High non-protective, long-lasting antibody levels in malaria are associated with haplotype shifting in MHC-peptide-TCR complex formation: a new mechanism for immune evasion.

An effective malarial vaccine must contain multiple immunogenic, protection-inducing epitopes able to block and destroy the P. falciparum malaria parasite, the most lethal form of this disease in the world. Our strategy has consisted in using conserved peptides blocking parasite binding to red blood cells; however, these peptides are non-immunogenic and non-protection-inducing. Modifying their critical residues can make them immunogenic. Such peptides induced antibody titers (determined by immunofluorescence antibody test, IFA) and made the latter reactive (determined by Western blot) and protection inducing against experimental challenge with a highly infective Aotus monkey adapted P. falciparum strain. Modified peptides also induce highly non-protective long-lasting antibody levels. Modifications performed might allow them to bind specifically to different HLA-DRbeta purified molecules. These immunological and biological activities are associated with modifications in their three-dimensional structure as determined by (1)H-NMR. It was found that modified, high non-protective long-lasting antibody level peptides bound to HLA-DR molecules from a different haplotype (to which immunogenic, protection-inducers bind) and had 4.6 +/- 1.4 A shorter distances between residues fitting into these molecules' Pocket 1 to Pocket 9, suggesting fitting into an inappropriate HLA-DR molecule. A multi-component, subunit-based, malarial vaccine is therefore feasible if modified peptides are suitably modified for an appropriate fit into the correct HLA-DRbeta1* molecule in order to form a proper MHC-II-peptide-TCR complex.

Determining the 3D structure of human ASC2 protein involved in apoptosis and inflammation.

ASC2 structure has been well defined by 1141 NOE experimental restraints. The model consists of five alpha helices. alpha-Helices are connected by short random structure loops. The sole exception is the loop connecting helices 2 and 3, which has a 20-residue length. Folding generally agrees with the folding of recently published death domain structures in which alpha-helix structures have been reported. In spite of structural similarity, amino acid sequence homology with the most similar protein (ASC1) is just 64%. DD, DED, and CASP protein structures present six helices along their sequences; ASC2 presents 5 well-defined helices due to long distance restraints. However, a helical fragment was observed between amino acids 38 and 42 (representing helix 3) in the death domains when constructing the model.

Elongating modified conserved peptides eliminates their immunogenicity and protective efficacy against P. falciparum malaria.

Plasmodium falciparum malaria protein peptides were synthesised in the search for more effective routes for inducing a protective immune response against this deadly parasite and this information has been associated with such molecules' three-dimensional structure. These peptides had high red blood cell binding activity and their carboxy- and amino-terminal extremes were elongated for determining their immunogenic and protection-inducing activity against this disease in the Aotus monkey experimental model. 1H-NMR was used for analysing their three-dimensional structure; FAST ELISA, immunofluorescence antibody test, and Western blot were used for identifying their antibody inducing capacity and these previously immunised Aotus were inoculated with a highly infective P. falciparum strain to determine whether these elongated peptides were able to induce protection. This was aimed at establishing an association or correlation between long peptides' three-dimensional structure and their immunogenic and protection-inducing response in these monkeys. Peptides 20026 (25 residue), 20028 (30 residue), and 20030 (35 residues) were synthesised based on elongating the amino-terminal region of the 10022 highly immunogenic and protection-inducing modified peptide. 1H-NMR studies revealed that the first three had Classical type III beta-turn structures, different from the 20-amino acid long modified peptide 10022 which had a distorted type III beta-turn. Humoral immune response analysis showed that even when some antibodies could be generated against the parasite, none of the immunised Aotus could be protected with elongated peptides suggesting that elongating them eliminated modified peptide 10022 immunogenic and protection-inducing capacity.

Two L1-peptides are excellent tools for serological detection of HPV-associated cervical carcinoma lesions.

A persistent high risk human papillomavirus (HR-HPV) infection causes cervical intraepithelial lesions and cervical carcinoma. There is evidence that detecting anti-L1 antibodies could be successfully used for discriminating between cervical lesion patients and women having normal cytology. It was found that peptides 18283 (55PNNNKILVPKVSGLQYRVFR74) and 18294 (284LYIKGSGSTANLASSNYFPT300) from the L1-surface exposed regions were specifically recognised by antibodies from the cervical lesion patient sera. These peptides were tested against 165 womens' normal cytology sera and 148 cervical lesion or cervical cancer patients' sera. Less than 3.6% of women's normal cytology sera recognised peptides 18283 or 18294; on the contrary, 91% to 96% of the cervical lesion (CIN I to CIN III) or cervical cancer patient sera recognised peptides 18283 and 18294. These data show that anti-peptide 18283 and 18294 antibodies in the patients' sera are strongly associated with the presence of HR-HPV associated cervical lesions, showing 92-97% sensitivity and 89-95% specificity in recognising precancerous and cervical cancer patients. These two peptides could be excellent tools for use in large-scale serological screening of women populations at risk of developing cervical carcinoma.

Protection against malaria induced by chirally modified Plasmodium falciparum's MSP-1 42 pseudopeptides.

The C-terminal portion of the Plasmodium falciparum blood stage MSP-1 antigen plays a key role in invasion of human erythrocytes. The MSP-1(1282-1301) non-polymorphic 1585 peptide, from the processed MSP-1(42) fragment, is poorly immunogenic and highly alpha-helical [Angew. Chem. Int. Ed. 40 (2001) 4654]. Assessing the alpha-carbon asymmetry and its implication in the host immune response is proposed in this work to overcome the 1585 peptide's immunological properties. Accordingly, the effect of incorporating single D-amino acids and psi-[CH(2)-NH] isoster bonds into the 1585 peptide was examined both at the immunogenic and 3D-structure levels. Therefore, specific binding to RBCs is promoted by site-directed chiral modifications on the native peptide as well as by simultaneously combining specific D-substitutions with psi-[CH(2)-NH] isoster bonds transforming this molecule into a high specific HLAbeta1*1101 allele binder. D-analog pseudopeptide immunized animals induced antibodies selectively recognizing a recombinant as well as native MSP-1(42) and MSP-1(33) fragments. Protection and low parasitemia levels were induced in Aotus monkeys immunized with the EVLYL(dK)PLAGVYRSLKKQLE analog. Peptide alpha-carbon chiral transformation is therefore an important target for structural modulation and, consequently, represents a novel approach towards designing multi-component subunit-based malarial vaccines.

Fitting modified HRP-I peptide analogue 3D structure into HLA-DR molecules induces protection against Plasmodium falciparum malaria.

Conserved, high-activity, red blood cell binding malaria peptide 6786, from the HRP-I protein, having a random 3D structure as determined by 1H-NMR, was non-immunogenic and non-protection inducing when used as an immunogen in Aotus monkeys. Modifications made in its amino acid sequence were thus performed to render it immunogenic and protection inducing. Non-immunogenic, non-protection inducing modified peptide 13852 presented A2-H8 and K14-L18 helix fragments. Immunogenic, non-protection inducing modified peptide 23428 presented a short, displaced helix in a different region, whilst immunogenic, protection inducing peptide 24224 had 2 displaced helical regions towards the central region giving more flexibility to its N- and C-terminals. Immunogenic and protection inducing peptides bound with high affinity to HLA-DRB1* 0301 whilst others did not bind to any HLA-DRB1* purified molecule. Structural modifications may thus lead to inducing immunogenicity and protection associated with their capacity to bind specifically to purified HLA-DRB1* molecules, suggesting a new way of developing multi-component, subunit-based malarial vaccines.

Mapping the anatomy of a Plasmodium falciparum MSP-1 epitope using pseudopeptide-induced mono- and polyclonal antibodies and CD and NMR conformation analysis.

Antigen structure modulation represents an approach towards designing subunit malaria vaccines. A specific epitope's alpha carbon stereochemistry, as well as its backbone topochemistry, was assessed for obtaining novel malarial immunogens. A variety of MSP-1(38-61) Plasmodium falciparum epitope pseudopeptides derived were synthesised, based on solid-phase pseudopeptide chemistry strategies; these included all-L, all-D, partially-D substituted, all-Psi-[NH-CO]-Retro, all-Psi-[NH-CO]-Retro-inverso, and Psi-[CH2NH] reduced amide surrogates. We demonstrate that specific recombinant MSP-1(34-469) fragment binding to red blood cells (RBCs) is specifically inhibited by non-modified MSP-1(42-61), as well as by its V52-L53, M51-V52 reduced amide surrogates and partial-D substitutions in K48 and E49. In vivo tests revealed that reduced amide pseudopeptide-immunised Aotus monkeys induced neutralising antibodies specifically recognising the MSP-1 N-terminus region. These findings support the role of molecular conformation in malaria vaccine development.

Orientating peptide residues and increasing the distance between pockets to enable fitting into MHC-TCR complex determine protection against malaria.

The erythrocyte binding antigen EBA-175 is a 175-kDa Plasmodium falciparum protein, which has been shown to be involved in the process of invasion of erythrocytes. It has been found that conserved peptide 1818 belonging to this protein has high red blood cell binding capacity and plays an important role in the invasion process. This peptide is neither immunogenic nor protective. Peptide 1818 analogues had some of their previously recognized critical red blood cell binding residues substituted for amino acids having similar volume or mass but different polarity to make them fit into HLA-DRbeta(1)*1101 molecules; these 1818 peptide analogues were then synthesized and inoculated into Aotus nancymaae monkeys, generating different immunogenic and/or protective immune responses. Short structures such as 3(10)-helix, classical, or distorted type-III beta-turns were found in the immunogenic and protective peptides once the secondary structure had been analyzed by NMR and its structure correlated with its immunological properties. These data suggest that peptide flexibility may lead to better fitting into immune system molecules, therefore making them excellent candidates for consideration as components of a subunit-based, multicomponent synthetic antimalarial vaccine.

Shortening and modifying the 1513 MSP-1 peptide's alpha-helical region induces protection against malaria.

Immunogenic and protective peptide sequences are of prime importance in the search for an anti-malarial vaccine. The MSP-1 conserved and semi-conserved sequences have been shown to contain red blood cell (RBC) membrane high affinity binding peptides (HABP). HABP 1513 sequence ((42)GYSLFQKEKMVLNEGTSGTA(61)), from this protein's N-terminal, has been shown to possess a T-epitope; however, it did not induce a humoral immune response or complete protection when evaluated in Aotus monkeys. Analogue peptides with critical binding residues replaced by amino acids with similar mass but different charge were synthesised and tested for immunogenicity and protectivity in monkey. NMR studies correlated structural behaviour with biological function. Non-immunogenic and non-protective 1513 native peptide presented a helical fragment between residues L(4) and E(14). C-terminal, 5-residue-shorter, non-immunogenic, non-protective peptide 17894 contained an alpha-helix from Q(6) to L(12) residues. Immunogenic and protective peptide 13946 presented a shorter alpha-helix between K(7) to N(13) residues. These data suggest that changing certain residues permits better peptide fit within the MHC class II-peptide-TCR complex, thus activating the immune system and inducing a protective immune response.

An alpha helix conformationally restricted peptide is recognized by cervical carcinoma patients' sera.

Human papillomavirus type 16 (HPV-16) represents the major cervical carcinoma associated virus among women, especially in Colombia. It has thus become important to develop reliable inexpensive tests for detecting the presence of this virus. It has been shown that HPV16-E7 oncoprotein structural features have three alpha-helical structures and a loop-like structure. The hydrazone link approach was used to mimic helix secondary substructures. Sera from women with invasive cervical carcinoma were tested against conformationally restricted peptides and their respective linear peptides to identify conformational epitopes. One peptide that was conformationally restricted to an alpha-helix showed very strong positive reaction with sera from women having invasive cervical carcinoma; there was no reaction with sera from patients with other carcinomas, children, or healthy women. NMR studies confirmed this peptide's alpha-helical structure. The observation that constrained protein substructure peptidomimetics can identify new conformationally sensitive antibodies in cervical carcinoma patients' sera is very important, since these antibodies are almost all generated by native proteins, providing a new selection of antibodies for diagnostic and vaccine studies.

Alpha helix shortening in 1522 MSP-1 conserved peptide analogs is associated with immunogenicity and protection against P. falciparum malaria.

1522 is a nonimmunogenic conserved high-activity binding peptide (HABP) belonging to Plasmodium falciparum MSP-1 protein N-terminal fragment. The key amino acids in binding to red blood cells (RBC) were identified and replaced by others having similar mass but different charge. Because conserved HABPs are not antigenic nor immunogenic, immunogenicity and protectivity studies were then conducted on them in the Aotus monkey. 1H-NMR studies included the lead peptide 1522 as well as the analogs 9782, 13446, 13448, and 13442 to relate their structure to biological function. All the peptides presented alpha-helical structure, with differences observed in helix location and extension. The nonprotective 1522 peptide was totally helical from the N- to the C-terminus, very similar to nonprotective 13442 and 13448 peptides whose extension was almost totally helical. The 9782 and 13446 protective peptides, however, possessed a shorter helical region where modified critical binding residues were not included. A more flexible region was generated at the C-terminus in those peptides with a shorter helical region, leading to a greater number of conformers. These data suggest that peptide flexibility results in increased interaction with immune system molecules, generating protective immunity.

NMR structure of Plasmodium falciparum malaria peptide correlates with protective immunity.

Apical membrane antigen-1 is an integral Plasmodium falciparum malaria parasite membrane protein. High activity binding peptides (HABPs) to human red blood cells (RBCs) have been identified in this protein. One of them (peptide 4313), for which critical binding residues have already been defined, is conserved and nonimmunogenic. Its critical binding residues were changed for amino acids having similar mass but different charge to change such immunological properties; these changes generated peptide analogues. Some of these peptide analogues became immunogenic and protective in Aotus monkeys.Three-dimensional models of peptide 4313 and three analogues having different immune characteristics, were calculated from nuclear magnetic resonance (NMR) experiments with distance geometry and restrained molecular dynamic methods. All peptides contained a beta-turn structure spanning amino acids 7 to 10, except randomly structured 4313. When analysing dihedral angle phi and psi values, distorted type III or III' turns were identified in the protective and/or immunogenic peptides, whilst classical type III turns were found for the nonimmunogenic nonprotective peptides. This data shows that some structural modifications may lead to induction of immunogenicity and/or protection, suggesting a new way to develop multicomponent, subunit-based malarial vaccines.