Protective efficacy of baculovirus dual expression system vaccine expressing Plasmodium falciparum circumsporozoite protein

Abstract

We have previously developed a new malaria vaccine delivery system based on the baculovirus dual expression system (BDES). In this system, expression of malaria antigens is driven by a dual promoter consisting of the baculovirus-derived polyhedrin and mammal-derived cytomegalovirus promoters. To test this system for its potential as a vaccine against human malaria parasites, we investigated immune responses against the newly developed BDES-based Plasmodium falciparum circumsporozoite protein vaccines (BDES-PfCSP) in mice and Rhesus monkeys. Immunization of mice with BDES-PfCSP induced Th1/Th2-mixed type immune responses with high PfCSP-specific antibody (Ab) titers, and provided significant protection against challenge from the bites of mosquitoes infected with a transgenic P. berghei line expressing PfCSP. Next, we evaluated the immunogenicity of the BDES-PfCSP vaccine in a rhesus monkey model. Immunization of BDES-PfCSP elicited high levels of anti-PfCSP Ab responses in individual monkeys. Moreover, the sera from the immunized monkeys remarkably blocked sporozoite invasion of HepG2 cells. Taken together with two animal models, our results indicate that this novel vaccine platform (BDES) has potential clinical application as a vaccine against malaria.

Figure 1. Schematic representation and expression analysis of BDES-PfCSP.

(A) Expression of the PfCSP gene cassette was driven by the dual promoter, which consisted of the CMV immediate early enhancer-promoter (pCMV), or the CAG promoter (pCAG) as well as the polyhedrin promoter (pPolh). Both full-length pfcsp and the N-terminal truncated pfcsp were fused to the N terminus of the AcNPV major envelope protein gp64 gene (gp64_21–512). All BDES constructs also had the endogenous gp64 gene in their genomes. SP, the gp64 signal sequence; FLAG, FLAG epitope tag; HIS, His epitope tag. (B) Western blot analysis of recombinant AcNPV particles. AcNPV-WT (lane 1), CMV-full (lane 2), CAG-full (lane 3) and CAG-205 (lane 4) particles were lysed, loaded onto an 8% gel and immunoblotted with the anti-VP39 Ab. The membrane was stripped and then reblotted with the anti-PfCSP mAb 2A10. (C) COS-7 cells and HepG2 cells were transduced with BDES-PfCSP at multiplicities of infection (MOI) of 500 and 100, respectively. After 48 hours incubation, such cells were stained with the 2A10 mAb conjugated to Alexa Fluor 594 (red). Nucleic acids in the cells were visualized by SYTO-13 (green). Bars, 50 µm.

Figure 2. Kinetics of anti-PfCSP humoral immune responses.

Groups of mice were injected with PBS or immunized intramuscularly with CMV-full, CAG-full or CAG-205 (n = 10). (A) Sera were collected from the immunized mice 3 weeks after the first and second immunizations and 2 weeks after the last immunization. Bars and error bars indicate the mean and SD of the values, respectively. Significant differences in each immunization group were evaluated using Student's t-tests. * p<0.05; ** p<0.01. (B) Individual sera after the last immunization were tested for total IgG, IgG1, IgG2a, IgG2b and IgG3 specific to PfCSP by ELISA. (C) Ab titers against the PfCSP NANP repeats in the sera after the last immunization. Statistically significant differences for each Ab titer among the three immunized groups were evaluated using the Kruskal-Wallis non-parametric tests (p<0.001). Data are representative of 3 independent experiments.

Figure 3. Intracellular cytokine staining of splenocytes from BDES-immunized mice.

Groups of mice were injected with PBS or immunized intramuscularly (i.m.) with CMV-full, AcNPV-WT or Adeno-COE/1–373 (A) (B) (n = 6, experiment 5 in Table S1), or CAG-full and CAG-205 (C) (n = 3, experiment 6 in Table S1). Two weeks after the last immunization, splenocytes from each group were stimulated with either NYDNAGTNL peptides or AcNPV-WT at a MOI of 1 in duplicate wells of the cultures. After 24 hours, the incubated cells were stained with Abs against CD3 and CD8, and then stained intracellularly with anti-IFN-γ or its isotype control. (A) Representative dot plots are shown. (B) (C) Bars and error bars show the mean values and standard errors for the percentage of IFN-γ positive cells, respectively. Significant differences between non-stimulated media controls and stimulated wells were evaluated using Student's t-tests. * p<0.05.

Figure 4. PfCSP expression in transgenic P. berghei.

(A) Confocal laser scanning microscopy PfCSP expression in transgenic P. berghei. Salivary glands from the infected mosquitoes as well as transgenic P. berghei sporozoites were probed with the 2A10 mAb conjugated to Alexa Flour 594 (red). Parasite nuclei and mosquito salivary glands were visualized by DAPI (blue). Bar  = 50 µm. (B) Salivary glands from uninfected mosquitoes or mosquitoes infected with the transgenic parasites were lysed and loaded onto 10% gels, and then immunoblotted with the 2A10 mAb and anti-AAPP mAb (28B8).

Figure 5. Parasitemia in the immunized mice after parasite challenge.

Giemsa-stained thin smears of tail blood were prepared at days 5, 7 and 9 after challenge. The percentage parasitemias and parasitemia reduction rates in the individual mice were compared with those from the PBS-control mice using the following formula: % reduction  =  [1–(% parasitemia of immune group)/(% parasitemia of PBS group]). Data from 3 independent experiments were pooled (n = 40), except for the CMV-full group (n = 15).