Vaccine potentials of an intrinsically unstructured fragment derived from the blood stage-associated Plasmodium falciparum protein PFF0165c

Abstract

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

FIG. 1.

Protein sequence and schematic representation of the PFF0165c protein. The protein sequence is 1,103 amino acids in length. Amino acids in blue form the unstructured region that was synthesized, shown by the blue block in the schematic representation. Residue 297, where a n E/G mutation occurs, is underlined. Amino acids in red form the α-helical coiled-coil region, shown in red in the schematic representation.

FIG. 2.

(A) Analytical HPLC of P27A. mAU, milli-absorbance unit, #, number. (B) Mass spectrum of P27A. Peaks at 12,361.10 and 6,182.07 represent the cytochrome c mass plus 1 or 2 hydrogen ions, respectively, taken as the standard. The peak at 11,929.66 represents the mass of P27A.

FIG. 3.

The CD spectrum of P27A was assessed over a 190- to 250-nm-wavelength range at 20°C, pH 7.3, in a 150-μg/ml solution, using a Jasco J-810 spectrometer. There is a single dip at around 200 nm, which is indicative of random coil conformation.

FIG. 4.

(A) Prevalence of recognition of P27A by sera of adults living in regions of Burkina Faso (BF, n = 37), Tanzania (TZ, n = 42), and Papua New Guinea (PNG, n = 56) where malaria is endemic. A serum sample was considered positive when the OD is greater than the average OD of naïve sera plus 3 SD. (B) Average OD values of donor samples with significant anti-P27A antibodies (i.e., serum samples with OD values greater than the average OD of negative controls plus 3 SD). BF, Burkina Faso (n = 28/37); TZ, Tanzania (n = 32/42); PNG, Papua New Guinea (n = 53/56). Bars represent mean OD values, while whikers indicate ±SD.

FIG. 5.

Immunofluorescence staining of malaria-infected erythrocytes. (A) Trophozoite-stage parasites were stained with mouse antibodies against P27A (red). Mouse antibodies recognize the parasite and structures of the parasite in the iRBC cytosol. (B, C) Polyclonal rabbit sera (B) and human affinity-purified sera (C) specific to P27A recognize identical structures, suggesting that all the sera recognize the same target (green), the parasite-derived PFF0165c protein. Nuclei are stained with DAPI (in blue). Right panels, transmission light microscopy images of the iRBC (differential interference contrast). α, anti.

FIG. 6.

Western blot analysis of 3D7 parasite lysate with mouse sera specific for P27A and affinity-purified rabbit sera specific for P27A. Late stages (trophozoites/schizonts) were enriched using a magnetic cell sorter. The enriched infected erythrocytes were fractionated into pellet fraction (lanes 1 and 3) and supernatant fraction (lanes 2 and 4) by saponin lysis in a 200-μl volume. P27A-specific rabbit sera (lanes 1 and 2) and P27A-specific mouse sera (lanes 3 and 4) recognize the protein at 160 kDa in the pellet fraction.

FIG. 7.

Immunofluorescence staining of malaria-infected erythrocytes. (A) Schizont-stage parasites were stained with mouse antibodies against P27A (red). Polyclonal mouse antibodies recognize structures in very close proximity to or at the RBC membrane. (B, C) Affinity-purified rabbit sera (B) and human affinity-purified sera (C) specific to P27A recognize identical structures (green). Nuclei are stained with DAPI (blue). Right panels, transmission light microscopy images of the iRBC (differential interference contrast). α, anti.

FIG. 8.

Results of ADCI assays with murine antibodies. Pooled sera of C3H (H-2^k) mice immunized with P27A formulated with Alhydrogel and EM005. The percent SGI is calculated with the formula 100 × [1 − (% parasitemia with MN and test antibodies/% parasitemia with test antibodies)/(% parasitemia with MN and IgGs from healthy donors/parasitemia with IgGs from healthy donors)].