Patent Application: US-201214360136-A

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 antigen or a fragment or fragments thereof , for use in raising immune responses in humans .

Description:
the present invention will now be described in detail with reference to the following figures which show : fig1 : 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 . fig2 : 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 ). fig3 : 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 fig2 c . c ) phagocytosis assay of opsonised it / par + infected erythrocytes co - incubated with the monocytic cell line thp - 1 [ 12 ]. data are shown as percentage of the positive control opsonised with a rabbit anti - human erythrocyte antibody . fig4 : antibodies to pfemp1 show cross - reactivity in surface recognition and rosette inhibition of p . falciparum clinical isolates . a ) flow cytometry of clinical isolate mal43 with 0 . 4 mg / ml of total igg from a non - immunised rabbit ( negative control , left panel ) and antibodies to tm284var1 ( middle panel ). infected erythrocytes stained with hoescht are in the right half , and antibody - positive infected erythrocytes stained with alexa fluor 488 are in the upper right quadrant . an overlay of histograms ( right panel ) shows a clear population of stained infected erythrocytes . b ) fresh clinical isolates tested with pfemp1 antibodies for surface reactivity ( ifa and flow cytometry at 0 . 4 mg / ml ) and rosette inhibition ( 1 mg / ml ). the percentage rosette inhibition is shown inside each rectangle for all isolate : antibody combinations with & gt ; 25 % rosette inhibition . the controls are as for fig2 c , and the anti - ros pool as for fig3 b . the anti - ros pool was tested for rosette inhibition only . c ) flow cytometry histograms to show negative controls , anti - pfemp1 positive and igm - positive infected erythrocytes for five clinical isolates . other isolates were tested by ifa only . the “ negative pfemp1 ab ” was antibody to tm180var1 and the igm negative control was a mouse igg1 isotype control . fig5 : 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 . fig6 : 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 . fig7 . 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 . fig8 : dbl domains from the itvar60 variant . the underlined regions indicate the recombinant proteins generated . abbreviations as for fig1 a . fig9 : 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 . fig1 : 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 . fig1 : 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 . fig1 : 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 . fig1 : 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 . fig1 : 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 . fig1 : 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 . fig1 : a : plasma from severe malaria cases tested on parasites expressing hb3var6 ; b : plasma from uncomplicated malaria controls tested on parasites expressing hb3var6 . fig1 : a : plasma from severe malaria cases tested on parasites expressing sa075var1 ; b : plasma from uncomplicated malaria controls tested on parasites expressing sa075var1 . fig1 : elisa showing polyclonal antibody response in mice immunised with tm284var1 . 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 ]. collection of clinical isolates ( blood samples ) from malaria patients was carried out in accordance with the declaration of helsinki . written informed consent was obtained from the patients &# 39 ; parents or guardians and was approved by the lothian regional ethical review committee ( lrec / 2002 / 4 / 34 ), the kemri ethical review committee , the gambia government / mrc laboratories joint ethics committee , the cameroon ministry of public health regional ethics committee and the university of bamako institutional review board . animal immunisations were carried out commercially by biogenes gmbh ( berlin , germany ) according to european union guidelines 86 / 609 / ewg of 24 . 11 . 1986 and the european agreement of 18 . 3 . 1996 for protection of animals used for scientific purposes . 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 . 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 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 ). 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 . 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 . 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 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 . the ability of pfemp1 antibodies to cross react with human igm was tested using purified human igm ( 5 μg / ml , rockland ) coated onto an elisa plate at 4 ° c . overnight . after blocking for 1 hour in pbs containing 0 . 05 % tween 20 ( pbst ) and 5 % milk , wells were incubated with 10 , 1 and 0 . 1 μg / ml of rabbit anti - nts dbl1α antibodies in pbst containing 1 % milk ( pbs ™). after 1 hour incubation at room temperature , wells were washed with pbst and incubated with 1 : 10 , 000 anti - rabbit igg - hrp ( sigma ) in pbs ™ for a further hour . after washing as above , reactions were developed by incubating the wells with substrate 3 , 3 ′, 5 , 5 ′- tetramethylbenzidinedihydrochloride ( sigma ) according to the manufacturer &# 39 ; s instructions and absorbance was measured at a wavelength of 450 nm . as a positive control , a rabbit anti - human igm f ( ab ′) 2 - hrp ( dako ) was used at 1 : 100 ( 10 μg / ml ), 1 : 1000 ( 1 g / ml ) and 1 : 10000 ( 0 . 1 μg / ml ). 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 . 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 ]. 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 ). 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 . 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 ( fig1 a ), defined by the presence of a conserved upstream sequence ( upsa ) and a characteristic n - terminal domain type ( called dblα1 or “ cys2 ”) that is associated with severe malaria [ 18 , 24 , 26 ]. the variants from the igm binding rosetting parasites form a distinct subset that share an unusual pfemp1 architecture , containing a triplet of domains that occur rarely in pfemp1 ( dble and dblz ) [ 6 ]. the binding site for non - immune igm lies within these dblε / ζ domains [ 43 , 44 ]( ag and jar , unpublished data ). the igm binding domain triplet is linked via at least one other domain ( dblγ ) to a typical group a pfemp1 head - structure [ 16 , 18 , 45 ]( fig1 a ). dblα domains from group a pfemp1 variants fall into eight subclasses ( dblα1 . 1 to dblα1 . 8 ) based on sequence homology [ 6 ]. the rosetting variants described previously ( itvar9 , palo alto varo and pf13 — 0003 ) [ 11 , 20 , 21 ] are all of the dblα1 . 6 subclass . the rosette - specific variants identified here are either dblα1 . 5 ( hb3var6 and muz12var1 ), dblα1 . 8 ( tm284 and itvar60 ) or dblα2 ( a non - group a type , tm180var1 ) [ 6 ]. 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 ( fig1 b ; shown previously for tm284 [ 44 ]). for each parasite strain , the rosette - specific pfemp1 probe detected a transcript in rosetting parasites ( arrowed ) that was absent / weak in isogenic non - rosetting parasites . the presence of other transcribed var genes in the non - rosetting parasites was shown using an exon ii probe that identifies all var genes ( fig1 b ). 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 ( fig1 c ). nts - dblα was chosen because it is the domain that binds erythrocytes to bring about rosetting [ 20 , 21 ], and variant - specific antibodies to this region were the most effective in inhibiting rosetting in previous studies [ 12 , 21 ]. 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 fig2 a ). rabbit pre - immune sera and non - immunised rabbit control sera were negative by ifa . titration of purified total igg from each antiserum showed specific surface reactivity against homologous parasites down to low concentrations ( end titres of 0 . 02 - 1 . 56 μg / ml of total igg , fig2 b and fig2 c , 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 & lt ; 10 μg / ml for heterologous antibody : parasite combinations ( fig2 c ). the non - igm binding rosetting strains ( muz12 , tm180 and it / r29 ) were also recognised by antibodies to igm binding rosetting variants , although higher concentrations were required ( 100 - 400 μg / ml of total igg , fig2 c ). although these concentrations are high , they still represent a considerable dilution of whole serum ( equivalent to 1 / 100 to 1 / 25 dilution ) therefore they are potentially achievable in vivo . the antibodies against rosetting pfemp1 variants did not recognise parasites selected for other adhesion phenotypes ( fig2 c ), including binding to cd36 or icam - 1 ( parasites expressing group b and c var genes ) or binding to brain endothelial cells ( parasites expressing an alternative sub - set of group a and b / a var genes ). thus , only parasites with a shared adhesion phenotype share epitopes that are recognised by cross - reactive antibodies to pfemp1 . 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 fig2 , 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 ( fig3 a , red curves ), except for tm180 , which was not inhibited ( fig3 a , brown curve ) despite good surface reactivity ( fig2 c ). parasite strains tm284 , it / par + and tm180 all showed rosette inhibition by heterologous antibodies ( fig3 a , blue curves ). at a higher concentration ( 1 mg / ml of total igg ) the cross - reactivity in rosette inhibition was even more marked , with all strains being inhibited by antibodies to at least one of the igm - binding rosetting pfemp1 variants ( fig3 b ). 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 ( fig3 c and fig7 a and b ). in contrast , antibodies to pfemp1 variants from non - igm binding rosetting parasites only effectively opsonised homologous parasites ( fig7 c 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 fig2 a ) and by flow cytometry ( fig4 a ). remarkably , antibodies to two pfemp1 variants ( hb3var6 and tm284var1 ) were sufficient to provide surface reactivity against all of the geographically diverse igm binding rosetting isolates ( fig4 b and c ). the proportions of anti - pfemp1 positive and igm positive cells were closely matched in each isolate ( pearson correlation r = 0 . 934 , p = 0 . 006 ; fig4 c , compare igm positive cells with the positive pfemp1 antibody stained cells ). rosette inhibition was also observed in four isolates , increasing to six isolates when a pool of anti - pfemp1 antibodies was used ( fig4 b ). the non - igm binding clinical isolate ( mal103 ) and two recently culture - adapted rosette - selected non - igm binding kenyan strains ( 9197 and sa075 ) were not recognized by the pfemp1 antibodies ( fig4 b ). 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 ( fig5 a ), and the surface reactivity with heterologous parasite strains was maintained when the parasites were grown in the absence of igm ( for example , it / par + parasites show surface reactivity with tm284var1 antibodies in the absence of igm as shown in fig5 b ). 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 ( fig4 b ), and selecting them for igm binding using magnetic beads coated with anti - human igm antibodies . after three rounds of selection , a population of igm binding rosetting parasites was obtained , which were recognised by antibodies to hb3var6 ( 9197 igm - selected , fig6 ) 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 ( fig6 b ). 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 , fig3 a ), confirming the role of these variants in rosette formation . furthermore , the antibodies against igm binding rosetting variants were cross - reactive , showing surface recognition of live infected erythrocytes and rosette inhibition with globally diverse parasite isolates sharing the same adhesion phenotype . in contrast , antibodies raised against group a pfemp1 variants from non - igm binding rosetting parasites were variant specific , as described previously [ 11 , 20 , 21 ]. these data indicate that not all group a pfemp1 variants induce cross - reactive antibodies , and that certain subsets of group a variants may be particularly cross - reactive . to our knowledge , this is the first report to describe the successful induction of broadly cross - reactive surface - recognising antibodies to pfemp1 variants implicated in severe childhood malaria . broadly cross - reactive antibodies against var2csa implicated in malaria in pregnancy have been described [ 47 , 48 ], however , var2csa is a unique case of a strain - transcendent var gene with much more limited sequence diversity than that seen in group a pfemp1 variants that are non - strain - transcendent [ 49 ]. 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 ( fig1 ). 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 ( fig6 ) 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 ( fig4 b ). these data are suggestive that these two main dblα1 types may underlie the igm binding rosetting phenotype in diverse field isolates , although clearly further sequence information is needed to substantiate this idea . tm180var1 differs from the other rosette - associated variants as it has an upsb sequence and a dblα2 subtype , and this may represent a distinct type of rosetting phenotype that requires further investigation . taking together the findings from this study and previously published work , we hypothesize that all group a pfemp1 variants with dblα1 . 5 , dblα1 . 6 or dblα1 . 8 domains may be rosette - mediating variants . if true , this would indicate that a substantial proportion ( approximately one third to one half ) of the group a var gene repertoire from every p . falciparum isolate may encode rosetting variants [ 6 ]. this would represent a substantial investment by the parasite in an adhesion phenotype whose benefit to parasite fitness remains unknown . 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 ( fig5 ). 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 . 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 ). 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 . antibodies were raised to all dbl domains from the itvar60 variant ( fig1 a and fig8 below regions shown in red ) and also to dbl4ε and dbl5e from the hb3var6 variant ( fig1 a ). 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 fig1 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 fig1 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 . 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 fig1 ). this recognition was specific to igm binding rosetting parasites , and was not seen with non - igm binding rosetting parasites ( see fig1 ). 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 . 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 - 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( 2011 ) allelic diversity of the plasmodium falciparum erythrocyte membrane protein 1 entails variant - specific red cell surface epitopes . plos one 6 : e16544 . parasites expressing itvar60 are specifically recognized by antibodies in plasma from children recovering from severe ( cerebral ) malaria 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 . recognition of surface antigens of live p . falciparum infected erythrocytes was tested by flow cytometry . parasite strains tested were : it / par + ( which are igm - positive rosetting parasites that express the itvar60 variant named in the patent ), which we predict should be recognized by the severe malaria children &# 39 ; s convalescent antibodies . it / r29 and sa075r +, which are igm - negative rosetting parasites that we predict should not be recognized by the severe malaria children &# 39 ; s convalescent antibodies . 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 ( fig1 , cm cases ). in contrast , age - matched control children with non - severe malaria do not show enhanced recognition of it / par + parasites in convalescent plasma ( fig1 , 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 ( fig1 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 ( fig1 a & amp ; b ), whereas , the control sa075var1 expressing parasites is not specifically recognized in this way ( fig1 a & amp ; b ). 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 ( fig1 ). in advance of screening for anti - pfemp1 monoclonal antibodies , fusions have been prepared from mouse spleen . 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 . 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