High-level expression of Plasmodium vivax apical membrane antigen 1 (AMA-1) in Pichia pastoris: strong immunogenicity in Macaca mulatta immunized with P. vivax AMA-1 and adjuvant SBAS2

Abstract

The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria. Correct conformation of the protein appears to be essential for the induction of parasite-inhibitory responses, and these responses appear to be primarily antibody mediated. Here we describe for the first time high-level secreted expression (over 50 mg/liter) of the Plasmodium vivax AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast Pichia pastoris. To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66Deltaglyc) lacking N-glycosylation sites was also developed. Expression of the PV66Deltaglyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66Deltaglyc43-487 was reactive with conformation-dependent monoclonal antibodies. With the SBAS2 adjuvant, Pichia-expressed PV66Deltaglyc43-487 was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000. This group of monkeys had a weak trend showing lower cumulative parasite loads following a Plasmodium cynomolgi infection than in the control group.

Figures

FIG. 1

SDS-PAGE analysis of PV66/AMA-1 expression in P. pastoris. (A) N-glycosidase F digestion of secreted recombinant PV66/AMA-143–487. PV66/AMA-143–487, present in the culture supernatant after methanol induction for 72 h, was digested with N-glycosidase F. Samples representing 10 μl of culture supernatant were analyzed. Lanes: 1, culture supernatant; 2, mock N-glycosidase F digestion; 3, N-glycosidase F-digested material. (B) Expression of PV66Δglyc43–487. Ten microliters of culture supernatant, at 48 h postinduction, was analyzed. Lanes: 1, PV66Δglyc43–487; 2, wild-type PV66/AMA-143–487. (C) Purified mid-scale-produced PV66Δglyc43–487. PV66Δglyc43–487 was purified by ion-exchange and gel filtration chromatographies from a mid-scale culture and chemostat fermentor supernatant. Ten micrograms of purified material was loaded on a nonreducing SDS gel. All gels were stained with Coomassie brilliant blue.

FIG. 2

Distribution of staining on immunofluorescence analysis of culture supernatants by MAbs 5G2 (A) and 3A9 (B) and of serum from rabbit 2 (diluted 1:1000) (C) and rhesus monkey VH2 (diluted 1:500) (D). Merozoites of P. vivax ONG strain parasites developing within schizont-infected erythrocytes (A, C, and D) and as free merozoites following release from schizonts (B) show the typical apically restricted pattern of staining consistent with AMA-1 localization in rhoptries (24). Identical patterns of distribution were seen for all serum samples from rhesus monkeys immunized with PV66Δglyc43–487.

FIG. 3

Results of assays to evaluate in vitro inhibitory effects of IgG isolated after immunization of rabbit 2 (PV66Δglyc43–487) and rabbit 3 (control HSA preparation). Ring-stage parasitemias are shown at 25 h after initiation of invasion cultures in two separate experiments (Exp 1 and Exp 2) in which no extra IgG was present (Ctrl) and in which IgG at 2.0 mg/ml from rabbit 2 (Rb2) and rabbit 3 (Rb3) was present. The standard deviations for triplicate samples are shown.

FIG. 4

Specific IgG levels in immunized rhesus macaques. Serum samples obtained over a 26-week period were tested at a 1:5,000 dilution in a PV66Δglyc43–487 IgG ELISA. Time points for immunization are indicated by solid arrows, and the day of infection with the P. cynomolgi M strain is indicated by the outline arrow. OD, optical density.

FIG. 5

Parasitemias of individual monkeys following P. cynomolgi infection in groups immunized with PV66/AMA-1 (solid lines) or an equivalent fraction from an HSA expression clone (dashed lines). Also shown are the cumulative (Cum) parasitemias for the entire group of PV66/AMA-1-immunized animals and for HSA-immunized animals.