In Vitro Studies with RecombinantPlasmodium falciparumApical Membrane Antigen 1 (AMA1): Production and Activity of an AMA1 Vaccine and Generation of a Multiallelic Response

Abstract

Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P. falciparum have been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P. falciparum strains is unknown. We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P. falciparum clones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage.

Addition of CpG ODN to recombinant Pseudomonas aeruginosa ExoProtein A conjugates of AMA1 and Pfs25 greatly increases the number of responders

“…Briefly, malarial antigens (AMA1-FVO or Pfs25 NF54) [11,12] were thiolated by DL-Nacetylhomocysteine thiolactone (Sigma Aldrich Inc., St Louis, MO) and carrier protein (rEPA) was modified with maleimide groups using Sulfo-EMCS (Pierce Inc., Rockford, IL). Conjugates were formed by incubation of the thiolated malarial antigens with the maleimide derivatized rEPA.…”

“…GIA was performed on total IgG purified from pooled immune sera [11,15]. The mouse sera from each immunization group were pooled and the total IgG purified.…”

Development and characterization of a standardized ELISA including a reference serum on each plate to detect antibodies induced by experimental malaria vaccines

“…AMA1 was produced and purified as described previously [15]. In brief, the synthetic AMA1 gene sequence was subcloned into the pPIC9K vector and transformed into P. pastoris.…”

Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

“…Since its initial discovery Ͼ15 years ago, AMA1 has received considerable attention as a malaria vaccine candidate (Deans et al, 1988;Collins et al, 1994;Anders et al, 1998;Kennedy et al, 2002;Stowers et al, 2002). AMA1 proteins are type I transmembrane proteins, with a short C-terminal cytoplasmic tail and a large N-terminal extracellular domain (ectodomain) containing 12-16 conserved cysteine residues (Waters et al, 1990;Hodder et al, 1996;Donahue et al, 2000;Hehl et al, 2000;Gaffar et al, 2004).…”