showed punctate fluorescence typical of PfEMP1, down to low concentrations (see Table 1) except the antibody to DBL3ζ which gave surface reactivity only at 100 μg/ml. The antibodies to other domains of ITvar60 inhibited rosetting, although none was as effective as the NTS-DBL1α antibody (see  FIG. 10  and Table 1). 
         [0139]    The ITvar60 antibodies were also tested for their ability to induce phagocytosis of infected erythrocytes (a function likely to be of importance in vivo). The antibodies to DBL2γ of ITvar60 were able to opsonize infected erythrocytes and induce phagocytosis similar to the NTS-DBL1α antibodies (see  FIG. 11  below). Interestingly, despite good surface reactivity and rosette inhibiting capabilities, the antibodies to DBL4ε and DBL5ε of ITvar60 were unable to opsonize and induce phagocytosis. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Effectiveness of ITvar60Abs in various 
               
               
                 assays against IT/PAR+ parasites. 
               
             
          
           
               
                   
                 Surface 
                 Rosette 
                 Phagocytosis ~50% 
               
               
                   
                 reactivity end 
                 Inhibition 
                 of positive 
               
               
                 Antibody 
                 titre (μg/ml) 
                 IC50 (μg/ml) 
                 control (μg/ml) 
               
               
                   
               
               
                 Negative control 
                 Negative 
                 Negative 
                 Negative 
               
               
                 rabbit IgG 
                 at 100 
                 at 500 
                 at 400 
               
               
                 Anti-NTS-DBL1α 
                 0.10 
                 0.08 
                 &lt;6.25 
               
               
                 Anti-DBL2γ 
                 0.02 
                 0.80 
                 &lt;6.25 
               
               
                 Anti-DBL3ζ 
                 100 
                 &gt;100 
                 &gt;400 
               
               
                 Anti-DBL4ε 
                 1.56 
                 25 
                 &gt;400 
               
               
                 Anti-DBL5ε 
                 1.56 
                 9 
                 &gt;400 
               
               
                   
               
             
          
         
       
     
       Tests of ITvar60 Antibodies Against Other Parasite Strains 
       [0140]    The antibodies were tested for surface reactivity with live infected erythrocytes from various rosetting parasite strains. Antibodies to DBL4ε of ITvar60 showed cross-reactivity against other IgM binding rosetting strains (see  FIG. 11 ). This recognition was specific to IgM binding rosetting parasites, and was not seen with non-IgM binding rosetting parasites (see  FIG. 12 ). 
       Discussion 
       [0141]    These data show that it is not only the NTS-DBL1α region of PfEMP1 that can induce cross-reactive antibodies, but other domains from IgM-binding rosetting variants show the same effect. This raises the possibility that other domains could be included in a vaccine. 
         [0142]    In terms of functional activity, antibodies to the NTS-DBL1α region are clearly the most effective in terms of rosette inhibition and phagocytosis (see Table 1). These activities are likely to be important for functional effectiveness of the antibodies in vivo, therefore these data argue for NTS-DBL1α being the most effective region to include in a vaccine. There are other possible mechanisms of antibody action in vivo (eg complement-mediated lysis of infected erythrocytes) that could be induced by all surface reactive antibodies. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE S1 
               
             
             
               
                   
               
               
                 Identification of a rosette-specific var gene by transcriptional profiling 
               
               
                 of isogenic rosetting (R+) and non-rosetting (R−) HB3 parasites. 
               
             
          
           
               
                 HB3R+ Rosette 
                 HB3R− Rosette 
               
               
                 frequency 58% 
                 frequency 2% 
               
             
          
           
               
                 Number of 
                   
                   
                 Number of 
                   
                   
               
               
                 recombinant 
                 Gene 
                 Upstream 
                 recombinant 
                 Gene 
                 Upstream 
               
               
                 plasmids 
                 name 
                 sequence 
                 plasmids 
                 name 
                 sequence 
               
               
                   
               
             
          
           
               
                 
                   14 
                 
                 
                   HB3var6 
                 
                 
                   A 
                 
                 11 
                 HB3var29 
                 C 
               
               
                 10 
                 HB3var3 
                 A 
                 7 
                 HB3var27 
                 B 
               
               
                 3 
                 HB3var31 
                 C 
                 5 
                 HB3var34 
                 C 
               
               
                 2 
                 HB3var29 
                 C 
                 2 
                 HB3var28 
                 C 
               
               
                 2 
                 HB3var34 
                 C 
                 2 
                 HB3var51 
                 C 
               
               
                 2 
                 HB3var27 
                 B 
                 2 
                 HB3var17 
                 B 
               
               
                 1 
                 HB3var1CSA 
                 A 
                 1 
                 HB3var3 
                 A 
               
               
                 1 
                 HB3var7 
                 B 
                 
                   1 
                 
                 
                   HB3var6 
                 
                 
                   A 
                 
               
               
                 1 
                 HB3var24 
                 B 
                 1 
                 HB3var11 
                 B 
               
               
                   
                   
                   
                 1 
                 HB3var14 
                 B 
               
               
                   
                   
                   
                 1 
                 HB3var19 
                 B 
               
               
                   
               
             
          
         
       
     
         [0143]    To identify the predominant rosette-specific PfEMP1 variant, the var gene transcriptional profiles of isogenic rosetting (R+) and non-rosetting (R−) parasites were compared. RNA was extracted from late ring stage parasites and var gene transcription assessed by reverse-transcriptase (RT)-PCR with degenerate primers to DBL1α (1, 2). The RT-PCR products were cloned by TA cloning (Invitrogen), and 40 colonies picked for mini-prep DNA extraction and sequencing (3). From the HB3R+ line (rosette frequency 58%), 36 recombinant plasmids with var gene inserts were obtained, and the most common sequence (39% of clones) was the group A var gene HB3var6 (shown in bold). This gene was found in only one out of 34 var gene inserts sequenced from the HB3R− line (rosette frequency 2%), whereas several group B and C var genes were detected commonly in the non-rosetting line. Another group A var gene was also common in the HB3R+ line (HB3var3, 10/36 clones) and rare in the HB3R− line (1/34 clones). A second independent selection starting from a different cryostabilate of HB3 parasites showed HB3var6 in 5/16 clones from R+ parasites and 0/15 clones from R− parasites, whereas HB3var3 was not detected in either R+ or R− populations. These data show that the predominant var gene transcribed in HB3 rosetting parasites is HB3var6. The same procedure was followed for other  P. falciparum  rosetting strains, with at least two independent selections and RT-PCRs indicating a predominant var gene in each case. 
       REFERENCES FOR TABLE S1 
       [0000]    
       
         1. Taylor H M, Kyes S A, Harris D, Kriek N, &amp; Newbold C I (2000) A study of var gene transcription in vitro using universal var gene primers. Mol Biochem Parasitol 105:13-23. 
         2. Bull P C, et al. (2005)  Plasmodium falciparum  Variant Surface Antigen Expression Patterns during Malaria. PLoS Pathog 1:e26. 
         3. Kyriacou H M, et al. (2006) Differential var gene transcription in  Plasmodium falciparum  isolates from patients with cerebral malaria compared to hyperparasitaemia. Mol Biochem Parasitol 150:211-218. 
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE S2 
               
             
             
               
                   
               
               
                 Pair-wise amino acid identities for NTS-DBLα, CIDR1 
               
               
                 and DBLγ from rosetting PfEMP1 variants a   
               
             
          
           
               
                   
                 HB3 
                 TM284 
                 IT 
                 Muz12 
                 TM180 
                 IT 
                 Palo 
                 3D7 
               
               
                   
                 var6 
                 var1 
                 var60 
                 var1 
                 var1 
                 var9 
                 AltoVarO 
                 PF13_0003 
               
               
                   
                   
               
             
          
           
               
                 Pair-wise amino acid identities for NTS-DBLα 
               
             
          
           
               
                 HB3var6 
                 100 
                 50.8 b   
                 51.3 
                 61.0 
                 41.8 
                 46.7 
                 47.3 
                 49.1 
               
               
                 TM284var1 
                   
                 100 
                 62.6 
                 47.0 
                 41.4 
                 58.7 
                 54.1 
                 47.5 
               
               
                 ITvar60 
                   
                   
                 100 
                 43.6 
                 38.9 
                 53.8 
                 55.3 
                 52.5 
               
               
                 Muz12var1 
                   
                   
                   
                 100 
                 43.0 
                 44.0 
                 47.3 
                 49.0 
               
               
                 TM180var1 
                   
                   
                   
                   
                 100 
                 40.8 
                 42.6 
                 39.3 
               
               
                 ITvar9 
                   
                   
                   
                   
                   
                 100 
                 61.6 
                 53.9 
               
               
                 PA varO 
                   
                   
                   
                   
                   
                   
                 100 
                 59.6 
               
               
                 3D7 PF13 
                   
                   
                   
                   
                   
                   
                   
                 100 
               
             
          
           
               
                 Pair-wise amino acid identities for CIDR1 
               
             
          
           
               
                 HB3var6 
                 100 
                 45.1 
                 48.6 
                 81.1 
                 24.9 
                 34.4 
                 40.4 
                 60.7 
               
               
                 TM284var1 
                   
                 100 
                 82.2 
                 44.6 
                 23.2 
                 37.2 
                 37.9 
                 45.6 
               
               
                 ITvar60 
                   
                   
                 100 
                 46.7 
                 25.3 
                 40.0 
                 38.6 
                 48.4 
               
               
                 Muz12var1 
                   
                   
                   
                 100 
                 24.6 
                 35.1 
                 37.9 
                 57.2 
               
               
                 TM180var1 
                   
                   
                   
                   
                 100 
                 21.4 
                 20.7 
                 24.6 
               
               
                 ITvar9 
                   
                   
                   
                   
                   
                 100 
                 35.8 
                 35.4 
               
               
                 PA varO 
                   
                   
                   
                   
                   
                   
                 100 
                 33.7 
               
               
                 3D7 PF13 
                   
                   
                   
                   
                   
                   
                   
                 100 
               
             
          
           
               
                 Pair-wise amino acid identities for DBLγ 
               
             
          
           
               
                 HB3var6 
                 100 
                 38.3 
                 38.1 
                 38.9 
                 46.6 
                 34.1 
                 40.9 
                 49.4 
               
               
                 TM284var1 
                   
                 100 
                 56.5 
                 38.3 
                 36.3 
                 29.3 
                 40.1 
                 37.8 
               
               
                 ITvar60 
                   
                   
                 100 
                 38.8 
                 34.9 
                 27.9 
                 41.0 
                 34.5 
               
               
                 Muz12var1 
                   
                   
                   
                 100 
                 45.4 
                 31.1 
                 43.8 
                 40.6 
               
               
                 TM180var1 
                   
                   
                   
                   
                 100 
                 28.8 
                 49.7 
                 35.3 
               
               
                 ITvar9 
                   
                   
                   
                   
                   
                 100 
                 29.9 
                 33.3 
               
               
                 PA varO 
                   
                   
                   
                   
                   
                   
                 100 
                 37.3 
               
               
                 3D7 PF13 
                   
                   
                   
                   
                   
                   
                   
                 100 
               
               
                   
               
               
                   a Rosetting variants described in this work plus ITvar9 [1], Palo Alto Var O [2] and PF13_0003 [3]. 
               
               
                   b Pair-wise amino acid identities between the IgM-binding rosetting strains shown in red References for Table S2 
               
               
                 1. Rowe J A, Moulds J M, Newbold C I, Miller L H (1997)  P. falciparum  rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature 388: 292-295. 
               
               
                 2. Vigan-Womas I, Guillotte M, Le Scanf C, Igonet S, Petres S, et al. (2008) An in vivo and in vitro model of  Plasmodium falciparum  rosetting and autoagglutination mediated by varO, a group A var gene encoding a frequent serotype. Infect Immun 76: 5565-5580. 
               
               
                 3. Vigan-Womas I, Guillotte M, Juillerat A, Vallieres C, Lewit-Bentley A, et al. (2011) Allelic diversity of the  Plasmodium falciparum  erythrocyte membrane protein 1 entails variant-specific red cell surface epitopes. PLoS One 6: e16544. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE S3 
               
             
             
               
                   
               
               
                 Pair-wise amino acid identities for DBLε from rosetting PfEMP1 variants 
               
             
          
           
               
                   
                 HB3 
                 HB3 
                 TM284 
                 TM284 
                 ITvar 
                 ITvar 
                 ITvar9 
                 Palo Alto 
               
               
                   
                 var6d3 a   
                 var6d6 
                 var1d3 
                 var1d5 
                 60 d4 
                 60 d5 
                 d3 
                 VarO d5 
               
               
                   
                   
               
             
          
           
               
                 HB3var6 d4 
                 100 
                 28.6 
                 43.5 
                 23.2 
                 23.7 
                 22.0 
                 24.5 
                 27.3 
               
               
                 HB3var6 d6 
                   
                 100 
                 30.5 
                 29.9 
                 30.5 
                 28.6 
                 38.8 
                 38.5 
               
               
                 TM284var1 d3 
                   
                   
                 100 
                 25.3 
                 22.7 
                 26.1 
                 26.3 
                 28.6 
               
               
                 TM284var1 d5 
                   
                   
                   
                 100 
                 23.7 
                 41.7 
                 28.9 
                 32.1 
               
               
                 ITvar60 d4 
                   
                   
                   
                   
                 100 
                 24.3 
                 32.1 
                 29.6 
               
               
                 ITvar60 d5 
                   
                   
                   
                   
                   
                 100 
                 27.6 
                 31.5 
               
               
                 ITvar9 d3 
                   
                   
                   
                   
                   
                   
                 100 
                 30.9 
               
               
                 PA varO d5 
                   
                   
                   
                   
                   
                   
                   
                 100 
               
               
                   
               
               
                   a d3: 3rd DBL domain from the N-terminus 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE S4 
               
             
             
               
                   
               
               
                 Pair-wise amino acid identities for 
               
               
                 DBLζ from rosetting PfEMP1 variants 
               
             
          
           
               
                   
                 HB3var6 
                 TM284var1 
                 ITvar60 
                 Palo Alto VarO 
               
               
                   
                   
               
             
          
           
               
                 HB3var6 
                 100 
                 42.7 
                 37.1 
                 36.1 
               
               
                 TM284var1 
                   
                 100 
                 35.6 
                 35.0 
               
               
                 ITvar60 
                   
                   
                 100 
                 36.8 
               
               
                 PA varO 
                   
                   
                   
                 100 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE S5 
               
             
             
               
                   
               
               
                 Pair-wise amino acid identities for 
               
               
                 DBLβ from rosetting PfEMP1 variants 
               
             
          
           
               
                   
                   
                   
                 Palo Alto 
                 3D7 
               
               
                   
                 Muz12var1 
                 TM180var1 
                 VarO 
                 PF13_0003 
               
               
                   
                   
               
             
          
           
               
                 Muz12var1 
                 100 
                 46.8 
                 46.9 
                 46.4 
               
               
                 TM180var1 
                   
                 100 
                 49.3 
                 49.2 
               
               
                 PA VarO 
                   
                   
                 100 
                 46.9 
               
               
                 3D7 PF13 
                   
                   
                   
                 100 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE S6 
               
             
             
               
                   
               
               
                 Pair-wise amino acid identities for DBLδ from rosetting PfEMP1 variants 
               
             
          
           
               
                   
                   
                   
                   
                 IT 
                 3D7 
               
               
                   
                 HB3var6 
                 Muz12var1 
                 TM180var1 
                 var9 
                 PF13_0003 
               
               
                   
                   
               
             
          
           
               
                 HB3var6 
                 100 
                 42.9 
                 37.0 
                 36.4 
                 48.1 
               
               
                 Muz12var1 
                   
                 100 
                 38.1 
                 40.7 
                 46.5 
               
               
                 TM180var1 
                   
                   
                 100 
                 47.5 
                 37.9 
               
               
                 ITvar9 
                   
                   
                   
                 100 
                 37.6 
               
               
                 3D7 PF13 
                   
                   
                   
                   
                 100 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE S7 
               
             
             
               
                   
               
               
                 Pair-wise amino acid identities for CIDR2 from rosetting PfEMP1 variants 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Palo Alto 
                 3D7 
               
               
                   
                 HB3var6 
                 Muz12var1 
                 TM180var1 
                 ITvar9 
                 VarO 
                 PF13 
               
               
                   
                   
               
             
          
           
               
                 HB3var6 
                 100 
                 28.1 
                 30.9 
                 26.7 
                 53.0 
                 32.3 
               
               
                 Muz12var1 
                   
                 100 
                 45.3 
                 50.2 
                 33.3 
                 41.8 
               
               
                 TM180var1 
                   
                   
                 100 
                 37.9 
                 34.4 
                 48.1 
               
               
                 ITvar9 
                   
                   
                   
                 100 
                 29.8 
                 36.8 
               
               
                 PAvarO 
                   
                   
                   
                   
                 100 
                 35.1 
               
               
                 3D7 PF13 
                   
                   
                   
                   
                   
                 100 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 3 
     Parasites Expressing ITvar60 are Specifically Recognized by Antibodies in Plasma from Children Recovering from Severe (Cerebral) Malaria 
     Materials and Methods 
     Plasma Samples 
       [0147]    Clinical plasma samples from 10 Kenyan cerebral malaria cases and 10 Kenyan non-severe controls (matched by age and date of admission) were collected at acute and convalescent stages. The acute sample (taken on hospital admission) reflects antibodies generated during prior malaria infections (i.e. this is the “baseline” sample); the convalescent sample (taken one month after admission) reflects antibodies generated to the parasites causing the recent clinical infection that resulted in hospitalization of the child. 
       Flow Cytometry 
       [0148]    Recognition of surface antigens of live  P. falciparum  infected erythrocytes was tested by flow cytometry. Parasite strains tested were: IT/PAR+ (which are IgM-positive rosetting parasites that express the ITvar60 variant named in the patent), which we predict SHOULD be recognized by the severe malaria children&#39;s convalescent antibodies. IT/R29 and SA075R+, which are IgM-negative rosetting parasites that we predict should NOT be recognized by the severe malaria children&#39;s convalescent antibodies. 
         [0149]    As predicted, IT/PAR+ parasites expressing the ITvar60 PfEMP1 variant are specifically recognized by antibodies in the plasma of children recovering from severe (cerebral) malaria ( FIG. 13 , CM cases). In contrast, age-matched control children with non-severe malaria do not show enhanced recognition of IT/PAR+ parasites in convalescent plasma ( FIG. 13 , CM-controls). 
         [0150]    These data support the hypothesis that ITvar60-like antigens are involved in the pathogenesis of severe malaria, and that antibodies formed against this variant will protect against future episodes of severe malaria (because epidemiological studies show that children rapidly become immune to severe malaria in the first few years of life, and rarely develop severe malaria more than once, although they remain susceptible to mild clinical disease for many years). 
         [0151]    In contrast to the above data, IgM-negative rosetting parasites (that we do not expect to be involved in severe malaria) are not specifically recognized by antibodies in the plasma of children recovering from severe malaria ( FIGS. 1 and 15 ). 
         [0152]    In a similar fashion, parasites expressing the HB3var6 antigen are specifically recognized by antibodies in the plasma of children recovering from severe malaria ( FIGS. 16A  &amp; B), whereas, the control SA075var1 expressing parasites is not specifically recognized in this way ( FIGS. 17  A &amp; B). 
       Antibodies 
       [0153]    Mice immunized with the TM284var1 antigen made excellent polyclonal antibody responses as shown by ELISA below. The polyclonal antibodies from each mouse also showed strong recognition of the native antigen on the surface of live infected erythrocytes down to at least 1/10,000 dilution, and completely disrupted rosettes at 1/1000 dilution ( FIG. 18 ). In advance of screening for anti-PfEMP1 monoclonal antibodies, fusions have been prepared from mouse spleen. 
       General References. 
       [0000]    
       
         1. Rappuoli R, Aderem A (2011) A 2020 vision for vaccines against HIV, tuberculosis and malaria. Nature 473: 463-469. 
         2. Hviid L (2010) The role of  Plasmodium falciparum  variant surface antigens in protective immunity and vaccine development. Hum Vaccin 6: 84-89. 
         3. Freitas-Junior L H, Bottius E, Pirrit L A, Deitsch K W, Scheidig C, et al. (2000) Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of  P. falciparum . Nature 407: 1018-1022. 
         4. Kraemer S M, Kyes S A, Aggarwal G, Springer A L, Nelson S O, et al. (2007) Patterns of gene recombination shape var gene repertoires in  Plasmodium falciparum : comparisons of geographically diverse isolates. BMC Genomics 8: 45. 
         5. Barry A E, Leliwa-Sytek A, Tavul L, Imrie H, Migot-Nabias F, et al. (2007) Population genomics of the immune evasion (var) genes of  Plasmodium falciparum . PLoS Pathog 3: e34. 
         6. Rask T S, Hansen D A, Theander T G, Gorm Pedersen A, Lavstsen T (2010)  Plasmodium falciparum  erythrocyte membrane protein 1 diversity in seven genomes—divide and conquer. PLoS Comput Biol 6. 
         7. Kyes S, Horrocks P, Newbold C (2001) Antigenic variation at the infected red cell surface in malaria. Annu Rev Microbiol 55: 673-707. 
         8. Chen D S, Barry A E, Leliwa-Sytek A, Smith T A, Peterson I, et al. (2011) A molecular epidemiological study of var gene diversity to characterize the reservoir of  Plasmodium falciparum  in humans in Africa. PLoS One 6: e16629. 
         9. Good M F, Doolan D L (2010) Malaria vaccine design: immunological considerations. Immunity 33: 555-566. 
         10. Newbold C I, Pinches R, Roberts D J, Marsh K (1992)  Plasmodium falciparum ; the human agglutinating antibody response to the infected red cell surface is predominantly variant specific. Experimental Parasitology 75: 281-292. 
         11. Vigan-Womas I, Guillotte M, Juillerat A, Vallieres C, Lewit-Bentley A, et al. (2011) Allelic diversity of the  Plasmodium falciparum  erythrocyte membrane protein 1 entails variant-specific red cell surface epitopes. PLoS One 6: e16544. 
         12. Ghumra A, Khunrae P, Ataide R, Raza A, Rogerson S J, et al. (2011) Immunisation with recombinant PfEMP1 domains elicits functional rosette-inhibiting and phagocytosis-inducing antibodies to  Plasmodium falciparum. PLoS One  6: e16414. 
         13. Langhorne J, Ndungu F M, Sponaas A M, Marsh K (2008) Immunity to malaria: more questions than answers. Nat Immunol 9: 725-732. 
         14. Bull P C, Kortok M, Kai O, Ndungu F, Ross A, et al. (2000)  Plasmodium falciparum -infected erythrocytes: agglutination by diverse Kenyan plasma is associated with severe disease and young host age. J Infect Dis 182: 252-259. 
         15. Nielsen M A, Staalsoe T, Kurtzhals J A, Goka B Q, Dodoo D, et al. (2002)  Plasmodium falciparum  variant surface antigen expression varies between isolates causing severe and nonsevere malaria and is modified by acquired immunity. J Immunol 168: 3444-3450. 
         16. Kraemer S M, Smith J D (2006) A family affair: var genes, PfEMP1 binding, and malaria disease. Curr Opin Microbiol 9: 374-380. 
         17. Rowe J A, Claessens A, Corrigan R A, Arman M (2009) Adhesion of  Plasmodium falciparum -infected erythrocytes to human cells: molecular mechanisms and therapeutic implications. Expert Rev Mol Med 11: e16. 
         18. Robinson B A, Welch T L, Smith J D (2003) Widespread functional specialization of  Plasmodium falciparum  erythrocyte membrane protein 1 family members to bind C D36 analysed across a parasite genome. Mol Microbiol 47: 1265-1278. 
         19. Janes J H, Wang C P, Levin-Edens E, Vigan-Womas I, Guillotte M, et al. (2011) Investigating the Host Binding Signature on the  Plasmodium falciparum  PfEMP1 Protein Family. PLoS Pathog 7: e1002032. 
         20. Rowe J A, Moulds J M, Newbold C I, Miller L H (1997)  P. falciparum  rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature 388: 292-295. 
         21. Vigan-Womas I, Guillotte M, Le Scanf C, Igonet S, Petres S, et al. (2008) An in vivo and in vitro model of  Plasmodium falciparum  rosetting and autoagglutination mediated by varO, a group A var gene encoding a frequent serotype. Infect Immun 76: 5565-5580. 
         22. Udomsangpetch R, Wahlin B, Carlson J, Berzins K, Torii M, et al. (1989)  Plasmodium falciparum -infected erythrocytes form spontaneous erythrocyte rosettes. Journal of Experimental Medicine 169: 1835-1840. 
         23. Kirchgatter K, Portillo Hdel A (2002) Association of severe noncerebral  Plasmodium falciparum  malaria in Brazil with expressed PfEMP1 DBL1 alpha sequences lacking cysteine residues. Mol Med 8: 16-23. 
         24. Kyriacou H M, Stone G N, Challis R J, Raza A, Lyke K E, et al. (2006) Differential var gene transcription in  Plasmodium falciparum  isolates from patients with cerebral malaria compared to hyperparasitaemia. Mol Biochem Parasitol 150: 211-218. 
         25. Rottmann M, Lavstsen T, Mugasa J P, Kaestli M, Jensen A T, et al. (2006) Differential Expression of var Gene Groups Is Associated with Morbidity Caused by  Plasmodium falciparum  Infection in Tanzanian Children. Infect Immun 74: 3904-3911. 
         26. Warimwe G M, Keane T M, Fegan G, Musyoki J N, Newton C R, et al. (2009)  Plasmodium falciparum  var gene expression is modified by host immunity. Proc Natl Acad Sci USA 106: 21801-21806. 
         27. Jensen A T, Magistrado P, Sharp S, Joergensen L, Lavstsen T, et al. (2004)  Plasmodium falciparum  associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes. J Exp Med 199: 1179-1190. 
         28. Carlson J, Helmby H, Hill A V, Brewster D, Greenwood B M, et al. (1990) Human cerebral malaria: association with erythrocyte rosetting and lack of anti-rosetting antibodies. Lancet 336: 1457-1460. 
         29. Treutiger C J, Hedlund I, Helmby H, Carlson J, Jepson A, et al. (1992) Rosette formation in  Plasmodium falciparum  isolates and anti-rosette activity of sera from Gambians with cerebral or uncomplicated malaria. American Journal of Tropical Medicine and Hygiene 46: 503-510. 
         30. Rowe A, Obeiro J, Newbold C I, Marsh K (1995)  Plasmodium falciparum  rosetting is associated with malaria severity in Kenya. Infect Immun 63: 2323-2326. 
         31. Rowe J A, Obiero J, Marsh K, Raza A (2002) Positive correlation between rosetting and parasitemia in  Plasmodium falciparum  clinical isolates. Am J Trop Med Hyg 66: 458-460. 
         32. Doumbo O K, Thera M A, Kone A K, Raza A, Tempest L J, et al. (2009) High levels of  Plasmodium falciparum  rosetting in all clinical forms of severe malaria in African children. Am J Trop Med Hyg 81: 987-993. 
         33. Le Scanf C, Vigan-Womas I, Contamin H, Guillotte M, Bischoff E, et al. (2008) Rosetting is associated with increased  Plasmodium falciparum  in vivo multiplication rate in the Saimiri sciureus monkey. Microbes Infect 10: 447-451. 
         34. Kaul D K, Roth EFJ, Nagel R L, Howard R I, Handunnetti S M (1991) Rosetting of  Plasmodium falciparum -infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions. Blood 78: 812-819. 
         35. Cockburn I A, Mackinnon M J, O&#39;Donnell A, Allen S J, Moulds J M, et al. (2004) A human complement receptor 1 polymorphism that reduces  Plasmodium falciparum  rosetting confers protection against severe malaria. Proc Natl Acad Sci USA 101: 272-277. 
         36. Rowe J A, Handel I G, Thera M A, Deans A M, Lyke K E, et al. (2007) Blood group O protects against severe  Plasmodium falciparum  malaria through the mechanism of reduced rosetting. Proc Natl Acad Sci USA 104: 17471-17476. 
         37. Scholander C, Treutiger C J, Hultenby K, Wahlgren M (1996) Novel fibrillar structure confers adhesive property to malaria-infected erythrocytes. Nature Medicine 2: 204-208. 
         38. Rowe J A, Shafi J, Kai O K, Marsh K, Raza A (2002) Nonimmune IgM, but not IgG binds to the surface of  Plasmodium falciparum -infected erythrocytes and correlates with rosetting and severe malaria. Am J Trop Med Hyg 66: 692-699. 
         39. Clough B, Abiola Atilola F, Black J, Pasvol G (1998)  Plasmodium falciparum : The Importance of IgM in the Rosetting of Parasite-Infected Erythrocytes. Experimental Parasitology 89: 129-132. 
         40. Somner E A, Black J, Pasvol G (2000) Multiple human serum components act as bridging molecules in rosette formation by  Plasmodium falciparum -infected erythrocytes. Blood 95: 674-682. 
         41. Barfod L, Dalgaard M B, Pleman S T, Ofori M F, Pleass R J, et al. (2011) Evasion of immunity to  Plasmodium falciparum  malaria by IgM masking of protective IgG epitopes in infected erythrocyte surface-exposed PfEMP1. Proc Natl Acad Sci USA 108: 12485-12490. 
         42. Claessens A, Ghumra A, Gupta A P, Mok S, Bozdech Z, et al. (2011) Design of a variant surface antigen-supplemented microarray chip for whole transcriptome analysis of multiple  Plasmodium falciparum  cytoadherent strains, and identification of strain-transcendent rif and stevor genes. Malar J 10: 180. 
         43. Semblat J P, Raza A, Kyes S A, Rowe J A (2006) Identification of  Plasmodium falciparum  var1 CSA and var2CSA domains that bind IgM natural antibodies. Mol Biochem Parasitol 146: 192-197. 
         44. Ghumra A, Semblat J P, McIntosh R S, Raza A, Rasmussen I B, et al. (2008) Identification of residues in the Cmu4 domain of polymeric IgM essential for interaction with  Plasmodium falciparum  erythrocyte membrane protein 1 (PfEMP1). J Immunol 181: 1988-2000. 
         45. Smith J D, Subramanian G, Gamain B, Baruch D I, Miller L H (2000) Classification of adhesive domains in the  Plasmodium falciparum  erythrocyte membrane protein 1 family. Mol Biochem Parasitol 110: 293-310. 
         46. Deans A M, Lyke K E, Thera M A, Plowe C V, Kone A, et al. (2006) Low multiplication rates of African  Plasmodium falciparum  isolates and lack of association of multiplication rate and red blood cell selectivity with malaria virulence. Am J Trop Med Hyg 74: 554-563. 
         47. Avril M, Kulasekara B R, Gose S O, Rowe C, Dahlback M, et al. (2008) Evidence for globally shared, cross-reacting polymorphic epitopes in the pregnancy-associated malaria vaccine candidate VAR2CSA. Infect Immun 76: 1791-1800. 
         48. Barfod L, Dobrilovic T, Magistrado P, Khunrae P, Viwami F, et al. (2010) Chondroitin sulfate A-adhering  Plasmodium falciparum -infected erythrocytes express functionally important antibody epitopes shared by multiple variants. J Immunol 185: 7553-7561. 
         49. Trimnell A R, Kraemer S M, Mukherjee S, Phippard D J, Janes J H, et al. (2006) Global genetic diversity and evolution of var genes associated with placental and severe childhood malaria. Mol Biochem Parasitol 148: 169-180. 
         50. Horrocks P, Pinches R, Christodoulou Z, Kyes S A, Newbold C I (2004) Variable var transition rates underlie antigenic variation in malaria. Proc Natl Acad Sci USA 101: 11129-11134. 
         51. Albrecht L, Moll K, Blomqvist K, Normark J, Chen Q, et al. (2011) var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive  Plasmodium falciparum  clone FCR3S1.2. Malar J 10: 17. 
         52. Joergensen L, Turner L, Magistrado P, Dahlback M A, Vestergaard L S, et al. (2006) Limited cross-reactivity among domains of the  Plasmodium falciparum  clone 3D7 erythrocyte membrane protein 1 family. Infect Immun 74: 6778-6784. 
         53. Marsh K, Howard R J (1986) Antigens induced on erythrocytes by  P. falciparum : expression of diverse and conserved determinants. Science 231: 150-153. 
         54. Chattopadhyay R, Sharma A, Srivastava V K, Pati S S, Sharma S K, et al. (2003)  Plasmodium falciparum  infection elicits both variant-specific and cross-reactive antibodies against variant surface antigens. Infect Immun 71: 597-604. 
         55. Gamain B, Miller L H, Baruch D I (2001) The surface variant antigens of  Plasmodium falciparum  contain cross-reactive epitopes. Proc Natl Acad Sci USA 98: 2664-2669. 
         56. Recker M, Gupta S (2006) Conflicting immune responses can prolong the length of infection in  Plasmodium falciparum  malaria. Bull Math Biol 68: 821-835. 
         57. Gupta S, Snow R W, Donnelly C A, Marsh K, Newbold C (1999) Immunity to non-cerebral severe malaria is acquired after one or two infections. Nat Med 5: 340-343. 
         58. Bull P C, Lowe B S, Kortok M, Marsh K (1999) Antibody recognition of  Plasmodium falciparum  erythrocyte surface antigens in Kenya: evidence for rare and prevalent variants. Infect Immun 67: 733-739. 
         59. Corrigan R A, Rowe J A (2010) Strain variation in early innate cytokine induction by  Plasmodium falciparum . Parasite Immunol 32: 512-527. 
         60. Handunnetti S M, David P H, Perera KLRL, Mendis K N (1989) Uninfected erythrocytes form “rosettes” around  Plasmodium falciparum  infected erythrocytes. American Journal of Tropical Medicine and Hygiene 40: 115-118. 
         61. Rowe J A, Scragg I G, Kwiatkowski D, Ferguson D J P, Carucci D J, et al. (1998) Implications of mycoplasma contamination in  Plasmodium falciparum  cultures and methods for its detection and eradication. Molecular and Biochemical Parasitology 92: 177-180. 
         62. Farnert A, Arez A P, Babiker H A, Beck H P, Benito A, et al. (2001) Genotyping of  Plasmodium falciparum  infections by PCR: a comparative multicentre study. Trans R Soc Trop Med Hyg 95: 225-232. 
         63. Kraemer S M, Gupta L, Smith J D (2003) New tools to identify var sequence tags and clone full-length genes using type-specific primers to Duffy binding-like domains. Mol Biochem Parasitol 129: 91-102. 
         64. Heddini A, Treutiger C J, Wahlgren M (1998) Enrichment of immunoglobulin binding  Plasmodium falciparum -infected erythrocytes using anti-immunoglobulin-coated magnetic beads. Am J Trop Med Hyg 59: 663-666. 
         65. Mu J, Awadalla P, Duan J, McGee K M, Joy D A, et al. (2005) Recombination hotspots and population structure in  Plasmodium falciparum . PLoS Biol 3: e335.