Recombinant Vaccinia Virus Expressing Plasmodium berghei Apical Membrane Antigen 1 or Microneme Protein Enhances Protection against P. berghei Infection in Mice

Abstract

Recombinant vaccinia viruses (rVV) are effective antigen delivery vectors and are researched widely as vaccine platforms against numerous diseases. Apical membrane antigen 1 (AMA1) is one of the candidate antigens for malaria vaccines but rising concerns regarding its genetic diversity and polymorphism have necessitated the need to search for an alternative antigen. Here, we compare the efficacies of the rVV vaccines expressing either AMA1 or microneme protein (MIC) of Plasmodium berghei in mice. Mice (BALB/c) were immunized with either rVV-AMA1 or rVV-MIC and subsequently challenge-infected with P. berghei. Compared to the control group, both antigens elicited elevated levels of parasite-specific antibody responses. Immunization with either one of the two vaccines induced high levels of T cells and germinal center B cell responses. Interestingly, rVV-MIC immunization elicited higher levels of cellular immune response compared to rVV-AMA1 immunization, and significantly reduced pro-inflammatory cytokine productions were observed from the former vaccine. While differences in parasitemia and bodyweight changes were negligible between rVV-AMA1 and rVV-MIC immunization groups, prolonged survival was observed for the latter of the two. Based on these results, our findings suggest that the rVV expressing the P. berghei MIC could be a vaccine-candidate antigen.

Keywords: Plasmodium berghei; apical membrane antigen 1; microneme protein; recombinant vaccinia virus (rVV); vaccine.

Figure A1

The diagram of the Plasmodium organism. The diagram describes the Plasmodium organism and the specified target gene (AMA1, MIC).

Figure A2

A schematic diagram depicting animal experimental schedule. Immunization intervals, blood collection, challenge infection, and euthanasia time points are indicated.

Figure A3

The gating strategy for CD4⁺ and CD8⁺ T cells. Lymphocytes were gated as indicated to analyze the frequencies of CD4⁺ and CD8⁺ T cells by flow cytometry.

Figure 1

Gene cloning. To generate P. berghei AMA1 and MIC recombinant vaccinia virus (rVV), gene cassettes with promoters and recombination sites were designed, shown as a schematic diagram (A,B). PbAMA1 or PbMIC genes were cloned into the pRB21 vector, and clones pRB21-AMA1 (C) and pRB21-MIC (D) were confirmed by restrictive enzyme digestion with EcoRⅠ and HindⅢ. The red color font denotes successful clones.

Figure 2

Transfection of CV-1 and confirming the production of rVV. The pRB21-PbAMA1 and pRB21-PbMIC DNA constructs were transfected into CV-1 cells, along with the vaccinia virus, to generate rVV-AMA1 and rVV-MIC. Representative images of rVV transfection under brightfield (A) and fluorescent microscope (B) are illustrated over the course of 3 days. AMA1 and MIC rVVs were identified by a Western blot using a P. berghei polyclonal antibody and a wild-type vaccinia virus (wt VV) control (C).

Figure 3

IgG antibody response in sera. On week 4 after prime, 1st boost, and 2nd boost immunization, the sera of mice were collected to investigate the levels of IgG antibody response. The 96-well immunoplates coated with P. berghei antigen (A), rVV-AMA1 (B), or rVV-MIC (C) were used to verify IgG antibody response by ELISA. Data are expressed as mean ± SD (** p < 0.01).

Figure 4

T cell and germinal center B cell response in blood and inguinal lymph nodes (ILN). Immunized mice (n = 8) were challenge-infected with P. berghei, and blood samples were collected at 6 dpi. Inguinal lymph nodes (ILN) from mice were collected at 6 dpi. After surface marker staining with the fluorophore-conjugated antibodies, CD4⁺ (A) and CD8⁺ (B) T cell responses in the blood and CD8⁺ (C) T cell, germinal center B cell (D) responses in ILN were assessed by flow cytometry. Data are expressed as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001; ns: no statistical significance).

Figure 5

Inflammatory cytokine production in the spleen. Inflammatory cytokine productions were assessed from the spleens of mice at 6 dpi with P. berghei. Splenic IFN-γ (A) and TNF-α (B) cytokines were investigated using the BD OptEIA IFN-γ, TNF-α ELISA kits. Data are expressed as mean ± SD (* p < 0.05; ns: no statistical significance).

Figure 6

Parasitemia, body weight, and survival rate. After immunization with the rVV-AMA1 or rVV-MIC vaccines, mice (n = 8) were infected intraperitoneally with 0.05% of P. berghei and monitored at regular intervals to assess changes in parasitemia, body weight, and survival. Parasitemia (A), body weight changes (B), and survival rate (C) from days 0 to 65 post-challenge infections have been monitored. The highest differences in parasitemia (D–H) and body weight (I) between immunized mice and non-immunized naïve control were observed at days 45 and 48 post-challenge infections, respectively. ns: no statistical significance.