Effect Of Yeast-mediated Delivery Of Myostatin And IL-21 Gene DNA Vaccines On Body Weight And Immune System In Mice

Myostatin(MSTN),also known as growth and differentiation factor 8(GDF8)negatively regulates skeletal muscle growth in mammals.Previously yeast-based myostatin specific recombinant protein vaccines were orally applied to enhance muscle growth in rabbits and mice S.cerevisiae yeast-based vaccines have achieved greater clinical responses for the treatment of cancer,viral diseases and it can deliver recombinant protein,exogenous DNA and functional shRNA to the intestinal DCs in mice by oral administration.Interleukin-21(IL-21)is a type I cytokine that plays vital role in the process of inflammatory,renal diseases,AIDS and cancers.IL-21 specific antibodies as treatment and IL-21 vaccine for prophylactic measures attract a great attention.In order to explore the modulating role of IL-21 in the intestinal mucous immune system of mice,its effect on MSTN specific DNA vaccine and to validate the feasibility of recombinant yeast containing special DNA antigens as the effective DNA-delivery system,we used(1)an effective yeast-based shRNA-delivery method to down regulate IL-21 expression in DCs and(2)MSTN yeast-based DNA vaccine to block the endogenous MSTN protein,by oral administration.The results will provide insight into the development of edible vaccine to prevent many muscle related diseases in human.For developing a better DNA vaccine(a cascade of shIL21-MSTN yeast-based DNA vaccine),we first constructed the target vectors,including CMV-driven MSTN expression vector and a shIL21-MSTN combined vector containing MSTN expression cassette and shIL-21(short hairpin RNA-IL21)expression cassette.To optimize RNAi effect for the selection of best shRNA,the psiCHECK? vector was used.After necessary validation,recombinant yeast vaccines harboring different vectors were well prepared.Subsequently,after two month administration,the MSTN-specific immune responses were detected with western blots.Commercial ELISA results indicated that MSTN-specific antibody titer was signiicantly higher in the shIL21-MSTN group than MSTN group,which was consistent with western blot result.ELISA assays also revealed that the production of IL-21 and IL-6 were decreased in experimental groups as compare to control groups.The most important finding was significant body mass increase after oral administration of these yeast-based DNA vaccines,in which the shIL21-MSTN vaccine was slightly higher than the sole MSTN vaccine in mice.Our results proved that the knocking down IL-21 by yeast delivery of shIL-21 expression cassette significantly enhanced the yeast-mediated MSTN-specific immune response in terms of increased antibody titers and weight gain and that suppression of IL-21 is safe and effective in mammal model mouse.These findings provided a good inspiration for livestock breeding through the new type of immunoregulatory method.On the other hand,we also detected the possible modulating role of shIL-21 on the DC-mediated immune function which needs more practical application and deeper exploration.To our best knowledge,this is the first report to be found that the oral yeast MSTN-specific DNA vaccine can significantly promote the body weight in mice.Based on these observations,we hope that the special yeast DNA vaccine could be extended to other large livestock species.This may lead to the development of edible DNA vaccine against autoimmune diseases and cancers in human.