Invasion-Inhibitory Antibodies Elicited by Immunization with Plasmodium vivax Apical Membrane Antigen-1 Expressed in Pichia pastoris Yeast

Abstract

h In a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparum apical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against Plasmodium vivax malaria, we expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous primeboost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussis monophosphoryl lipid A (MPLA), flagellin FliC from Salmonella enterica serovar Typhimurium, saponin Quil A, or incomplete Freund's adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under "conditions of good laboratory practice" provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasioninhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastoris is a promising antigen for use in future preclinical and clinical studies.

Main B‐cell epitopes of PvAMA‐1 and PvMSP‐9 are targeted by naturally acquired antibodies and epitope‐specific memory cells in acute and convalescent phases of vivax malaria

“…High levels of naturally acquired antibodies to PvAMA‐1 are associated with improved control of parasitaemia in P vivax infection . Moreover, murine anti‐PvAMA‐1 antibodies induced by immunization can partially block erythrocyte invasion by P vivax merozoites in vitro . These findings are consistent with the demonstration of a key role of AMA‐1 binding to rhoptry neck protein 2 (RON2) in red blood cell invasion by P falciparum .…”

Development of vaccines for Plasmodium vivax malaria

“…The 'Protein/Ad' group (protein formulated with Montanide ISA720 followed by Ad5PvAMA1) elicited long-lasting PvAMA1-specific antibody responses, as well as memory T cell responses [68]. PvAMA-1 has also been expressed as a secreted protein in Pichia pastoris [69]. Formulations with saponin Quil A and incomplete Freund's adjuvant elicited higher antibody titers when compared to monophoshoryl lipid A (MPLA) or Alum plus MPLA.…”

“…Formulations with saponin Quil A and incomplete Freund's adjuvant elicited higher antibody titers when compared to monophoshoryl lipid A (MPLA) or Alum plus MPLA. Importantly, PvAMA-1 elicited invasion inhibitory antibodies against diverse P. vivax strains [69].…”

Plasmodium vivax Antigen Discovery Based on Alpha-Helical Coiled Coil Protein Motif

“…Most P. vivax antigens considered to have vaccine potential have been tested in in vitro studies as well as in preliminary preclinical studies in mice and primates [9][10][11][12][13]. Only a few of these antigens further selected by classical immuno-serological methods have undergone phase I clinical trials [14][15][16].…”