Claudia Reyes

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

Peptides from the Plasmodium falciparum STEVOR putative protein bind with high affinity to normal human red blood cells

Synthetic 20-mer long non-overlapped peptides, from STEVOR protein, were tested in RBC binding assays for identifying STEVOR protein regions having high RBC binding activity and evaluating whether these regions inhibit Plasmodium falciparum in vitro invasion. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay with high activity binding peptides (HABPs). HABP binding assays using RBCs previously treated with enzymes were carried out to study the nature of the receptor. The molecular weight of RBC surface proteins interacting with HABPs was determined by cross-linking assays and SDS-PAGE analysis. RBC binding assays revealed that peptides 30561 (41MKSRRLAEIQLPKCPHYNND60), 30562 (61PELKKIIDKLNEERIKKYIE80) and 30567 (161ASCCKVHDNYLDNLKKGCFG180) bound saturably and with high binding activity, presenting nanomolar affinity constants. HABP binding activity to RBCs previously treated with neuraminidase and trypsin decreased, suggesting that these peptides bound to RBC surface proteins and that such binding could be sialic acid dependent. Cross-linking and SDS-PAGE assays showed that the three HABPs specifically bound to 30 and 40 kDa molecular weight RBC membrane proteins. Peptides 30561, 30562 and 30567 inhibited P. falciparum in vitro invasion of red blood cells in a concentration-dependent way. Goat sera having STEVOR protein polymeric peptides antibodies inhibit parasite in vitro invasion depending on concentration. Three peptides localized in STEVOR N-terminal and central regions had high, saturable, binding activity to 30 and 40 kDa RBC membrane proteins. These peptides inhibited the parasites in vitro invasion, suggesting that STEVOR protein regions are involved in P. falciparum invasion processes during intra-erythrocyte stage.

The Mycobacterium tuberculosis membrane protein Rv2560 − biochemical and functional studies

The characterization of membrane proteins having no identified function in Mycobacterium tuberculosis is important for a better understanding of the biology of this pathogen. In this work, the biological activity of the Rv2560 protein was characterized and evaluated. Primers used in PCR and RT‐PCR assays revealed that the gene encoding protein Rv2560 is present in M. tuberculosis complex strains, but transcribed in only some of them. Sera obtained from rabbits inoculated with polymer peptides from this protein recognized a 33 kDa band in the M. tuberculosis lysate and a membrane fraction corresponding to the predicted molecular mass (33.1 kDa) of this protein. Immunoelectron microscopy analysis found this protein on the mycobacterial membrane. Sixteen peptides covering its entire length were chemically synthesized and tested for their ability to bind to A549 and U937 cells. Peptide 11024 (121VVALSDRATTAYTNTSGVSS140) showed high specific binding to both cell types (dissociation constants of 380 and 800 nm, respectively, and positive receptor–ligand interaction cooperativity), whereas peptide 11033 (284LIGIPVAALIHVYTYRKLSGG304) displayed high binding activity to A549 cells only. Cross‐linking assays showed the specific binding of peptide 11024 to a 54 kDa membrane protein on U937. Invasion inhibition assays, in the presence of shared high‐activity binding peptide identified for U937 and A549 cells, presented maximum inhibition percentages of 50.53% and 58.27%, respectively. Our work highlights the relevance of the Rv2560 protein in the M. tuberculosis invasion process of monocytes and epithelial cells, and represents a fundamental step in the rational selection of new antigens to be included as components in a multiepitope, subunit‐based, chemically synthesized, antituberculosis vaccine.

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite–erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive‐stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti‐RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high‐activity binding peptides (HABPs) were characterized by having α‐helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid‐dependent and/or glycoprotein‐dependent nature, as evidenced in enzyme‐treated erythrocyte binding assays and further corroborated by cross‐linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 μM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.

Identification and evaluation of universal epitopes in Plasmodium vivax Duffy binding protein

Selected PvDBP-derived synthetic peptides were tested in competition assays with HLA molecules in order to identify and evaluate their binding to a wide range of MHC class II molecules. Binding was evaluated as the peptides ability to displace the biotinylated control peptide (HA(306-318)) and was detected by a conventional ELISA. Thus, one epitope for the HLA-DR1 molecule, two epitopes for the HLA-DR4 molecule, six epitopes for the HLA-DR7 molecule and three epitopes for the HLA-DR11 molecule displaying a high binding percentage (above 50%) were experimentally obtained. The in vitro results were compared with the epitope prediction results. Two peptides behaved as universal epitopes since they bound to a larger number of HLA-DR molecules. Given that these peptides are located in the conserved PvDBP region II, they could be considered good candidates to be included in the design of a synthetic vaccine against Plasmodium vivax malaria.