A Recombinant Chimera Vaccine Composed of LTB and Mycoplasma hyopneumoniae Antigens P97R1, mhp390 and P46 Elicits Cellular Immunologic Response in Mice

Abstract

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), leading to a mild and chronic pneumonia in swine. Relative control has been attained through active vaccination programs, but porcine enzootic pneumonia remains a significant economic challenge in the swine industry. Cellular immunity plays a key role in the prevention and control of porcine enzootic pneumonia. Therefore, the development of a more efficient vaccine that confers a strong immunity against M. hyopneumoniae is necessary. In this study, a multi-antigen chimera (L9m6) was constructed by combining the heat-labile enterotoxin B subunit (LTB) with three antigens of M. hyopneumoniae (P97R1, mhp390, and P46), and its immunogenic and antigenic properties were assessed in a murine model. In addition, we compared the effect of individual administration and multiple-fusion of these antigens. The chimeric multi-fusion vaccine induced significant cellular immune responses and high production of IgG and IgM antibodies against M. hyopneumoniae. Collectively, our data suggested that rL9m6 chimera exhibits potential as a viable vaccine candidate for the prevention and control of porcine enzootic pneumonia.

Keywords: Mycoplasma hyopneumoniae; chimeric vaccine; immune responses; multi-antigen.

Figure 1

Schematic representation for the construction of rL9m6 chimera. The P46, mhp390, and R1 repeat region of P97 was amplified from Mhp168 genomic DNA. The recombinant L9m6 corresponds to the pET-30a vector backbone. The P46 gene was inserted between the Not I and Xho I site, and then the mhp390 gene was cloned into Sal I and Not I site. The P97R1 was cloned into BamH I and Sal I site, while the LTB was amplified from the genomic DNA of E. coli and cloned into Nco I and BamH I site.

Figure 2

SDS-PAGE analysis of purified recombinant protein. M, Protein Marker (kDa); lane 1, purified rP46 by Ni-NTA column; lane 2, purified rP97R1 by Ni-NTA column; lane 3, purified rMhp390 by Ni-NTA column; lane 4, purified rL9m6 by Ni-NTA column. * indicate the individual recombinant protein.

Figure 3

Study design and IFN-γ production from each vaccinated group. (A) Overview of experimental design. Forty-two female BALB/c mice were randomly allocated to 7 groups. Each animal was immunized 35 days post-primary inoculation, according to Table 2. Blood samples were obtained from the tail of mice at three time points. (B) The production of IFN-γ in the supernatant of splenocytes harvested from immunized mice after in vitro restimulation. Splenocytes were isolated 3 weeks after secondary immunization and stimulated with 10 μg/mL extract protein of M. hyopneumoniae for 72 h. The presence of IFN-γ was evaluated using mouse IFN-γ ELISA kits (Beyotime Biotechnology, Shanghai, China) in accordance with the manufacturer’s instructions. The concentrations of IFN-γ were quantified using standard curves. The presented values represent means ± standard errors (SE) from three independent experiments. *** p < 0.001 indicate statistically significant difference among various groups.

Figure 4

Proportion of splenic CD4⁺ T cells measured by intracellular cytokine staining. (A) The count of CD3⁺, CD4⁺, and CD8⁺ T lymphocytes in the spleen was measured by flow cytometry. Representative flow cytometric dot plot demonstrates the gating strategy employed for analyzing subsets of T lymphocytes based on forward and sidelight scatter. Dot plots exhibiting double-positive staining indicate the CD4⁺ T cells (Q-UR). (B) The CD4⁺ T cells rates in the spleen, shown as a percentage, were assessed by flow cytometry. *** p < 0.001 indicate statistically significant difference among various groups.

Figure 5

Proportion of splenic CD8⁺ T cells measured by intracellular cytokine staining. (A) The count of CD3⁺, CD4⁺, and CD8⁺ T lymphocytes in the spleen was quantified by flow cytometry. Representative flow cytometric dot plot demonstrates the gating strategy employed for analyzing subsets of T lymphocytes based on forward and sidelight scatter. Dot plots exhibiting double-positive staining indicate the CD8⁺ T cells (Q-UR). (B) The CD8⁺ T cells rates in the spleen, shown as a percentage, were assessed by flow cytometry. * p < 0.05, and *** p < 0.001 indicate statistically significant differences among various groups.

Figure 6

The IgG antibody response in mice immunized with each vaccine formulation. Serum samples were collected 5, 6, and 8 weeks after primary immunization. The IgG level of seroconversion induced by each vaccinated group was determined via indirect ELISA utilizing M. hyopneumoniae whole cell extract.

Figure 7

The IgM antibody response in mice immunized with each vaccine formulation. Serum samples were collected 5, 6, and 8 weeks after primary immunization. The IgM level of seroconversion induced by each vaccinated group was measured by indirect ELISA using the whole cell extract of M. hyopneumoniae.