ABSTRACT
Surface exposed protein antigens of the malaria parasite Plasmodium falciparum frequently harbor multiple dimorphic amino acid positions. These are associated with parasite immune evasion and represent a major obstacle for subunit vaccine design. Here, we have analyzed the flexibility of the humoral immune response against a semiconserved sequence (YX(44)LFX(47)KEKMX(52)L) of the key malaria blood stage vaccine candidate merozoite surface protein-1 (MSP-1). Monoclonal antibodies (mAbs) raised against one of the six described natural sequence variants of MSP-1(43-53) were analyzed for cross-reactivity with the other allelic forms, which differ in one to three positions from the immunizing sequence. Enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) spectroscopy demonstrated marked differences in mAb binding avidity to the variant sequences and isothermal titration calorimetry (ITC) provided evidence for a very low affinity of some of the interactions. In immunofluorescence analysis (IFA) and Western blotting analysis, the mAbs nevertheless stained all analyzed parasite clones expressing MSP-1(43-53) variant sequences. When used for the evaluation of humoral immune responses in clinical malaria vaccine trials, these two commonly used methods may thus not be suitable to distinguish biologically functional high affinity antibody responses from irrelevant low-affinity cross-reactivities.
Subject(s)
Antibodies, Protozoan/immunology , Antigenic Variation , Epitopes/immunology , Merozoite Surface Protein 1/immunology , Peptide Fragments/immunology , Plasmodium falciparum/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibody Affinity , Base Sequence , Blotting, Western , Calorimetry , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Epitopes/genetics , Fluorescent Antibody Technique , Merozoite Surface Protein 1/chemistry , Merozoite Surface Protein 1/genetics , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Polymorphism, Genetic , Sequence Alignment , Surface Plasmon ResonanceABSTRACT
Immunopotentiating reconstituted influenza virosomes (IRIV) were used as a delivery system for the synthetic peptide-based malaria vaccine SPf66. The reduced SPf66 peptide molecules containing terminal cysteine residues were covalently attached to phosphatidylethanolamine with the heterobifunctional crosslinker gamma-maleimidobutyric acid N-hydroxysuccinimide ester. The SPf66-phosphatidylethanolamine was incorporated into IRIV and BALB/c mice were immunized twice by intramuscular injection with peptide-loaded virosomes. Titres of elicited anti-SPf66 IgG were determined by ELISA. These titres were significantly higher and the required doses of antigen were lower, when mice had been preimmunized with a commercial whole virus influenza vaccine. After preimmunization with the influenza vaccine, SPf66-IRIV elicited far more consistently anti-SPf66 antibody responses than SPf(66)n adsorbed to alum. MoAb produced by four B cell hybridoma clones derived from a SPf66-IRIV-immunized mouse cross-reacted with Plasmodium falciparum blood stage parasites in immunofluorescence assays. All four MoAbs were specific for the merozoite surface protein-1 (MSP-1)-derived 83.1 portion of SPf66. Sequencing of their functionally rearranged kappa light chain variable region genes demonstrated that the four hybridomas were generated from clonally related splenic B cells. Biomolecular interaction analyses (BIA) together with these sequencing data provided evidence for the selection of somatically mutated affinity-matured B cells upon repeated immunization with SPf66-IRIV. The results indicate that IRIV are a suitable delivery system for synthetic peptide vaccines and thus have a great potential for the design of molecularly defined combined vaccines targeted against multiple antigens and development stages of one parasite, as well as against multiple pathogens.
Subject(s)
Drug Delivery Systems , Hemagglutinin Glycoproteins, Influenza Virus , Influenza A virus , Malaria Vaccines/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Recombinant Proteins , Vaccines, Synthetic/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Protozoan/immunology , Base Sequence , Cross Reactions , DNA, Complementary , Liposomes , Malaria Vaccines/administration & dosage , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptides/immunology , Tumor Cells, Cultured , Vaccines, Synthetic/administration & dosageABSTRACT
Lithography can be performed with beams of neutral atoms in metastable excited states to pattern self-assembled monolayers (SAMs) of alkanethiolates on gold. An estimated exposure of a SAM of dodecanethiolate (DDT) to 15 to 20 metastable argon atoms per DDT molecule damaged the SAM sufficiently to allow penetration of an aqueous solution of ferricyanide to the surface of the gold. This solution etched the gold and transformed the patterns in the SAMs into structures of gold; these structures had edge resolution of less than 100 nanometers. Regions of SAMs as large as 2 square centimeters were patterned by exposure to a beam of metastable argon atoms. These observations suggest that this system may be useful in new forms of micro- and nanolithography.
