Table 1.
Bacterial strains and plasmids.
Table 2.
Primers used for PCR amplification.
Table 3.
Immunization groups.
Fig 1.
Plasmids for the expression of recombinant VP1 antigen and western blotting of expressed WCFS1-pSIP411-VP1 and NC8-pSIP411-VP1 proteins.
(A) The pSIP411-VP1 plasmid was constructed as described in the article and the purple arrow stands for optimized VP1. (B) The VP1 proteins expressed in WCFS1-pSIP411-VP1 and NC8-pSIP411-VP1 were detected by western blotting. Lane 1: WCFS1-pSIP411 induced by SppIP; Lane 2: WCFS1-pSIP411-VP1 without inducer; Lane 3: WCFS1-pSIP411-VP1 induced by SppIP; Lane 4: NC8-pSIP411-VP1 induced by SppIP; Lane 5: FMDV as positive control; Lane 6: NC8-pSIP411-VP1 without inducer.; Lane 7: NC8-pSIP411 induced by SppIP. Abbreviations: sh71rep: replication origin for lactobacillus; ermL: erythromycin-resistance marker; PsppIP: inducible promoters; sppK: histidine protein kinase; sppR: response regulator.
Fig 2.
Titers of VP1-specific antibodies and neutralizing antibody.
(A) sIgA antibody in feces detected by capture ELISA on days 0, 5, 7, 10, 15, 17, 20, 25, 27 and 30. (B) sIgA antibody in saliva detected by capture ELISA at 0, 5, 7, 10, 15, 17, 20, 25, 27 and 30d. (C) IgA antibody in serum detected by capture ELISA on days 0, 10, 20 and 30. (D) IgG antibody in serum detected by indirect ELISA on days 0, 10, 20 and 30. (E) IgM antibody in serum detected by capture ELISA on days 0, 10, 20 and 30. (F) Titers of FMDV-specific neutralizing antibodies. Data are expressed as mean of optical density (OD) ± SD (n = 3). “*” stands for statistically significant differences relative to the milk control and “#” represents statistically significant differences relative to the WCFS1/NC8-pSIP411 control (P<0.05). Abbreviations: Ig, immunoglobulin; SD, standard deviations.
Fig 3.
Flow cytometry for percentages of lymphocyte subpopulations between experimental and control groups.
Heparinized blood from immunized guinea pigs on day 30 were double-stained with anti-CD4-RPE and anti-CD8-FITC, or incubated with the corresponding isotype controls (RPE and FITC conjugated mouse IgG1 antibodies as negative controls) for 30 minutes at room temperature. CD4+ and CD8+ T cells were analyzed as fluorescence profiles. Data are expressed as mean ± SD (n = 3). Abbreviations: Ig, immunoglobulin; FITC, Fluorescein isothiocyanate; RPE, R. Phycoerythrin; SD, standard deviations.
Fig 4.
Proliferation of guinea pigs PBMCs stimulated with FMDV.
PBMCs isolated from guinea pigs 30 days after the first immunization were labeled with CFSE and stimulated in vitro with inactivated FMDV, ConA (positive control), or RPMI-1640 (negative control), and plated in three replicate cultures for 60 hours. Data were acquired with FCM and analyzed with CellQuest™ software. For determination of proliferation of CFSE-stained cells, 10,000 events were captured. Data are expressed as mean percentages of proliferated cells ± SD (n = 3). Abbreviations: CFSE, carboxyfluorescein diacetate succinimidyl ester; ConA, concanavalin A; FCM, flow cytometry; FITC, fluorescein isothiocyanate; FMDV, foot-and-mouth disease virus; PBMCs, peripheral blood mononuclear cells; RPMI-1640, Roswell Park Memorial Institute 1640; SD, standard deviation.
Table 4.
Protection of guinea pigs (n = 5) against FMDV challenge.