Baculovirus-based nasal drop vaccine confers complete protection against malaria by natural boosting of vaccine-induced antibodies in mice

Abstract

Blood-stage malaria parasites ablate memory B cells generated by vaccination in mice, resulting in diminishing natural boosting of vaccine-induced antibody responses to infection. Here we show the development of a new vaccine comprising a baculovirus-based Plasmodium yoelii 19-kDa carboxyl terminus of merozoite surface protein 1 (PyMSP1(19)) capable of circumventing the tactics of parasites in a murine model. The baculovirus-based vaccine displayed PyMSP1(19) on the surface of the virus envelope in its native three-dimensional structure. Needle-free intranasal immunization of mice with the baculovirus-based vaccine induced strong systemic humoral immune responses with high titers of PyMSP1(19)-specific antibodies. Most importantly, this vaccine conferred complete protection by natural boosting of vaccine-induced PyMSP1(19)-specific antibody responses shortly after challenge. The protective mechanism is a mixed Th1/Th2-type immunity, which is associated with the Toll-like receptor 9 (TLR9)-dependent pathway. The present study offers a novel strategy for the development of malaria blood-stage vaccines capable of naturally boosting vaccine-induced antibody responses to infection.

FIG. 1.

Construction and analysis of recombinant AcNPV expressing PyMSP1₁₉. (A) Schematic diagram of AcNPV-PyMSP1₁₉surf genome. PyMSP1₁₉ was expressed as a PyMSP1₁₉-gp64 fusion protein under the control of the polyhedron promoter. Numbers indicate the amino acid positions of the PyMSP1₁₉-gp64 fusion protein and the endogenous gp64 protein. pPolh, polyhedrin promoter; SP, gp64 signal sequence; FLAG, FLAG epitope tag; pgp64, gp64 promoter. (B) Western blot analysis of AcNPV-PyMSP1₁₉surf. AcNPV-WT (lanes 1, 3, and 5) and AcNPV-PyMSP1₁₉surf (lanes 2, 4, and 6) were treated with loading buffer containing 1% 2-ME and examined using anti-FLAG MAb (lanes 1and 2), P. yoelii-hyperimmune serum (lanes 3 and 4), and anti-gp64 MAb (lanes 5 and 6). Arrows indicate the positions of PyMSP1₁₉-gp64 fusion protein and endogenous gp64. (C and D) Structural analysis of PyMSP1₁₉-gp64 fusion protein. AcNPV-PyMSP1₁₉surf was treated with loading buffer containing various concentrations of 2-ME. The reactivity of the PyMSP1₁₉-gp64 fusion protein was examined using either P. yoelii-hyperimmune serum (C) or anti-FLAG MAb (D). The concentrations of 2-ME are shown at the top. (E and F) Electron micrographs of AcNPV-PyMSP1₁₉surf displaying PyMSP1₁₉ on the viral envelope. AcNPV-PyMSP1₁₉surf was treated with either mouse anti-GST-PyMSP1₁₉ antiserum (E) or normal mouse serum (F) followed by labeling with anti-mouse-gold conjugate. The surfaces of the virions were strongly labeled with gold particles (arrows). Bars, 100 nm.

FIG. 2.

PyMSP1₁₉-specific antibody responses. Sera were collected from individual mice (10 mice/group) 3 weeks after the last immunization. (A) The individual sera were tested for total IgG, IgG1, IgG2a, IgG2b, and IgG3 specific for PyMSP1₁₉ by ELISA. The data represent one of two experiments, which had similar results. Data are presented as means ± standard deviations (SD) for the groups. Significant differences in total IgG titers between different groups were evaluated using two-tailed Fisher's exact probability test. *, P < 0.01. (B and C) Confocal fluorescence micrographs of sera obtained from mice immunized with AcNPV-PyMSP1₁₉surf. Free merozoites (B) and mature schizonts (C) were clearly stained (green) by serum (1:500 dilution) obtained from one mouse immunized i.n. with AcNPV-PyMSP1₁₉surf. Cell nuclei were visualized by DAPI (4′,6-diamidino-2-phenylindole) staining (blue). Similar results were observed for all sera obtained from mice immunized i.m. and i.n. with AcNPV-PyMSP1₁₉surf. Bar, 10 μm. (D) PyMSP1₁₉-specific antibody responses induced by mucosal immunization regimens. Groups of mice (n = 10) were immunized either i.n. with GST-PyMSP1₁₉ plus AcNPV-WT, i.n. with GST-PyMSP1₁₉ alone, or orally with AcNPV-PyMSP1₁₉surf. Sera were collected from individual mice 3 weeks after the last immunization and tested for PyMSP1₁₉-specific antibodies and IgG isotypes by ELISA. Ig63 was undetectable in all groups. Data are presented as means ± SD for the groups. *, P < 0.01.

FIG. 3.

Antibody induction in response to infection. (A) Correlation between PyMSP1₁₉-specific antibody titer and parasitemia. Mice were ranked based on maximal parasitemia (Spearman's rank correlation; r = −0.616; P = 0.0009). Crosses, death. (B) Comparison of antibody induction between days 0 and 29 postchallenge in surviving mice. A total of 22 surviving mice were from the i.m. (n = 5) and i.n. (n = 10) AcNPV-PyMSP1₁₉surf groups and the GST-PyMSP1₁₉-plus-alum (n = 7) group. PyMSP1₁₉-specific antibody titers of each group were compared between days 0 and 29. Data are means ± standard errors (SE) for the groups. Significant differences between each group were evaluated using the Mann-Whitney U test. *, P < 0.01.

FIG. 4.

Antibody and cytokine responses during the course of infection. Groups of mice were immunized either i.n. with AcNPV-PyMSP1₁₉surf or i.p. with GST-PyMSP1₁₉ in alum and then challenged i.v. with 10³ P. yoelii-parasitized RBC. Parasitemia was monitored daily for 4 days after challenge, and sera were collected periodically postchallenge to measure antibody titers and cytokine production. (A) Kinetics of PyMSP1₁₉-specific antibody titers and parasitemia during the course of infection. Each point for antibody titers (solid lines) represents the mean ± SD (n = 3). One representative parasitemia level for each group (dotted lines) is shown. i.n. AcNPV-PyMSP1₁₉surf data are shown in orange, and GST-PyMSP1₁₉-plus-alum data are shown in green. (B and C) IgG (B) and IgM (C) responses to blood-stage parasites at days 0 and 29. Data are means ± SE for the groups (n = 3). Significant differences between different groups were evaluated using two-tailed Fisher's exact probability test. **, P < 0.01; *, P < 0.05. (D to F) Kinetics of cytokine responses. Each point represents the mean ± SD (n = 3). (D) TNF-α; (E) IL-5; (F) IL-10. i.n. AcNPV-PyMSP1₁₉surf data are shown in orange, and GST-PyMSP1₁₉-plus-alum data are shown in green. Data for nonimmunized mice are shown in blue.

FIG. 5.

PyMSP1₁₉-specific antibody response in TLR9-deficient mice. Sera were collected from individual TLR9-deficient mice (8 mice/group) 3 weeks after the last immunization and tested for PyMSP1₁₉-specific antibodies and IgG isotypes by ELISA. (A) Comparison of PyMSP1₁₉-specific antibody responses between TLR9-deficient mice and normal BALB/c mice. Data are means ± SE for the groups. Significant differences between each group were evaluated using the Mann-Whitney U test. **, P < 0.01; *, P < 0.05. (B) Isotype profiles of PyMSP1₁₉-specific antibodies. IgG3 was undetectable in all groups. The data represent one of two experiments, which had similar results. Data are means ± SD for the groups.