A subunit vaccine based on rH-NS induces protection against Mycobacterium tuberculosis infection by inducing the Thl immune response and activating macrophaqes

Mycobacterium tuberculosis （Mtb） is a Gram-positive pathogen which causes tuberculosis in both animals and humans. All tested rH-NS formulations induced a specific Thl response, as indicated by increased production of interferon y （IFN-γ） and interleukin 2 （IL-2） by lymphocytes in the spleen of mice which were immunized with rH-NS alone or with rH-NS and the adjuvant cyclic GMP-AMP （cGAMP）. Serum from mice immunized with rH-NS with or without adjuvant also had higher levels of IL-12p40 and TNF-α, compared with those from control mice immunized with phosphate-buffered saline. Both vaccines increased protective efficacy in mice which were challenged with Mtb H37Rv, as measured by reduced relative CFU counts in the lungs. We found that rH-NS induced the produc- tion of TNF-α, IL-6, and IL-12p40, which relied on the activation of mitogen-activated protein kinases by stimulating the rapid phosphorylation of ERK1/2, p38, and JNK, and on the activation of tran- scription factor NF-κB in macrophages. Additionally, we also found that rH-NS could interact with TLR2 but not TLR4 in pull-down assays. The rH-NS-induced cytokine production from TLR2-silenced RAW264.7 cells was lower than that from BALB/c macrophages. Prolonged exposure （〉24h） of RAW264.7 cells to rH-NS resulted in a significant enhancement in IFN-γ-induced MHC II expression, which was not found in shTLR2-treated RAW264.7 cells. These results suggest that rH-NS is a TLR2 agonist which induces the production of cytokines by macrophages and up-regulates macrophage function.