| Allergy is a hypersensitivity disorder of the immune system. Although the mortality of allergy is not very high, some epidemiologists believe that it would be one greatest threat to public heath in 21 st century. The number of allergen is huge, in which allergic diseases induced by HDM allergen is one of the commonest allergy. Clinical studies indicated that more than 80% of HDM allergic patients had a reaction with Der p2. So far, there is no effective way for the therapy and prophylaxis of allergy. Compared to traditional method, probiotics treatment is considered to be a new and potential treatment strategy for allergy without adverse side-effects, but its clinical effect could not live up to expectations. Some researchers believe that the beneficial function of probiotics in vivo could be enhanced by molecular modification. In this study, we sought to find an effective way for the prevention of allergy via the study of recombinant LAB vaccine. The main content of this paper is as follows:Current studies indicate that the location of heterologous protein in recombinant LAB strain could have a significant effect on its immune effect in vivo. To determine the most effective way, Der p2 was expressed in different cellular locations of recombinant LAB strain and then their prophylactic effect was evaluated in mice models. The result showed that three different strains could significantly suppress lung inflammatory responses by reducing the levels of Th2 cytokines IL-4 and IL-5 in BALF. In addition, recombinant LAB strains could increase the production of Ig G2 a to inhibit specific Ig E response via the modulation of cellular immune response. These protective effects could be closely associated with the induction of Tregs in the MLN. Taken together, different recombinant LAB strains had similar immune protective effect against allergy in vivo, suggesting that the location of Der p2 in recombinant strain had no significant effect its immunoregulation in vivo.During the treatment or prophylaxis of allergy, natural Der p2 always cause unavoidable side-effects induced by the combination of Der p2 and specific Ig E. It has been demonstrated that this Ig E response could be reduced or eliminated by disrupting the structure of Der p2. Based on the sequence of Der p2, Der p2 derivative DM was engineered by changing the amino acids sequence and deleting the protein loci related with the protein folding. This derivative DM contains most of the amino acids sequence of Der p2 but its spatial structure is different from Der p2. The in vitro study showed that DM have no significant reaction with specific Ig E, but have a similar response with T cells in comparision with Der p2. To verify the immune characteristics of DM, recombinant strain LL-DM was engineered to produce Der p2 derivative DM. It was demonstrated that recombinant strain LL-DM could inhibit allergic responses in mice model, the protective effect and mechanisms involved were similar with recombinant Der p2-expressing strain.Compare with macromolecular allergen derivatives, micromolecular allergen fragment could not only avoid Ig E-mediated side-effects, but also reduce the untoward reaction caused by the proliferation of effector cell. Based on Der p2 sequence, five Der p2 peptides comprsing the major T-cell epitopes were synthetized by chemical method. The in vitro study showed that these Der p2 peptides have no significant reaction with specific Ig E as well as DM, but their reaction with T cells was significantly reduced compared with DM. To verify the immune characteristics of Der p2 peptides, recombinant strains were engineered to produce Der p2 peptides respectively. It was demonstrated that mucosal delivery of Der p2 peptides could inhibit allergic response by the same way as LL-DM, but their inhibitory effect was a bit lower than DM. Considering the long-term of allergy treatment, allergen peptides could be better than macromolecular allergen derivatives.Previous study showed that Lactococcus lactis used for the expression of heterologous protein is not considered as probiotics with anti-allergy property. To further enhance the immune effect of recombinant LAB strain, probiotics with anti-allergy property was screened via an in vitro cell model. Compared with other strains, Streptococcus thermophilus CCFM218(ST218) could induce the expression of IL-10 to inhibit IL-4 production, which is considered to be an anti-allergy strain. Although ST218 did not reduce the level of specific Ig E in the sensitized mice, it could modulate the production of Ig G2 a to alleviate allergic responses. The examination of histology and m RNA expression level in lung tissues demonstrated that ST218 could significantly alleviate lung inflammatory response, which could be associated with the modulation of intestinal microbiota and mucosal immune response induced by ST218.To evaluate the effect of ST218 on anti-allergy role of recombinant LAB strains, their inhibitory effect on allergic responses was analyzed in vivo. It was demonstrated that ST218 could increase the inhibitory effect of recombinant strain on the level of specific Ig E, which could be associated with the modulation of Tregs in the MLN. The level of cytokines in BALF demonstrated that the mixture of ST218 and recombinant strains could inhibit lung inflammatory response, but there was no significant difference between the mixture and recombinant strains, which could be attributed to lower inflammation level.To effectively prevent the prevalence of dust mite allergy, we designed and constructed a new kind of recombinant LAB vaccine, in which some factors which influence its immune effect in vivo was studied. This paper could provide important theoretical basis and reference value for the application of recombinant LAB in the future. |
PDF Full Text Request