Evaluation of a Recombinant Mouse X Pig Chimeric Anti-Porcine DEC205 Antibody Fused with Structural and Nonstructural Peptides of PRRS Virus

Abstract

Activation of the immune system using antigen targeting to the dendritic cell receptor DEC205 presents great potential in the field of vaccination. The objective of this work was to evaluate the immunogenicity and protectiveness of a recombinant mouse x pig chimeric antibody fused with peptides of structural and nonstructural proteins of porcine respiratory and reproductive syndrome virus (PRRSV) directed to DEC205⁺ cells. Priming and booster immunizations were performed three weeks apart and administered intradermally in the neck area. All pigs were challenged with PRRSV two weeks after the booster immunization. Immunogenicity was evaluated by assessing the presence of antibodies anti-PRRSV, the response of IFN-γ-producing CD4⁺ cells, and the proliferation of cells. Protection was determined by assessing the viral load in the blood, lungs, and tonsils using qRT-PCR. The results showed that the vaccine exhibited immunogenicity but conferred limited protection. The vaccine group had a lower viral load in the tonsils and a significantly higher production of antibodies anti-PRRSV than the control group (p < 0.05); the vaccine group also produced more CD4⁺IFN-γ⁺ cells in response to peptides from the M and Nsp2 proteins. In conclusion, this antigenized recombinant mouse x pig chimeric antibody had immunogenic properties that could be enhanced to improve the level of protection and vaccine efficiency.

Keywords: DEC205; PRRSV; antigen targeting; chimeric antibody; dendritic cells; recombinant antibodies.

Figure 1

Graphic representation of the recombinant antigenized chimeric mouse x pig antibody utilized. In blue: variable region of a mouse anti-DEC205 monoclonal antibody. In gray: constant region of a pig IgG. The antigenized part consists of a chimeric protein based on selected immunogenic peptides from PRRSV (red).

Figure 2

Analysis of CD4⁺IFN-γ-secreting cells. (A) Representative experiment with PBMCs stimulated with individual porcine reproductive and respiratory syndrome virus (PRRSV) peptides, the whole virus, phytohaemagglutinin (PHA) (positive control) or medium along (unstimulated control). For extracellular labeling, an anti-CD4 antibody conjugated with fluorescein isothiocyanate (FITC) was used, and for intracellular staining, an anti-pig IFN-γ antibody conjugated with phycoerythrin (PE) was used. The selected population corresponded to CD4⁺IFN-γ⁺ cells. (B) Frequency of CD4⁺ IFN-γ-secreting cells in immunized pigs. The results were obtained by subtracting the percentage of CD4⁺IFN-γ⁺ cells in the unstimulated cell group from the percentage of double-positive cells in the experimental group.

Figure 3

Proliferation of PBMCs from immunized pigs in response to structural and nonstructural peptides and PRRSV. The results were obtained by subtracting the percentage of proliferating cells observed with the unstimulated cells from the percentage observed with the stimulated cells.

Figure 4

Antibody response to the anti-PRRSV antibody. (A) Evaluation of antibodies against the anti-PRRSV antibody at the beginning of the experiment (Day 0), at the time of the booster immunization (Day 21), at the time of the challenge (Day 35), and at the end of the experiment (Day 56) by ELISA. Gray bars represent the control group, and blue bars represent the vaccine group. Bars represent the mean ± standard error (SE) of three animals. (B) Antibody response evaluated by a commercial kit from IDEXX to confirm seroconversion in the challenged pigs.

Figure 5

Viremia and the viral load in the lungs and tonsils of challenged pigs. (A) Viremia evaluated on the day of the challenge and every week thereafter until the end of the experiment (three weeks postchallenge). The viral load in the lungs (B) and tonsils (C) at the end of the experiment. The results are expressed as cycle threshold (Ct) values, and the dotted line represents the cut-off for qPCR. Each point represents the Ct value of one pig.

Figure 5

Viremia and the viral load in the lungs and tonsils of challenged pigs. (A) Viremia evaluated on the day of the challenge and every week thereafter until the end of the experiment (three weeks postchallenge). The viral load in the lungs (B) and tonsils (C) at the end of the experiment. The results are expressed as cycle threshold (Ct) values, and the dotted line represents the cut-off for qPCR. Each point represents the Ct value of one pig.

Figure 6

Microscopic lung lesion evaluation of immunized pigs at the end of the experiment. The scoring system utilized to evaluate the severity of the lung lesions found in pigs was as follows: 0—no significant lesions; 1—mild significant lesions; 2—moderate significant lesions; and 3—severe significant lesions. Tissue samples were magnified at 10×.