Preliminary study on recombinant Lactococcus lactis expressing thymosin and interferon fusion protein as an immune adjuvant for chicken Newcastle disease virus live vaccine
To evaluate the potential of recombinant Lactococcus lactis expressing chicken thymosin and chicken interferon fusion protein as an immune adjuvant for Newcastle disease virus(NDV)live vaccine and its effect on immune efficacy,14-day-old SPF chickens were intranasally immunized with recombinant Lactococcus lactis expressing chicken thymosin and chicken gamma interferon fusion protein(cTα1-cIFN-γ,abbreviated as Tα1-IFN)mixed with live NDV vaccine(Tα1-IFN/r-L.lactis+live NDV vaccine),and the live NDV vaccine group,L.lactis+live NDV vaccine group and PBS negative control group were set up.Blood samples were collected at 3,7,14,21 and 28 days post-immunization(dpi),and the HI antibody titers in the serum of chickens in each group were detected at 14,21 and 28 dpi,respectively;the secretion levels of major cytokines in the serum of chickens in each group at 3,7 and 14 dpi were determined by indirect ELISA kits;peripheral blood mononuclear(PBMC)and peripheral blood leukocytes were isolated from chickens in each group at 21 and 28 dpi,and the proliferative activities of T-lymphocytes in PBMC and antigen-presenting cells(APCs)in leukocytes were detected by CCK-8 assay.The results showed that there was no significant difference in NDV HI antibody titers between Tα1-IFN/r-L.lactis+NDV live vaccine group and NDV live vaccine group as well as L.lactis+NDV live vaccine group at 14 and 21 dpi,respectively.However,at 28 dpi,the NDV HI antibody titer in Tα1-IFN/r-L.lactis+NDV live vaccine group was extremely significantly higher than that of the other two groups(P<0.0001).The results of cytokine assay showed that the secretion levels of IFN-γ,IL-2(Th1 cytokine)and IL-4(Th2 cytokine)in chicken sera of the Tα1-IFN/r-L.lactis+NDV group were significantly higher than those of the NDV group,the L.lactis+NDV group and the negative control group at the corresponding timepoints after immunization.The secretion levels of IL-1β(pro-inflammatory factor)and IL-10(anti-inflammatory factor)in chicken sera of the Tα1-IFN/r-L.lactis+NDV live vaccine group were highly significantly higher than those in the remaining groups at 3,7 and 14 dpi(P<0.0001).The proliferation assay of cells showed that the proliferative activities of chicken PBMC of the Tα1-IFN/r-L.lactis+NDV live vaccine group were extremely significantly higher than that in the rest of the groups at 21 and 28 dpi;At 28 dpi,the proliferative activity of chicken PBMC of this group was highly significantly higher than those in the L.lactis+NDV live vaccine group and the negative control group(P<0.0001),but there was no significant difference compared to the NDV live vaccine group(P>0.05);the proliferative activity of APC in peripheral blood leukocytes of chickens in the Tα1-IFN/r-L.lactis+NDV live vaccine group was highly significantly higher than that in the rest of the groups at 21 and 28dpi(P<0.0001);At 28 dpi,the proliferative activity of APCs in chicken leukocytes in this group was highly significantly higher than that in the live NDV vaccine group(P<0.01),but was not significantly different from that in either the L.lactis+NDV live vaccine group or the negative control group.The above results indicated that the combination of NDV live vaccine and recombinant Lactococcus lactis expressing Tα1-IFN in this study could induce high levels of humoral and cellular immune responses in SPF chickens after intranasal immunization,enhance the response ability of SPF chickens immune system,while maintaining the immune homeostasis in chickens,which is expected to be a new and potential immunoadjuvant,and provides important experimental data and reference basis for the development of new immunoadjuvants.
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