Mauricio Urquiza

Identification of Plasmodium falciparum MSP-1 peptides able to bind to human red blood cells.

To determine amino acid sequences of the Plasmodium falciparum MSP‐1 protein that interact with red blood cell membranes in a specific receptor‐ligand interaction, 78 sequential peptides, 20 amino acids long and spanning the entire length of the molecule, were synthesized and analysed with a specific binding assay developed for this purpose. Results show that peptides based on conserved and dimorphic regions of MSP‐1, interact with human red blood cells (RBCs). This interaction occurs predominantly with peptides contained within the MSP‐1 proteolytic fragments of 83 kDa, 38 kDa, 33 kDa and 19 kDa. Affinity constants of these peptides were between 140 and 250 nM. Peptide‐RBC binding post enzyme treatment showed that the RBC receptors are not sialic acid dependent and appear to be proteic in nature. Some of these peptides inhibited merozoite invasion of RBCs yet did not inhibit intra‐erthrocytic development. These peptides, in conjunction with those from other merozoite surface proteins, may be used to rationally design a second generation of synthetic peptide‐based malaria vaccines.

Intimate Molecular Interactions of P. falciparum Merozoite Proteins Involved in Invasion of Red Blood Cells and Their Implications for Vaccine Design

3. Merozoite Surface Protein (MSP) Family 3662 3.1. Merozoite Surface Protein-1 (MSP-1) 3664 3.2. Merozoite Surface Protein-2 (MSP-2) 3675 3.3. Merozoite Surface Protein-3 (MSP-3) 3677 3.4. Merozoite Surface Protein-4 (MSP-4) 3677 3.5. Merozoite Surface Protein-5 (MSP-5) 3678 3.6. Merozoite Surface Protein-6 (MSP-6) 3678 3.7. Merozoite Surface Protein-7 (MSP-7) 3678 3.8. Merozoite Surface Protein-8 (MSP-8) 3679 3.9. Merozoite Surface Protein-9 (MSP-9) or Acid Basic Repeat Antigen (ABRA) 3679

Plasmodium falciparum AMA-1 erythrocyte binding peptides implicate AMA-1 as erythrocyte binding protein

The role of AMA-1 during merozoite invasion has not yet been determined. However, reported experimental evidence suggests that this protein can be used, in particular as erythrocyte-binding protein, since, Fab fragments against this protein are able to block merozoite invasion. Using a previously described methodology, eight peptides with high binding activity to human erythrocyte, scattered along the different domains and having around 130 nM affinity constants, were identified in the Plasmodium falciparum AMA-1 protein. Their binding activity was sialic acid independent. Some of these peptides showed homology with the erythrocyte binding domains of one of the apical organelle protein family, MAEBL, identified in rodent malarial parasites. One of these peptides shares amino acid sequence with a previously reported B-cell epitope which induces antibodies to block parasite growth. The critical residues were identified for erythrocyte binding conserved peptides 4313 (DAEVAGTQYRLPSGKCPVFG), 4321 (VVDNWEKVCPRKNLQNAKFG), 4325 (MIKSAFLPTGAFKADRYKSH) and 4337 (WGEEKRASHTTPVLMEKPYY). All conserved peptides were able to block merozoite invasion of new RBC and development, suggesting that these peptides are involved in P. falciparum invasion.

Plasmodium falciparum acid basic repeat antigen (ABRA) peptides: erythrocyte binding and biological activity

Non overlapping 20-mer peptides, covering the complete sequence of acid basic repeat antigen (ABRA) of Plasmodium falciparum, were synthesised and tested in binding assays to erythrocytes. Five peptides localised in the N-terminal region coded 2148 (121LQSHKKLIKALKKNIESYQN(140)), 2149 (141KKHLIYKNKSYNPLLLSCVK(160)), 2150 (161KMNMLKENVDYIQKNQNLFK(180)), 2152 (201YKSQGHKKETSQNQNENNDN(220)) and 2153 (221QKYQEVNDEDDVNDEEDTND(240)) specifically bind to erythrocytes. These peptides bind independently of the peptide and erythrocyte charge, with high affinity (Kd between 70 and 180 nM) and the hydrophobic interaction is important for this binding ( approximately 30% hydrophobic critical residues). These results allow us define a specific erythrocyte binding region (residues 121-240), which may bound to at least three different binding sites on erythrocytes. Peptide 2153 shares the underlined sequence 221QKYQEVNDEDDVNDEEDTND(240) with an earlier 18-mer peptide recognised by human exposed sera. Peptides number 2148 and 2149 in vitro inhibit erythrocyte invasion by merozoites. We found that 2149 peptide and some of its glycine analogues show specific haemolytic and/or antimicrobial activity. We discuss a possible role of ABRA or its regions in the merozoite invasion of erythrocyte.

Hepatitis C virus (HCV) E1 and E2 protein regions that specifically bind to HepG2 cells

BACKGROUND/AIMS Identify hepatitis C virus (HCV) sequences in E1 and E2 protein binding to HepG2. METHODS Synthetic 20-mer long, ten-residue overlapped peptides, from E1 and E2 proteins, were tested in HepG2 or Raji cell-binding assays. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay for high activity binding peptides (HABPs). Receptors for HepG2 cell were determined by cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. RESULTS Twelve HABPs were found in HCV genotype 1a, allowing six hepatocyte-binding sequences (HBSs) to be defined: two peptide-binding regions in E1 HABPs 4913 (YQVRNSTGLYHVTNDCPNSS) and 4918 (MTPTVATRDGKLPATQLRRHY). Four hepatocyte-binding regions were defined in E2: region-I, peptide 4931 (ETHVTGGSAGHTVSGFVSLLY); region-II, 4937-4939 (HHKFNSSGCPERLASCRPLTDFDQGWGPISYANGSGPDQR); region-III, 4943-4945 (PVYCFTPSPVVVGTTDRSGAPTYSWGENDTDVFVLNNTR) and region-IV, 4949-4952 (CGAPPCVIGGAGNNTLHCPTDCFRKHPDATYSRCGSGPWITPRCLVDYPY). The underlined sequences are most relevant in the binding process. HABPs 4913 and 4938 also bind to CD81 positive Raji cells. Region-II 4938 HABPs bind to 50 and 60kDa HepG2 cell membrane surface proteins. CONCLUSIONS Six HVRs to the HepG2 were identified. Some HABPs have been previously found to be antigenic and immunogenic. HABPs, 4918 (from E1), 4938, 4949, 4950, 4951 and 4952 (from E2) have not been previously recognised. These HABPs could be relevant to HCV invasion of hepatocytes.

Plasmodium vivax MSP-1 peptides have high specific binding activity to human reticulocytes

Plasmodium vivax merozoites have high preferential ability to interact with and invade reticulocytes, although these cells correspond to only 2% of the red blood cells (RBC) population. P. vivax merozoite surface protein-1 (Pv-MSP-1) is believed to have an important role in attachment and invasion process. Using 88 non-overlapping 20-mer peptides, covering the entire Pv-MSP-1 Belem strain sequence, RBC and reticulocyte binding assays were performed. Fourteen sequences were identified with high specific binding activity to reticulocytes, but only three had high specific binding activity to mature erythrocytes. These peptides showed affinity constant values between 20 and 150nM, indicating a strong interaction between these sequences and reticulocyte receptors. Critical residues in binding to reticulocytes for these peptides were determined by competition binding assays with glycine scanning analogues. All high binding peptides bind to reticulocyte surface proteins having a molecular mass of around 18-20kDa which are not present in mature RBC. Interestingly, some high activity binding peptides (HABPs) are located close to the hypothesised 42 and 19kDa fragment cleavage sites for this protein, suggesting that these sequences have an important role in target cell attachment and invasion process by Pv-MSP-1.HABPs may be clustered in two regions, with region I being located between amino acids 280-719, and region II between amino acids 1060-1599 with higher than 25% identity level. A P. falciparum MSP-1 antigenic domain binds to RBCs and inhibits parasite invasion. Peptides 1721 and 1724 bind with high activity to reticulocytes in homologous Pv-MSP-1, suggesting similar functions for these two sequences.

Serine repeat antigen peptides which bind specifically to red blood cells.

It has been reported that serine repeat antigen (SERA) binds directly to human erythrocyte membranes, inside-out vesicles and intact mouse erythrocytes. Similarly, mAbs specific against SERA are effective in blocking red blood cell (RBC) invasion by P. falciparum merozoites. Furthermore, the N-terminal recombinant SERA fragment inhibits the merozoite invasion of erythrocyte. In this study of 49 non-overlapping 20-residue-long peptides encompassing the whole SERA protein FCR3 strain, seven peptides having high RBC binding activity were found. Six of these peptides (three from the SERA N-terminal domain) are located in conserved regions and show affinity constants between 150 and 1100 nM, Hill coefficients between 1.5 and 3.0 and 30000-120000 binding sites per cell. Some of these peptides inhibited in vitro merozoite invasion of erythrocyte and intra-erythrocytic development. Residues which are critical in the binding to erythrocytes (in bold face), i.e. 6725 (YLKETNNAISFESNSGSLEKK), 6733 (YALGSDIPEKCDTLASNCFLS), 6737 (YDNILVKMFKTNENNDKSELI), 6746 (DQGNCDTSWIFASKYHLETI), 6754 (YKKVQNLCGDDTADHAVNIVG) and 6762 (NEVSERVHVYHILKHIKDGK), were determined by means of competition assays with high-binding peptide glycine analogues. The identification of peptides which bind to erythrocyte membrane is important in understanding the process of RBC invasion by P. falciparum merozoites.

Plasmodium vivax Duffy binding protein peptides specifically bind to reticulocytes

Plasmodium vivax Duffy Binding Protein (Pv-DBP) is essential during merozoite invasion of reticulocytes. Reticulocyte binding region identification is important for understanding Pv-DBP reticulocyte recognition. Fifty 20 mer non-overlapping peptides, spanning Pv-DBP sequences, were tested in erythrocyte and reticulocyte binding assays. Ten HARBPs, mainly located in region II (Kd 50-130 nM), were High Activity Reticulocyte Binding Peptides (HARBPs); one bound to erythrocytes. Reticulocyte trypsin-, chymotrypsin- or neuraminidase- treatment affects HARBP binding differently, suggesting that these peptides have different reticulocyte-binding-sites. Some peptides bound to a Coomasie non-stainable 40 Kda band. Some HARBPs were able to block recombinant PvRII binding (Pv-DBP region II) to Duffy positive reticulocytes.

Gene Immunotherapy of Chronic Lymphocytic Leukemia: A Phase I Study of Intranodally Injected Adenovirus Expressing a Chimeric CD154 Molecule

New therapies for chronic lymphocytic leukemia (CLL) are needed, particularly those that can eradicate residual disease and elicit anti-CLL immune responses. CD40 ligation on CLL cells, which can be achieved using adenovirus encoding chimeric CD154 (Ad-ISF35), enhances their ability to function as antigen-presenting cells and increases their sensitivity to clearance by immune-effector mechanisms. In this study, we report the results of a first-in-man phase I trial of intranodal direct injection (IDI) of Ad-ISF35 in patients with CLL to evaluate toxicity, safety, and tolerability. Fifteen patients received a single IDI of 1 × 10(10) to 33 × 10(10) Ad-ISF35 viral particles (vp), with a defined maximum tolerated dose as 1 × 10(11) vp. Although the most common adverse events were transient grade 1 to 2 pain at the injection site and flu-like symptoms following IDI, some patients receiving the highest dose had transient, asymptomatic grade 3 to 4 hypophosphatemia, neutropenia, or transaminitis. Increased expression of death receptor, immune costimulatory molecules, and Ad-ISF35 vector DNA was detected in circulating CLL cells. Notably, we also observed preliminary clinical responses, including reductions in leukemia cell counts, lymphadenopathy, and splenomegaly. Six patients did not require additional therapy for more than 6 months, and three achieved a partial remission. In conclusion, Ad-ISF35 IDI was safely delivered in patients with CLLs and induced systemic biologic and clinical responses. These results provide the rationale for phase II studies in CLLs, lymphomas, and CD40-expressing solid tumors.

Amino terminal peptides of the ring infected erythrocyte surface antigen of Plasmodium falciparum bind specifically to erythrocytes.

The Ring-Infected Erythrocyte Surface Antigen (Pf155/RESA) sequence was chemically synthesized in fifty four 20-mer sequential peptides, covering the entire protein, each of which was tested in erythrocyte binding assays. Peptides 6671 and 6673, corresponding to residues 141-160 and 181-200, respectively, presented a high specific binding activity to erythrocytes with affinity constants of 190 nM and 105 nM respectively. Their binding was sensitive to previous enzymatic treatment of erythrocytes. A region of peptide 6673 has been identified, very recently, as a B-cell epitope, target of neutralizing antibodies (Siddique AB, Iqbal J, Ahlborg N, Wâhlin FB, Perlmann P, Berzins K. Antibodies to nonrepeat sequences of antigen Pf155/RESA of Plasmodium falciparum inhibit parasite growth in vitro. Parasitol Res 1998;84:485-91). The critical residues for erythrocyte binding for peptide 6671 (MTDVNRYRYSNNYEAIPHIS) and for peptide 6673 (LGRSGGDIIKKMQTLWDEIM) were recognized. Based on these data, the presence of five functional regions of RESA is postulated.