Induction of strain-transcending immunity against Plasmodium chabaudi adami malaria with a multiepitope DNA vaccine

Abstract

A major goal of current malaria vaccine programs is to develop multivalent vaccines that will protect humans against the many heterologous malaria strains that circulate in endemic areas. We describe a multiepitope DNA vaccine, derived from a genomic Plasmodium chabaudi adami DS DNA expression library of 30,000 plasmids, which induces strain-transcending immunity in mice against challenge with P. c. adami DK. Segregation of this library and DNA sequence analysis identified vaccine subpools encoding open reading frames (ORFs)/peptides of >9 amino acids [aa] (the V9+ pool, 303 plasmids) and >50 aa (V50+ pool, 56 plasmids), respectively. The V9+ and V50+ plasmid vaccine subpools significantly cross-protected mice against heterologous P. c. adami DK challenge, and protection correlated with the induction of both specific gamma interferon production by splenic cells and opsonizing antibodies. Bioinformatic analysis showed that 22 of the V50+ ORFs were polypeptides conserved among three or more Plasmodium spp., 13 of which are predicted hypothetical proteins. Twenty-nine of these ORFs are orthologues of predicted Plasmodium falciparum sequences known to be expressed in the blood stage, suggesting that this vaccine pool encodes multiple blood-stage antigens. The results have implications for malaria vaccine design by providing proof-of-principle that significant strain-transcending immunity can be induced using multiepitope blood-stage DNA vaccines and suggest that both cellular responses and opsonizing antibodies are necessary for optimal protection against P. c. adami.

FIG. 1.

Segregation of the P. c. adami DS VR1020 genomic expression library and evaluation of vaccine efficacy. The original library construction and evaluation of the ability of the library to induce survival after homologous challenge with virulent P. c. adami DS are described down to the level of the 3KA subpools (46, 58). Subpool 3KA-4 induced the highest protection (63% survival). Following DNA sequencing of the 733 plasmids in the 3KA-4 subpool, the library was subdivided based on the size of the predicted ORF in-frame with the TPA leader sequence. The V9+ pool encodes ORFs of >9 aa: the V50+ pool encodes ORFs of >50 aa. The V9+ and V50+ pools were evaluated for efficacy against heterologous P. c. adami DK challenge, assessed as reduction in cumulative parasitemia.

FIG. 2.

Kinetics of infection and cumulative parasitemia of mice immunized with the VR1020/30K pool or the control VR1020 empty vector. Panels A-B and C-D represent the results of two independent experiments. Mice (n = 6) were vaccinated by the i.e.d. route (A-D) or the i.m. route (E, F). Two weeks after the third DNA dose, mice were challenged by intraperitoneal infection with 5 × 10⁴ P. c. adami DK IRBC. Daily parasitemia is shown as % parasitemia (mean ± standard error of the mean [SEM]). Cumulative parasitemia values represent the sum of daily parasitemia throughout the period of patent infection. Groups were compared using a Student t test: *, P < 0.001; **, P < 0.01.

FIG. 3.

Kinetics of infection and cumulative parasitemia of mice immunized with the VR1020/V9+ pool or the control VR1020/OOF pool by the i.e.d. route. Panels A-B and C-D represent the results of two independent experiments. Two weeks after the third DNA dose, the mice (n = 6) were challenged by intraperitoneal infection with 5 × 10⁴ P. c. adami DK IRBC. Daily parasitemia is shown as % parasitemia (mean ± standard error of the mean [SEM]). Cumulative parasitemia represents the sum of daily parasitemia values throughout the period of patent infection. Groups were compared using a Student t test: *, P < 0.001; **, P < 0.01.

FIG. 4.

Kinetics of infection and cumulative parasitemia of mice immunized with the VR1020/V50+ pool or the control VR1020 empty vector by the i.e.d. route. Panels A-B and C-D represent the results of two independent experiments. Two weeks after the third DNA dose, the mice (n = 6) were challenged by intraperitoneal infection with 5 × 10⁴ P. c. adami DK IRBC. Daily parasitemia is shown as % parasitemia (mean ± standard error of the mean [SEM]). Cumulative parasitemia represents the sum of daily parasitemia values throughout the period of patent infection. Groups were compared using a Student t test: *, P < 0.05.

FIG. 5.

Sequence homology and blood-stage expression levels of the 38 ORFs orthologous to Plasmodium sequences in the V50+ vaccine. A: homology with other Plasmodium species; B: the annotation of these sequences; C: stacked bar chart showing the blood-stage expression patterns (in P. falciparum) of 31 sequences demonstrating significant orthology with P. falciparum defined by the maximal hour of expression (see reference 5) and by the overall level of blood-stage expression, using 150 units as the cutoff value to differentiate between high and low expression (; Le Roch and Winzeler, personal communication). No sequence showed maximal expression in the merozoite stage. Twenty-eight sequences showed direct orthology to P. falciparum by BLASTP analysis (no data were available for gene X95276). Three additional sequences were orthologous to a P. yoelii sequence that was defined as orthologous to P. falciparum in PlasmoDB (see Table 1).

FIG. 6.

Phagocytosis of P. c. adami IRBC preincubated with sera from mice immunized with the VR1020/30K, VR1020/V50+, or VR1020/V9+ vaccines and from mice that had received the control VR1020 empty vector. A. Uptake of P. c. adami DS IRBC. B. Uptake of P. c. adami DK IRBC. C. In a separate experiment, sera from mice vaccinated with the V9+ plasmids were assessed using P. c. adami DS and DK IRBC. DS immune and DK immune: sera from immune mice that had resolved a primary infection with P. c. adami DS and DK parasites were included as positive controls. Each analysis used sera from four individual mice. In each panel, the opsonization observed with the control VR1020 serum is compared individually with each vaccine serum (30K, V50+, or V9+) using a nonparametric Mann-Whitney t test. *, P < 0.03.

FIG. 7.

IFN-γ secretion by splenocytes from individual BALB/c mice vaccinated i.e.d. with the gene gun. Mice were immunized with the VR1020/V9+ and VR1020/V50+ vaccines. Control values (CTRL) represent splenocyte cultures from mice that received the VR1020 empty vector. Splenocytes were kept either unstimulated (UNST.) or stimulated with 2 ×10⁶ IRBC (P. c. adami DK and/or DS) for 17 h. The supernatants from the splenocyte cultures were harvested, and IFN-γ was measured by ELISA. The responses in splenic cells from mice given empty VR1020 plasmid DNA stimulated with IRBC (CTRL + IRBC) were compared with the responses with splenic cells from mice given vaccine plasmid DNA also stimulated with IRBC (V9⁺ or V50⁺ IRBC), using a nonparametric Mann-Whitney t test. Panel A: *, P = 0.0159. Panel B: *, P = 0.0079. Panel C: *, P = 0.0002. Panel D: *, P = 0.0286).