# Patent

## Title
Malaria vaccine

## 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.

## Background
RELATED APPLICATIONS
This application is a 35 U.S.C. §371 national stage application of PCT Application No. PCT/GB2012/052893, filed on Nov. 22, 2012, which claims priority from British Application No. 1120109.2, filed on Nov 22, 2011, the contents of which are incorporated herein by reference in their entireties. The above-referenced PCT International Application was published as International Publication No. WO 2013/076492 A1 on May 30, 2013.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 9013-134TS—ST25.txt, 175,973 bytes in size, generated on May 20, 2014 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.
FIELD OF THE INVENTION
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
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.
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).
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.
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].
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
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. 
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.
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.
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.
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.
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.
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”.
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. 
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.
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.
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.
    
    


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.
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.
The PfEMP1 variant designated HB3var6 has the following amino acid sequence:







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






Moreover, the HB3var6 PfEMP1 variant is encoded by the following nucleic acid sequence:







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






PfEMP1 variant designated TM284var1 has the following amino acid sequence:







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






Moreover, the TM284var1 PfEMP1 variant is encoded by the following nucleic acid sequence:







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






The PfEMP1 variant designated ITvar60 has the following amino acid sequence:







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






Moreover, the ITvar60 PfEMP1 variant is encoded by the following nucleic acid sequence:







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






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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
    
    


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.
In other embodiments, the DBL4ε domain is obtained from the PfEMP1 variant ITvar60.
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.
    
    


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.
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).
    
    


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.
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.
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.
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.
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'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.
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's adjuvant.
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.
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.
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.
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).
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.
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.
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.
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.
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, Fab2, scFv, VH domain and/or VLDomain 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 & Bethell, 2000, Taylor & 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.
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.
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.
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
The present invention will now be described in detail with reference to the following figures which show:
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.
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).
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. 2c. 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.
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 >25% rosette inhibition. The controls are as for FIG. 2c, and the Anti-Ros Pool as for FIG. 3b. 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.
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.
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.
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.
FIG. 8: DBL domains from the ITvar60 variant. The underlined regions indicate the recombinant proteins generated. Abbreviations as for FIG. 1a. 
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.
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.
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.
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.
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.
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.
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.
FIG. 16: A: Plasma from severe malaria cases tested on parasites expressing HB3var6; B: plasma from uncomplicated malaria controls tested on parasites expressing HB3var6.
FIG. 17: A: Plasma from severe malaria cases tested on parasites expressing SA075var1; B: plasma from uncomplicated malaria controls tested on parasites expressing SA075var1.
FIG. 18: ELISA showing polyclonal antibody response in mice immunised with TM284var1.



EXAMPLE 1
Materials and Methods
Parasites and Parasite Culture.
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.
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' 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.
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.
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.
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).
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.
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.
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.
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.
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 (PBSTM). After 1 hour incubation at room temperature, wells were washed with PBST and incubated with 1:10,000 anti-rabbit IgG-HRP (Sigma) in PBSTM 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'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.
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.
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.
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).
Therapeutic mAbs.
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
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. 1a), 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. 1a). 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].
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).
Correct identification of rosette-specific variants was confirmed by Northern blotting (FIG. 1b; 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. 1b).
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. 1c). 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].
Functional Activity and Cross-Reactivity of PfEMP1 Antibodies Against Laboratory-Adapted P. falciparum Strains
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. 2a). 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. 2b and FIG. 2c, rectangles in bold).
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 <10 μg/ml for heterologous antibody:parasite combinations (FIG. 2c). 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. 2c). 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.
The antibodies against rosetting PfEMP1 variants did not recognise parasites selected for other adhesion phenotypes (FIG. 2c), 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.
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. 3a, red curves), except for TM180, which was not inhibited (FIG. 3a, brown curve) despite good surface reactivity (FIG. 2c). Parasite strains TM284, IT/PAR+ and TM180 all showed rosette inhibition by heterologous antibodies (FIG. 3a, 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. 3b).
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. 3c and FIG. 7a and b). In contrast, antibodies to PfEMP1 variants from non-IgM binding rosetting parasites only effectively opsonised homologous parasites (FIG. 7c and d).
Functional Activity and Cross-Reactivity of Anti-PfEMP1 Antibodies Against P. falciparum Clinical Isolates
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].
Surface reactivity with the panel of PfEMP1 antibodies was detected by punctate fluorescence of live infected erythrocytes by IFA (similar to FIG. 2a) and by flow cytometry (FIG. 4a). 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. 4b 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. 4c, 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. 4b). 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. 4b). Therefore, in clinical isolates the cross-reactivity of PfEMP1 antibodies was only seen amongst parasites showing the IgM binding rosetting phenotype.
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. 5a), 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. 5b).
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. 4b), 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. 6b).
Discussion
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. 3a), 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].
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.
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. 4b). 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.
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.
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].
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.
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
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 & Methods
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:
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+)
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).
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.







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
<6.25


Anti-DBL2γ
0.02
0.80
<6.25


Anti-DBL3ζ
100
>100
>400


Anti-DBL4ε
1.56
25
>400


Anti-DBL5ε
1.56
9
>400









Tests of ITvar60 Antibodies Against Other Parasite Strains

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
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.
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.







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









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


    1. Taylor H M, Kyes S A, Harris D, Kriek N, & 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.









TABLE S2







Pair-wise amino acid identities for NTS-DBLα, CIDR1


and DBLγ from rosetting PfEMP1 variantsa
















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
 
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





aRosetting variants described in this work plus ITvar9 [1], Palo Alto Var O [2] and PF13_0003 [3].


bPair-wise amino acid identities between the IgM-binding rosetting strains shown in bold


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.













TABLE S3







Pair-wise amino acid identities for DBLε from rosetting PfEMP1 variants
















HB3
HB3
TM284
TM284
ITvar
ITvar
ITvar9
Palo Alto



var6d3a
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





ad3: 3rd DBL domain from the N-terminus













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
















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
















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
















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


PA varO




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
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
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'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's convalescent antibodies.
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).
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).
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).
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 & B), whereas, the control SA075var1 expressing parasites is not specifically recognized in this way (FIGS. 17 A & B).
Antibodies
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.


    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.
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## Claims

1. A method of raising an immune response in a human, the method comprising a step of administering an immunogenic amount of one or more Erythrocyte Membrane Protein-1 (PfEMP1) antigen(s) from Plasmodium falciparum (P. falciparum), to a human subject, wherein the PfEMP1 antigen(s) is:
(i) one or more PfEMP1 antigens having an amino acid sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; or
(ii) one or more PfEMP1 antigens having an amino acid sequence exhibiting at least 95% homology or identity to an amino acid sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

2. The method of claim 1, wherein the PfEMP1 antigen comprises a PfEMP1 Duffy Binding Like (DBL) domain, and wherein the DBL domain is one or more selected from the group consisting of:
(i) an NTS-DBL1α a domain from the PfEMP1 antigen having an amino acid sequence of SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5; and
(ii) a DBL4ε domain from the PfEMP1 antigen having an amino acid sequence of SEQ ID NO:5.

3. The method of claim 1, wherein the immune response:
(i) is effective against diverse strains and isolates of the malaria parasite, P. falciparum; and
(ii) comprises antibodies which exhibit a degree of affinity, selectivity and/or specificity to the PfEMP1 antigens having an amino acid sequence of SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5, and a degree of affinity, selectivity and/or specificity to PfEMP1 antigens other than the PfEMP1 antigens having an amino acid sequence of SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5.

4. A method of raising an immune response in a human comprising administering to a subject or a subject in need thereof, a vaccine or vaccine composition comprising one or more PfEMP1 antigens, or a DBL domain from one or more PfEMP1 antigens, selected from the group consisting of:
(i) a PfEMP1 antigen having an amino acid sequence of SEQ ID NO:1;
(ii) a PfEMP1 antigen having an amino acid sequence of SEQ ID NO:3;
(iii) a PfEMP1 antigen having an amino acid sequence of SEQ ID NO:5;
an NTS-DBL1α domain of the PfEMP1 antigen having an amino acid sequence of SEQ ID NO:1;
(v) an NTS-DBL1α domain of the PfEMP1 antigen having an amino acid sequence of SEQ ID NO:3:
(vi) an NTS-DBL1α domain of the PfEMP1 antigen having an amino acid sequence of SEQ ID NO:5; and
(vii) a DBL4ε domain of the PfEMP1 antigen having an amino acid sequence of SEQ NO:5.

5. The method of claim 4, wherein the immune response is raised in a human or a juvenile human.

6. method of raising an immune response in a human comprising administering to a subject or a subject in need thereof, an antibody exhibiting a degree of affinity, specificity and/or selectivity for one or more of the PfEMP1 antigens, or a DBL domain from one or more PfEMP1 antigens, selected from the group consisting of:
(i) a PfEMP1 antigen having an amino acid sequence of SEQ ID NO:1;
(ii) a PfEMP1 antigen having an amino acid sequence of SEQ ID NO:3:
(iii) a PfEMP1 antigen having an amino acid sequence of SEQ ID NO:5;
(iv) an NTS-DBL1α domain of the PfEMP1 antigen having an amino acid sequence of SEQ ID NO:1;
(v) an NTS-DBL1═ domain of the PfEMP1 antigen having an amino acid sequence of SEQ ID NO:3;
(vi) an NTS-DBL1α domain of the PfEMP1 antigen having an amino acid sequence of SEQ NO:5; and
(vii) a DBL4ε domain of the PfEMP1 antigen having as amino acid sequence of SEQ ID NO:5.

7. The method of claim 6, wherein the immune response is raised in a human or a juvenile human.