Enhanced immunity to Plasmodium falciparum circumsporozoite protein (PfCSP) by using Salmonella enterica serovar Typhi expressing PfCSP and a PfCSP-encoding DNA vaccine in a heterologous prime-boost strategy

Abstract

Two Salmonella enterica serovar Typhi strains that express and export a truncated version of Plasmodium falciparum circumsporozoite surface protein (tCSP) fused to Salmonella serovar Typhi cytolysin A (ClyA) were constructed as a first step in the development of a preerythrocytic malaria vaccine. Synthetic codon-optimized genes (t-csp1 and t-csp2), containing immunodominant B- and T-cell epitopes present in native P. falciparum circumsporozoite surface protein (PfCSP), were fused in frame to the carboxyl terminus of the ClyA gene (clyA::t-csp) in genetically stabilized expression plasmids. Expression and export of ClyA-tCSP1 and ClyA-tCSP2 by Salmonella serovar Typhi vaccine strain CVD 908-htrA were demonstrated by immunoblotting of whole-cell lysates and culture supernatants. The immunogenicity of these constructs was evaluated using a "heterologous prime-boost" approach consisting of mucosal priming with Salmonella serovar Typhi expressing ClyA-tCSP1 and ClyA-tCSP2, followed by parenteral boosting with PfCSP DNA vaccines pVR2510 and pVR2571. Mice primed intranasally on days 0 and 28 with CVD 908-htrA(pSEC10tcsp2) and boosted intradermally on day 56 with PfCSP DNA vaccine pVR2571 induced high titers of serum NANP immunoglobulin G (IgG) (predominantly IgG2a); no serological responses to DNA vaccination were observed in the absence of Salmonella serovar Typhi-PfCSP priming. Mice primed with Salmonella serovar Typhi expressing tCSP2 and boosted with PfCSP DNA also developed high frequencies of gamma interferon-secreting cells, which surpassed those produced by PfCSP DNA in the absence of priming. A prime-boost regimen consisting of mucosal delivery of PfCSP exported from a Salmonella-based live-vector vaccine followed by a parenteral PfCSP DNA boosting is a promising strategy for the development of a live-vector-based malaria vaccine.

FIG. 1.

(A) Schematic diagram showing B- and T-cell epitopes in tCPS2. aa, amino acids. (B) Genetic map of pSEC10tcsp1 or pSEC10tcsp2. Alleles 1 and 2 are not specified as the two expression plasmids were constructed in similar ways. P_ompC is a modified osmotically controlled ompC promoter from E. coli; clyA encodes cytolysin A from Salmonella serovar Typhi; clyA::tcsp encodes prokaryotic codon-optimized truncated CSP fused to the carboxyl terminus of ClyA; tetA encodes a promoterless tetracycline efflux protein from pBR322; aph encodes the aminoglycoside 3′-phosphotransferase conferring resistance to kanamycin; ori101 is the origin of replication from pSC101 providing an expected expression plasmid copy number of ∼5 copies per chromosomal equivalent; repA encodes the replication protein essential for replication of ori101; par is the passive partitioning locus from pSC101; T1 is the transcriptional terminator from the rrnB rRNA operon of E. coli; hok-sok is the postsegregational killing locus from the multiple-antibiotic-resistance R-plasmid pR1; parA encodes the active partitioning system from pR1. (C) Western immunoblot analysis of whole bacterial lysates of CVD 908-htrA (lane 2), CVD 908-htrA(pSEC10) (lane 3), CVD 908-htrA(pSEC10tcsp1) (lane 4), and CVD 908-htrA(pSEC10tcsp2) (lane 5). P. falciparum sporozoite crude lysate (7.5 ng) was included in lane 1 as a positive control. The membrane was probed with MAb 2A10 specific for the NANP repeat region of CSP. M, molecular mass (in kilodaltons). (D) Western immunoblot analysis of filtered culture supernatants from CVD 908-htrA (lane 1), CVD 908-htrA(pSEC10) (lane 2), CVD 908-htrA(pSEC10tcsp1) (lane 3), and CVD 908-htrA(pSEC10tcsp2) (lane 4). The membrane was probed with MAb 2A10. The arrows indicate the ClyA-tCSP fusion protein; although the expected position was 54 kDa, a band at a slightly higher molecular mass was observed both in whole bacterial lysates and in culture supernatants.

FIG. 2.

Immune responses induced by Salmonella serovar Typhi expressing ClyA-tCSP1 followed by PfCSP DNA vaccine pVR2510 in a heterologous prime-boost strategy. (A) NANP-specific IgG titers measured by ELISA. Mice were primed i.n. with two doses (two asterisks) of either CVD 908-htrA or CVD 908-htrA(pSEC10tcsp1) on days 0 and 28 and boosted i.m. on day 62 with 100 μg pVR2510, which encodes the full-length PfCSP. Mice primed and boosted with pVR2510 and mice primed and boosted with PBS served as positive and negative controls, respectively. An additional control group received CVD 908-htrA carrying empty pSEC10 followed by pVR2510; these mice did not elicit NANP-specific IgG (data not shown). The arrows indicate each immunization. The data are titers from individual animals measured on days 0, 28, 56, and 85 postimmunization; the dashed line was plotted upon the GMT. (B) Frequency of PfCSP-specific IFN-γ-secreting cells measured by the ELISPOT assay. Mice were immunized as described above. Spleens were harvested 2 months after the boost, and splenocytes were restimulated in vitro with irradiated MHC-matched P815 cells infected with PfCSP-encoding vaccinia virus (solid bars) or a vaccinia virus control (cross-hatched bars) as described in Materials and Methods. The bars indicate the mean numbers of IFN-γ SFC per 10⁶ cells, and the error bars indicate standard deviations. Statistically significant differences in responses between groups are indicated.

FIG. 3.

Immune responses to PfCSP DNA vaccine pVR2571 obtained using different routes and delivery systems. (A) NANP-specific IgG titers. Mice were immunized on days 0, 28, and 56 (indicated by arrows) with 100 μg of pVR2571, which contained the mammalian codon-optimized csp, i.d using the Biojector 2000 needle-free jet injector or i.m. using a needle and syringe. The solid circles indicate the titers of individual mice; the dashed lines were plotted upon the GMT. (B) Frequency of PfCSP-specific IFN-γ SFC. Mice were immunized with pVR2571 as described above. Spleens were harvested 2 months after the last immunization, and IFN-γ responses were measured by the ELISPOT assay as described in the legend to Fig. 2. The bars indicate the mean numbers of IFN-γ SFC per 10⁶ cells, and the error bars indicate standard deviations. Statistically significant differences are indicated.

FIG. 4.

Dose-response analysis of antibody and IFN-γ responses induced by PfCSP DNA vaccine pVR2571. (A) NANP-specific IgG titers. Mice were immunized with increasing doses of pVR2571 by the i.d. route using the Biojector 2000. Mice immunized with a control plasmid expressing an unrelated antigen (pVR2576) or PBS were included as negative controls; no antigen-specific antibody titers were observed (data not shown). The solid circles indicate the titers of individual mice, and the dashed lines were plotted upon the GMT. Arrows indicate each immunization. (B) Frequency of PfCSP-specific IFN-γ SFC. Mice were immunized with increasing doses of DNA vaccine pVR2571 as indicated above. IFN-γ responses were measured as described in the legend to Fig. 2 at 2 months after the last immunization. The bars indicate the mean numbers of IFN-γ SFC per 10⁶ cells, and the error bars indicate standard deviations. Statistically significant differences are indicated.

FIG. 5.

Immune responses in mice primed with Salmonella serovar Typhi expressing ClyA-tCSP2 and boosted with PfCSP DNA vaccine pVR2571. (A) NANP-specific IgG titers. Mice were primed with one dose (one asterisk) (day 28) or two doses (two asterisks) (days 0 and 28) of CVD 908-htrA, CVD908-htrA(pSEC10tcsp1), or CVD908-htrA(pSEC10tcsp2) and boosted i.d. with 20 μg of pVR2571. Mice that received three doses of PBS served as negative controls. The solid circles indicate the antibody titers in individual mice, and antibody production curves are plotted upon the GMT. Arrows indicate each immunization. (B) Frequency of PfCSP-specific IFN-γ SFC. Mice were immunized as described above. Spleens were harvested 2 months after the boost, and IFN-γ responses were measured by the ELISPOT assay as described in the legend to Fig. 2. The bars indicate the mean numbers of SFC per 10⁶ cells, and the error bars indicate standard deviations. Statistically significant differences between IFN-γ responses in mice primed twice with CVD 908-htrA(pSEC10tcsp2) and IFN-γ responses in mice primed with CVD 908-htrA or PBS are indicated.

FIG. 6.

Binding of PfCSP antibodies elicited by Salmonella serovar Typhi ClyA-tCSP priming and PfCSP DNA boosting to P. falciparum sporozoites. Antibodies recognizing PfCSP in the P. falciparum sporozoite were detected by an immunofluorescence assay using FITC-labeled anti-mouse IgG (upper panels); parasite nuclei were stained with DAPI (superimposed images in lower panels). MAb 2A10 was used as a positive control. The images were obtained with a ×100 objective and a ×40 objective (insets).