Comparison of analytical methods for the evaluation of antibody responses against epitopes of polymorphic protein antigens.
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.
Swiss Tropical Institute, Socinstrasse 57, CH 4002, Basel, Switzerland.
A
A
Helg
M S
MS
Mueller
A
A
Joss
F
F
Pöltl-Frank
F
F
Stuart
J A
JA
Robinson
G
G
Pluschke
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000913:Antibodies, Protozoan / Q000276:immunology*; D000915:Antibody Affinity; D000940:Antigenic Variation; D001483:Base Sequence; D015153:Blotting, Western; D002151:Calorimetry; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D020066:Merozoite Surface Protein 1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D010963:Plasmodium falciparum / Q000276:immunology*; D011110:Polymorphism, Genetic; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000939:Epitopes; C474776:MSP-1(43-53); D020066:Merozoite Surface Protein 1; D010446:Peptide Fragments