Protective humoral immunity elicited by a needle-free malaria vaccine comprised of a chimeric Plasmodium falciparum circumsporozoite protein and a Toll-like receptor 5 agonist, flagellin

Abstract

Immunization with Plasmodium sporozoites can elicit high levels of sterile immunity, and neutralizing antibodies from protected hosts are known to target the repeat region of the circumsporozoite (CS) protein on the parasite surface. CS-based subunit vaccines have been hampered by suboptimal immunogenicity and the requirement for strong adjuvants to elicit effective humoral immunity. Pathogen-associated molecular patterns (PAMPs) that signal through Toll-like receptors (TLRs) can function as potent adjuvants for innate and adaptive immunity. We examined the immunogenicity of recombinant proteins containing a TLR5 agonist, flagellin, and either full-length or selected epitopes of the Plasmodium falciparum CS protein. Mice immunized with either of the flagellin-modified CS constructs, administered intranasally (i.n.) or subcutaneously (s.c.), developed similar levels of malaria-specific IgG1 antibody and interleukin-5 (IL-5)-producing T cells. Importantly, immunization via the i.n. but not the s.c. route elicited sporozoite neutralizing antibodies capable of inhibiting >90% of sporozoite invasion in vitro and in vivo, as measured using a transgenic rodent parasite expressing P. falciparum CS repeats. These findings demonstrate that functional sporozoite neutralizing antibody can be elicited by i.n. immunization with a flagellin-modified P. falciparum CS protein and raise the potential of a scalable, safe, needle-free vaccine for the 40% of the world's population at risk of malaria.

Fig 1

(A) Schematic diagram of P. falciparum CS protein showing locations of the T1 and B cell epitopes within the central repeat region and the universal Th cell epitope, T*, in the C terminus. (B) Schematic diagrams of E. coli-expressed recombinant proteins comprised of full-length flagellin (STF2) combined with P. falciparum T1BT* epitopes as either a single copy [STF2.(T1BT*)_1X] or four copies [STF2.(T1BT*)_4X]. A second type of construct, STF2Δ.CS, was comprised of a truncated flagellin protein (STF2Δ) and nearly full-length P. falciparum CS protein.

Fig 2

TLR5 signaling by flagellin-modified CS constructs. Human TLR5-transfected RAW 264.7 cells (closed symbols) or untransfected cells (open symbols) were stimulated with 10-fold dilutions of (A) STF2.(T1BT*)_1X or a positive-control protein, flagellin-modified ovalbumin (STF2.OVA), or (B) STF2Δ.CS, and levels of TNF-α in culture supernatants were measured by ELISA. (C) Flow cytometry of TLR5 expression in human monocyte-derived DC that were immature, matured with a cytokine cocktail, or stimulated with STF2 flagellin (10 μg/ml). (D) Flow cytometry of TLR5 expression in murine D1 cells, an immature DC line.

Fig 3

DC maturation following stimulation with flagellin-modified CS constructs. (A) Confocal microscopy of monocyte-derived human DC expression of costimulatory molecule CD86 (green) following incubation with unmodified CS protein or STF2Δ.CS. The DC nuclei were stained with Hoechst stain (blue). (B) Percentages of CD86⁺ human DC after 6 or 24 h of stimulation with the flagellin-modified CS construct STF2.(T1BT*)_4X or STFΔ.CS or with full-length or truncated flagellin (STF2 and STF2Δ). Means with SD for replicates are shown. Data are representative of two independent experiments. (C) Percentages of murine D1 cells expressing costimulatory and MHC II molecules following stimulation with 10 μg/ml of flagellin or flagellin-modified CS proteins. Means with SD for replicates are shown. Data are representative of three independent experiments.

Fig 4

DC uptake of flagellin-modified CS constructs. (A) FACS histogram of murine BMDC following 24 h of incubation with flagellin or flagellin-modified CS (10 μg/ml). Intracellular CS was detected by labeling permeabilized cells with biotinylated MAb 2A10 followed by streptavidin-conjugated Qdots. (B) MAb 2A10-positive murine BMDC measured after incubation for various times with flagellin-modified CS constructs or flagellin-only controls (STF2 or STF2Δ). Means with SD for replicates are shown. Data are representative of three independent experiments. (C) Dose-dependent increase in MAb 2A10 staining of human DC following 24 h of incubation with flagellin-modified CS constructs or flagellin-only controls. Means with SD for replicates are shown. Data are representative of three independent experiments.

Fig 5

Kinetics and fine specificities of antibody responses in mice immunized with flagellin-modified CS constructs. (A) Kinetics of IgG response of C57BL/6 mice (3 mice/group) immunized s.c. or i.n. with STF2Δ.CS (50 μg/dose), as measured by ELISA using CS repeat peptide, flagellin (STF2Δ), or STF2Δ.CS as the coating antigen. Results shown are geometric mean titers (GMT) with 95% confidence intervals. (B) IgG GMT in tlr5^−/− mice and WT controls (3 mice/group) immunized s.c. or i.n. with four doses of STF2.(T1BT*)_4X (10 μg/dose). Following four immunizations, all WT controls seroconverted. In contrast, none of the tlr5^−/− mice immunized s.c. had detectable antibodies to CS repeats or flagellin, with one mouse having detectable antibodies to the immunogen. Two of three tlr5^−/− mice immunized i.n. had detectable antibodies to CS repeats, with one mouse having detectable antibodies to flagellin and immunogen. The tlr5^−/− mice that did not seroconvert were assigned a value of 20 for calculation of GMT.

Fig 6

IgG subtypes in mice immunized with flagellin-modified CS. Antibodies specific for CS repeats (A) or flagellin (B) were measured in pooled immune sera (1:80 dilution) from mice immunized i.n. or s.c. with four doses of STF2Δ.CS or flagellin (50 μg/dose). OD, optical density.

Fig 7

Flagellin-modified CS constructs elicit Th2-type cytokine responses. Spleen cells were obtained from mice (A) immunized s.c. or i.n. with four doses of STF2Δ-CS (50 μg/dose) or (B) immunized i.n. with five doses of either STF2.(T1BT*)_4X or STF2Δ.CS (10 μg/dose). Malaria-specific cells were expanded in vitro with T1BT* peptide for 7 days prior to ELISPOT assay. Results are shown as numbers of spot-forming cells (SFC)/10⁶ cells following restimulation with T1BT* peptide, flagellin (STFΔ), or the respective immunogen [STF2Δ-CS or STF2.(T1BT*)_4X]. IL-5 (C) or IL-6 (D) in pooled cell culture supernatants were measured using multiplex CBA. Cytokine concentrations are shown in pg/ml, after subtraction (Δ) of levels in medium-only cultures.

Fig 8

Sporozoite-neutralizing antibody and protection in mice immunized with flagellin-modified CS constructs. (A) TSNA was carried out using a 1:5 dilution of pooled sera (3 mice/group) obtained after the 3rd to 6th dose of STF2Δ-CS, STF2.(T1BT*)_4X, or unmodified T1BT* peptide (10 μg/dose). Results measured by qPCR analysis of hepatoma cell extracts obtained at 48 h postinfection are shown as mean P. berghei 18S rRNA copy numbers. MAb 2A10, specific for P. falciparum CS repeats, and MAb 3D11, specific for P. berghei CS repeats, served as positive and negative controls, respectively. (B) TSNA was carried out using individual sera (1:5 dilution) from 3 mice/group immunized i.n. or s.c. with five doses of STF2Δ.CS (50 μg/dose). 18S rRNA copy numbers in cultures with i.n. immune sera, but not s.c. immune sera, were significantly reduced compared to naive control levels. (C) In vivo protection was measured in the mice used for panel B following challenge by exposure to the bites of 5 to 15 PfPb-infected mosquitoes. Results shown are mean parasite 18S rRNA levels in livers harvested at 40 h postinfection, as measured by qPCR. Consistent with in vitro TSNA, the liver parasite burden was reduced in i.n. immunized but not s.c. immunized mice compared to naive controls following challenge. *, P < 0.05 by the Kruskal-Wallis test; ns, nonsignificant.

Fig 9

Antibody-mediated protection in mice immunized i.n. with flagellin-modified CS protein. Mice (n = 6 mice/group) were immunized i.n. with STF2Δ.CS or STF2 only (50 μg/dose). (A) TSNA was carried out using a 1:5 or 1:10 dilution of individual sera obtained after the fifth dose. Results shown are mean 18S rRNA copy numbers in 48-h extracts of HepG2 cell cultures as measured by qPCR. (B) In vivo protection measured in immunized mice that were nondepleted (3 mice/group; solid bars) or depleted of T cells (3 mice/group; hatched bars) by injection of anti-CD4 and anti-CD8 MAbs (100 μg of each MAb) for 3 days prior to challenge by exposure to the bites of PfPb-infected mosquitoes. Results shown are mean 18S rRNA copy numbers in liver extracts obtained at 40 h postchallenge, as measured by qPCR. *, P < 0.05; **, P < 0.01 by the Kruskal-Wallis test; ns, nonsignificant.