The Malaria Parasite ' s Achilles ' Heel : Functionally-relevant Invasion Structures

Malaria parasites have their Achilles' heel; they are vulnerable in small parts of their relevant molecules where they can be wounded and killed. These are sporozoite and merozoite protein conserved high activity binding peptides (cHABPs), playing a critical role in binding to and invasion of host cells (hepatocytes and erythrocytes, respectively). cHABPs can be modified by specific amino acid replacement, according to previously published physicochemical rules, to produce analogues (mHABPs) having left-handed polyproline II (PPIIL)-like structures which can modulate an immune response due to fitting perfectly into the HLA-DRβ1* peptide binding region (PBR) and having an appropriate presentation to the T-cell receptor (TCR). Achilles' heel of microbes According to the historian Statius, the Nereid Thetis, Achilles' mother dipped him headfirst into the River Styx when he was born to make him immortal and so that no arrow could wound him; not having anywhere else to hold him by, she held him by his right heel, this therefore becoming the only vulnerable site for the hero of the Iliad. Microbes also have their Achilles' heel, particularly the parasite P. falciparum, the causal agent of the lethal form of malaria; the conserved high activity binding peptides (cHABPs) from sporozoite (Spz) and merozoite (Mrz) proteins directly participate in binding to and invasion of host cells (hepatocytes and erythrocytes, respectively) (Garcia et al., 2006; Rodriguez et al., 2008). These conserved functionally relevant sequences, common to all the parasite's genetic variants in the world, are highly vulnerable to blocking parasite cell binding, cell lysis and death. When properly modified, these structures become mHABPs (Patarroyo and Patarroyo, 2008; Curtidor et al., 2011; Patarroyo et al., 2011) containing segments similar to native protein structures while some other portions, modif ied according to previously demonstrated physicochemical rules, allow their fit into the HLA-DRβ1* peptide binding region (PBR), converting mHABPs into strain-transcending, immune protection-inducing structures (IMPIPS). IMPIPS induce very high, strong, specific antibody and protective immune responses, as has been thoroughly demonstrated, establishing a new methodology for vaccine development. These cHABPs are thus the Achilles' heel of microbes, particularly the malaria parasite. The malaria parasite's Achilles' heel revealed by 3D structural analysis Analysing the Spz proteins studied CSP region I cHABP 4383 contains the RxLxE Plasmodium falciparum export element (PEXEL) motif (shadowed) (Table 1) (Hiss et al., 2008) mediating parasite protein membrane transport and Kappa B factor activation to induce Spz differentiation into Mrz. CSP 4388 (Table 1), considered to link repeats region to RII was localized 15 residues upstream the high content heparan sulphate proteoglycan (HSPG) binding site on hepatocytes where the parasite will become arrested and reproduce (Mota et al., 2001; Sibley, 2004). This CSP region I 3D structure has not yet been determined, therefore, these cHABP localizations are not shown in Figure 1. However, the 3D structure for the C-terminal CSP region III (named the α thrombospondin related (TSR) domain containing highly polymorphic T-helper epitopes Th2R, Th3R and CS.T3 has recently been determined by X-ray crystallography (Doud et al., 2012), an unexpected folding being found where the N and C termini are extremely close to each other. Intermediate conserved binding capacity peptide 4397 (Figure 1A, Table 1) in the β1 strand of this structure establishes a network of Hbonds between 323I and 333P with 319Y from 4394, 324Q with 355K, 328S with 346I, in non-binding 4400 and 4398 peptides respectively, and 341G with 369C in highly variable nonbinding sequence 4403 (J. E. Suarez et al., 2001). The CS.T3 conserved region associated with CD4* T-cell response in our 4405 peptide does not bind to hepatocytes. Sporozoite TRAP cHABP 3243 (Table 1) plays a main role in cell entry, cHABP 3271 (Table 1) contains the residues forming the metal ion-dependent adhesion site (MIDAS, highlighted) and 3279 (Figure 1B) which is the HSPG binding site located in the vWA domain (Pihlajamaa et al., 2013). It was also seen that bridges are formed between intermediate binding peptide 3277 (Figure 1B) amino acids 201F, 202L, 203V and 205C with 197A, 198F, 199N and 200R (Figure 1B). Similarly, cHABP 3287 (Figure 1C) located in the TSR region formed by one ripped and two anti-parallel and β-strands forms a groove conformed by π cation interactions between 247W, 250W and 262R, 264R (respectively), plus 3 H-bonds established between cHABP 3289 243S, 248D and 251S with 266R, 263S and 264R (Figure 1C). Positively-charged residues from this structure bind to negatively-charged receptors like heparan sulphate proteoglycans (HSPG) from hepatocytes and endothelial cells (Tossavainen et al., 2006) for mediating cell traversal act iv i ty and invas ion. Meanwhi le , TRAP 3347 (541YAGEPAPFVEPLGEE555) located 15 residues upstream the aldolase binding site, mediates this protein's binding to Curr. Issues Mol. Biol. (2016) 18: 11-20. horizonpress.com/cimb $11 The Malaria Parasite's Achilles' Heel: Functionallyrelevant Invasion Structures Malaria Parasite's Achilles' Heel Patarroyo et al. Curr. Issues Mol. Biol. (2016) 18: 11-20. horizonpress.com/cimb $12 Figure 1. 3D structure ribbon representation of malarial proteins (determined by X-crystallography) displaying cHABP localization and (on the right-hand side) isolated cHABPs establishing H-bonds amongst themselves to create the niche, trough, channel or cavity where receptors bind. (A). CSP the C-terminal region III (PDB 3VDK) (Doud et al., 2012). (B). TRAP vWA domain (PDB 4F1J) (Pihlajamaa et al., 2013). (C) TRAP TSR domain (PDB 2BBX) (Tossavainen et al., 2006). (D) SPECT-1 protein (PDB 4U5A) (Hamaoka and Ghosh, 2014). (E and F) SPECT-2 C8α-MACPF domain (PDB 2QQH) (Hadders et al., 2007). (G) P. falciparum AMA-1 (PDB 1Z40) (Bai et al., 2005). (H) P. vivax AMA-1 (PDB 1W81) (Pizarro et al., 2005). (I) EBA-175 RII dimeric fragment (PDB 1ZRO) (Tolia et al., 2005). (J) EBA-140 protein (PDB 4JNO) (Lin et al., 2012). (K) Pf EMP-1 DBL1α domain (PDB 2XU0) (Juillerat et al., 2011). (L) Pf Rh5 (PDB 4WAT) (Chen et al., 2014). (M) MSP-1 19kDa fragment (PDB 1OB1) (Pizarro et al., 2003). (N) MSP-2 1H-NMR 3D structure cHABP 4044 structure; dodecylphosphocholine-2H38 (DPC) binding residues shown in green. (O) SERA-5 fragment (PDB 3CH2) (Hodder et al., 2009). Malaria Parasite's Achilles' Heel Patarroyo et al. Curr. Issues Mol. Biol. (2016) 18: 11-20. horizonpress.com/cimb $13 Table 1. Peptides having gauche+ and gaucheconformation. Amino acid sequences for P. falciparum Spz and Mrz cHABPs (their critical binding residues are shown in bold) and their corresponding mHABPs below (in bold), with their critical binding and replaced residues highlighted in bold. The same amino acids in their corresponding peptide analogues are represented by a dashed line. Colors in the Table show the HLA-DRβ1* PBR register region (pink) and structural features (green). mHABPs' reciprocal antibody titers were determined by IFA (shown in brackets). Prot indicates total number of protected Aotus after experimental challenge. DR: HLA-DRβ1* allele with which each HABP experimentally displayed high HLA-DRβ1* purified molecule binding capacity. Peptide *24238 having gauche– orientation. ND: not determined by Spz challenge due to weird and non-reproducible results obtained with the only Aotus monkey-adapted P. falciparum strain (Santa Lucia). Malaria Parasite's Achilles' Heel Patarroyo et al. the actin-myosin motor of apicomplexa which are critical for Spz motility (Sultan et al., 1997). The very recently described SPECT-1 3D structure (Hamaoka and Ghosh, 2014) provides very strong support for our findings, since hypothetical RBC membrane cholesterol molecule (630 Å3) could fit in such very spacious interior cavity having around 750 Å3. cHABPs 33372 and 33375 are components of this cavity (Figure 1D, Table 1). On the other hand, cHABPs 33375 and 33374 (122LISNLSKRQQKLKGDKIKKV141) established H-bonds between themselves. SPECT-2 containing a 40kDa membrane attack complex pore forming (MACPF) domain or cholesteroldependent cytolysins (CDC) (Gilbert et al., 2014) is formed by D1, D2, D3 and D4 domains, where D4 binds to the cell membrane and D2 binds to D1 and D3 to mediate insertion into the membrane and pore formation (Hadders et al., 2007). A series of important cHABPs are found in the (MACPF) domain, such as cHABP 34938 (Table 1) and 34936, establishing H-bonds via 335Y with 301Y (Figure 1E) to generate a niche where a still unrecognized receptor binds to mediate cell traversal activity. By contrast, SPECT-2 34949 cHABP in D3 (Figure 1F, Table 1) (Hadders et al., 2007) interacts with heparin-like and chondroitin sulphate receptors on cells. After alignment with the complement component C8 alpha chain, it was found that 552D (34949) is involved in oligomerisation of perforin due to its negative charge and location (Baran et al., 2009). SPECT-2 cHABP 34959 (Table 1) outside the MACPF domain contains the 744TTL(I)Y747(W) cholesterol binding motif on membrane surface (Farrand et al., 2010) which is critical in pore formation and cell traversal activity. STARP 20546 (41VIKHNRFLSEYQSNFLGGGY60) displays the PEXEL motif RXLXE (shadowed), suggesting this protein is transported from the micronemes to the Spz membrane where it can be identified by immunofluorescence (Bermudez et al., 2010). Regarding Mrz invasion of RBC, the thoroughly analyzed AMA-1 protein, deeply involved in Mrz reo r i en ta t i on , mov ing j unc t i on fo rma t ion and parasitophorous vacuole resealing during RBC invasion (Yap et al., 2014), cHABPs 4313 (Table 1) in domain I and 4325 (Table 1) in domain II of this protein form the niche or trough via H-bonds between 134D and 143R with 390Y and 391K wh

The malaria parasite's Achilles' heel revealed by 3D structural analysis Analysing the Spz proteins studied CSP region I cHABP 4383 contains the RxLxE Plasmodium falciparum export element (PEXEL) motif (shadowed) (Table 1) (Hiss et al., 2008) mediating parasite protein membrane transport and Kappa B factor activation to induce Spz differentiation into Mrz.CSP 4388 (Table 1), considered to link repeats region to RII was localized 15 residues upstream the high content heparan sulphate proteoglycan (HSPG) binding site on hepatocytes where the parasite will become arrested and reproduce (Mota et al., 2001;Sibley, 2004).This CSP region I 3D structure has not yet been determined, therefore, these cHABP localizations are not shown in Figure 1.However, the 3D structure for the C-terminal CSP region III (named the α thrombospondin related (TSR) domain containing highly polymorphic T-helper epitopes Th2R, Th3R and CS.T3 has recently been determined by X-ray crystallography (Doud et al., 2012), an unexpected folding being found where the N and C termini are extremely close to each other.Intermediate conserved binding capacity peptide 4397 (Figure 1A, Table 1) in the β1 strand of this structure establishes a network of Hbonds between 323 I and 333 P with 319 Y from 4394, 324 Q with 355 K, 328 S with 346 I, in non-binding 4400 and 4398 peptides respectively, and 341 G with 369 C in highly variable nonbinding sequence 4403 (J.E. Suarez et al., 2001).The CS.T3 conserved region associated with CD4* T-cell response in our 4405 peptide does not bind to hepatocytes.Sporozoite TRAP cHABP 3243 (Table 1) plays a main role in cell entry, cHABP 3271 (Table 1) contains the residues forming the metal ion-dependent adhesion site (MIDAS, highlighted) and 3279 (Figure 1B) which is the HSPG binding site located in the vWA domain (Pihlajamaa et al., 2013).It was also seen that bridges are formed between intermediate binding peptide 3277 (Figure 1B) amino acids 201 F, 202 L, 203 V and 205 C with 197 A, 198 F, 199 N and 200 R (Figure 1B).Similarly, cHABP 3287 (Figure 1C) located in the TSR region formed by one ripped and two anti-parallel and β-strands forms a groove conformed by π cation interactions between 247 W, 250 W and 262 R, 264 R (respectively), plus 3 H-bonds established between cHABP 3289 243 S, 248 D and 251 S with 266 R, 263 S and 264 R (Figure 1C).Positively-charged residues from this structure bind to negatively-charged receptors like heparan sulphate proteoglycans (HSPG) from hepatocytes and endothelial cells (Tossavainen et al., 2006) for mediating cell traversal a c t i v i t y a n d i n v a s i o n .M e a n w h i l e , T R A P 3 3 4 7 ( 541 YAGEPAPFVEPLGEE 555 ) located 15 residues upstream the aldolase binding site, mediates this protein's binding to The Malaria Parasite's Achilles' Heel: Functionallyrelevant Invasion Structures   (Doud et al., 2012).(B).TRAP vWA domain (PDB 4F1J) (Pihlajamaa et al., 2013).(C) TRAP TSR domain (PDB 2BBX) (Tossavainen et al., 2006).(D) SPECT-1 protein (PDB 4U5A) (Hamaoka and Ghosh, 2014).(E and F) SPECT-2 C8α-MACPF domain (PDB 2QQH) (Hadders et al., 2007).(G) P. falciparum AMA-1 (PDB 1Z40) (Bai et al., 2005).(H) P. vivax AMA-1 (PDB 1W81) (Pizarro et al., 2005).(I) EBA-175 RII dimeric fragment (PDB 1ZRO) (Tolia et al., 2005).(J) EBA-140 protein (PDB 4JNO) (Lin et al., 2012).
The very recently described SPECT-1 3D structure (Hamaoka and Ghosh, 2014) provides very strong support for our findings, since hypothetical RBC membrane cholesterol molecule (630 Å 3 ) could fit in such very spacious interior cavity having around 750 Å 3 .cHABPs 33372 and 33375 are components of this cavity (Figure 1D, Table 1).On the other hand, cHABPs 33375 and 33374 ( 122 LISNLSKRQQKLKGDKIKKV 141 ) established H-bonds between themselves.
SPECT-2 containing a 40kDa membrane attack complex pore forming (MACPF) domain or cholesteroldependent cytolysins (CDC) (Gilbert et al., 2014) is formed by D1, D2, D3 and D4 domains, where D4 binds to the cell membrane and D2 binds to D1 and D3 to mediate insertion into the membrane and pore formation (Hadders et al., 2007).A series of important cHABPs are found in the (MACPF) domain, such as cHABP 34938 (Table 1) and 34936, establishing H-bonds via 335 Y with 301 Y (Figure 1E) to generate a niche where a still unrecognized receptor binds to mediate cell traversal activity.By contrast, SPECT-2 34949 cHABP in D3 (Figure 1F, Table 1) (Hadders et al., 2007) interacts with heparin-like and chondroitin sulphate receptors on cells.After alignment with the complement component C8 alpha chain, it was found that 552 D ( 34949) is involved in oligomerisation of perforin due to its negative charge and location (Baran et al., 2009).SPECT-2 cHABP 34959 (Table 1) outside the MACPF domain contains the 744 TTL(I)Y 747 (W) cholesterol binding motif on membrane surface (Farrand et al., 2010) which is critical in pore formation and cell traversal activity.STARP 20546 ( 41 VIKHNRFLSEYQSNFLGGGY 60 ) displays the PEXEL motif RXLXE (shadowed), suggesting this protein is transported from the micronemes to the Spz membrane where it can be identified by immunofluorescence (Bermudez et al., 2010).
Regarding Mrz invasion of RBC, the thoroughly analyzed AMA-1 protein, deeply involved in Mrz r e o r i e n t a t i o n , m o v i n g j u n c t i o n f o r m a t i o n a n d parasitophorous vacuole resealing during RBC invasion (Yap et al., 2014), cHABPs 4313 (Table 1) in domain I and 4325 (Table 1) in domain II of this protein form the niche or trough via H-bonds between 134 D and 143 R with 390 Y and 391 K where a still unrecognized receptor binds (Bai et al., 2005) (Figure 1G).A very informative finding for invasion of host cells was shown by the H-bond established between 49 T some residues downstream AMA-1 cHABP 4310 (Table 1) localized in this molecule's prodomain and 474 Y in cHABP 4332 (Table 1) localized 450 residues downstream cHABP 4310 in domain III, containing this molecule's cleavage site (arrow) (Figure 1H).After cleavage, cHABP 4332 remains anchored to the Spz membrane and is the only cHABP found inside infected RBC, demonstrating different domains' functional compartmentalization and cooperation to perform a set function (4313 and 4325 involved in RBC invasion present in domains I and II, respectively; and 4310 and 4332 in the prodomain and domain III, respectively, involved in hepatocyte invasion).Concerning the most relevant of the Duffy binding-like (DBL) family of proteins (McHenry and Adams, 2006) binding to neuraminidase sensitive receptors EBA-175 583 K and 586 D, mHABP 1783 ( 580 HRN 583 KKN 586 DKLYRDEW 594 WK 596 VIKK 599 ) bind to glycophorin A receptor glycan 5 on RBC while 594 W induces fold stabilization and 580 H establishes H-bonds with its contralateral 596 V to allow the dimerization of this protein to mediate RBC attachment and invasion (Tolia et al., 2005;Ambroggio et al., 2013) (Figure 1I).EBA-175 cHABPs 1815 ( 1220 YTNQNINISQERDLQKHG FH 1239 ) and 1818 ( 1280 NNNFNNIPSRYNLYDKKLDL 1299 ) are located in regions IV-V of this protein and antibodies against this region neutralize multiple P. falciparum strains in vitro (Ambroggio et al., 2013); however, no 3D structure has been determined so far for EBA-175 regions IV-V.
In another member of the DBL family, cHABP 26147 (Table 1) from the relevant EBA-140 protein (which also binds to neuraminidase sensitive receptor) establishes three H-bonds via 618 Q with cHABP 26144 541 D, 550 D and 551 I, the latter exposing the α-helix where 556 Y interacts with the acetamide group in sialic acid in RBC glycophorin C (Figure 1J) (Lin et al., 2012).
Regarding another member of this DBL family, cHABP 6510 (Table 1) from Pf-EMP-1, expressed on infected erythrocyte (iE) membrane, establishes an H-bond between 132 C and 161 E in cHABP 6512 (Patarroyo et al., 2014) (Figure 1K), forming the niche where the A1 blood group terminal α-1,3 linked N-acetylgalactosamine (Vigan-Womas et al., 2012) binds through residues 138 Q and 140 E in cHABP 6510 to form rosettes with non-infected erythrocytes binding to small vessel endothelial cells to induce severe placental and cerebral malaria.
The Pf-RH5 protein, a member of the erythrocyte binding ligand (EBL) family, mediates a critical nonredundant interaction with human RBC surface protein basigin (present in other tissues) during invasion (Crosnier et al., 2011;Wright et al., 2014).cHABP 36735 amino acids 377 N, 378 K, 379 D and 380 L interact with cHABP 36736 381 S, 382 D and 384 T by H-bond formation (Figure 1L) to create a niche where RBC bind and where a single amino acid replacement determines the preference for Aotus or human RBC binding.
SERA-5 cHABP 6754 (Table 1) forms H-bonds via 755 H and 756 A with 6746 (Table 1) 588 S, forming the noncanonical enzymatic triad (Figure 1O) for this relevant molecule in malarial protein processing and egress (Hodder et al., 2009).No function has been assigned yet for SERA 6737 ( 401 YDNILVKMFKTNENNDKSELI 421 ) but it is located 20 residues downstream the cleavage site of this protein during Mrz and Spz maturation and processing by subtisilin 1 (Kanodia et al., 2014), suggesting it is only exposed after SERA processing.

Achilles can also die
All the foregoing, striking data clearly shows that invasion by P. falciparum Spz and Mrz requires this parasite to create a niche, trough, channel or cavity formed by one or two cHABPs localized in different molecule regions or domains for binding to the receptors on host cells (Patarroyo et al., 2010a).These cHABPs, due to their critical function during infection, cannot display variations in their amino acid sequences or in their 3D structure, therefore being immunologically silent and making them the malaria parasite's Achilles' heel.
Consequently, these cHABPs are excellent targets for inducing a protective, strain-transcending immune response to impede, block or destroy parasite function.
When some critical host cell binding residues in cHABPs have been appropriately modified (mHABP) as determined by glycine analogue scanning, their altered structures allow them to fit into the HLA-DRβ* PBR.This makes them high specific antibody titer inducers against the protein and the parasite (determined by ELISA, IFA (Figure 2A) and WB).This also makes them capable of inducing protection against experimental infection in the Aotus monkey experimental model (Figure 2B) which has an almost identical immune system to that of humans (C.F. Suarez et al., 2006).Since the Santa Lucia strain gives very weird and irreproducible results (this being the only Aotus-adapted P. falciparum one), testing Spz-induced immunity means that analysis of Spz-derived mHABPs is limited to determining very high, long-lasting, antibody induction (VHLLAI), as assessed by IFA (Table 1) and WB.Changes in mHABP secondary structure would include αhelices becoming displaced, shortened or modified as in 22814 (1783) (Table 1) (Cifuentes et al., 2003a), modified β-turns, as in 24246 (3289), 10008 (4044) and 24112 (4044) (Cifuentes et al., 2003b) and when they have random structures they acquire specific conformations, as in 10022 (4313) (distorted type III' turn in T7 to F10 ) or 23426 (6754) (type V turn between V3-L6) (Purmova et al., 2002;Bermudez et al., 2012) (Table 1).
Throughout these years we have established that most modifications must be made in mHABP unstructured regions (Table 1) and, according to previously published rules, that 2 to 4 replacements are needed to convert such regions into PPIIL structures, thereby facilitating mHABP binding to the HLA-DRβ1* PBR, leaving the rest of the molecule unmodified.This would suggest that mHABPs have both T-cell binding sequences able to interact with the TCR as well as B-cell sequences able to induce antibody production against the whole molecule.
To the best of our knowledge, these are the first structural-functional T-and B-cell epitopes (tailor-made) forming components of a complete, fully protective, anti-P.falciparum malaria vaccine, as thoroughly demonstrated (Curtidor et al., 2011;Patarroyo et al., 2011).

Rotamer orientation for appropriate TCR interaction
Once firmly anchored to the HLA-DRβ* PBR via H-bonds, mHABPs have to be presented to the TCR to induce an appropriate immune response.The TCR scans the topochemical characteristics of the HLA-DRβ*-mHABP complex which, due to the peculiar diagonal canonical TCR orientation, allows ±60° freedom (Rudolph et al., 2006).Therefore, mHABP rotamer orientation must ensure their solvent-exposed or upwardly-orientated TCR contacting residues is specific stereo-electronic and topochemical.In pure atomic molecular architecture, the χ1 angles of residues localized in positions p3 and p7 (position is designated by p in the rest of the paragraph) must have gauche+ orientation (Table 2) as well as the some χ2 angle in p5, p2 must be polar and orientated towards the righthand side-chain of an mHABP backbone, p3 must be apolar and towards the left, p5 perpendicular to a peptide's backbone structure, p7 towards the right and p8 towards the left (Bermudez et al., 2014).These physicochemical parameters have allowed the development of principles and rules for a logical and rational methodology for minimal subunit-based, multi-epitope, multi-stage, fully-protective, complete, definitive chemically-synthesised vaccines which can also be used for developing vaccines against other infectious diseases, such as tuberculosis, Ebola, etc., and even cancer associated with or induced by microbes, providing strong support for our functional-structural approach to developing new vaccines.
Most parts of microbes (including malaria), just like Achilles' body, are invulnerable or have many shields or tricks to protect themselves but, like the hero of the Iliad, very small parts of their structure (body/heels) are vulnerable and this is where can be hurt and killed.Table 2. IMPIPS Φ, ψ and χ1 angles.The colors of residues whose position is horizontally displayed on top follow the code: p1 (fuchsia), p2 (red), p3 (pale blue), p4 (dark blue), p5 (pink), p6 (orange), p7 (grey), p8 (yellow) and p9 (green).The colors displayed in the Table correspond to structure conformations present in these mHABPs: (grey: PPIIL), (orange: αR), (lilac: αL).Residues in p3 (dark blue) and p7 (yellow) have gauche+ orientation, while χ1 angles (red) have gauche− orientation, blocking, interfering or suppressing mHABPs.

Figure 2 .
Figure 2. (A) Sporozoite and merozoite protein immunofluorescence patterns and location recognized by Aotus monkey sera, having high antibody titers when immunized with mHABPs (shown in bold) from corresponding proteins (shown in bold letters) and cHABPs (shown in parentheses).Spz IFA (first row) Aotus antibodies against CSP 25608 (4383) were used for these triple-labeling studies as reference, using anti-Aotus IgG coupled to fluorescein isothiocyanate (FITC) (showing green fluorescence on Spz membrane).Immune sera reactivity with SPECT-1 and -2 proteins was detected by using anti-Aotus IgG coupled to rhodamine (RITC) (showing red under a fluorescence microscope).Nuclear DNA was stained with 4,6diamidino-2-phenylindole (DAPI) showing bright blue fluorescence.Regarding Mrz protein localization, sera from Aotus monkeys immunized with mHABPs (in bold), derived from proteins (also in bold letters), led to corresponding cHABPs (in parentheses) completely agreeing with the localization of the P. falciparum proteins from which these mHABPs had been derived.(B) This Figure gives some examples of the parasitaemia course in Aotus monkeys immunized with fully protectioninducing peptides.The course of parasitaemia displayed on a semi-logarithmic scale was quantified daily by the highly specific and sensitive acridine orange staining method by fluorescence microscope.

Table 1 .
Peptides having gauche+ and gauche-conformation.Amino acid sequences for P. falciparum Spz and Mrz cHABPs (their critical binding residues are shown in bold) and their corresponding mHABPs below (in bold), with their critical binding and replaced residues highlighted in bold.The same amino acids in their corresponding peptide analogues are represented by a dashed line.Colors in the Table show the HLA-DRβ1* PBR register region (pink) and structural features (green).mHABPs' reciprocal antibody titers were determined by IFA (shown in brackets).Prot indicates total number of protected