Protection against Mycobacterium tuberculosis infection offered by a new multistage subunit vaccine correlates with increased number of IFN-γ+ IL-2+ CD4+ and IFN-γ+ CD8+ T cells.

Xiaochun Wang,Jingyan Zhang,Xuefeng Yuan,Jinping Liang,Xindong Teng,Ying Zhang,Xionglin Fan

doi:10.1371/journal.pone.0122560

Xiaochun Wang, Jingyan Zhang + Show 5 more

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https://doi.org/10.1371/journal.pone.0122560

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Abstract

Protein subunit vaccines present a compelling new area of research for control of tuberculosis (TB). Based on the interaction between Mycobacterium tuberculosis and its host, five stage-specific antigens of M. tuberculosis that participate in TB pathogenesis—Rv1813, Rv2660c, Ag85B, Rv2623, and HspX—were selected. These antigens were verified to be recognized by T cells from a total of 42 whole blood samples obtained from active TB patients, patients with latent TB infections (LTBIs), and healthy control donors. The multistage polyprotein A1D4 was developed using the selected five antigens as a potentially more effective novel subunit vaccine. The immunogenicity and protective efficacy of A1D4 emulsified in the adjuvant MTO [monophosphoryl lipid A (MPL), trehalose-6,6′-dibehenate (TDB), components of MF59] was compared with Bacillus Calmette-Guerin (BCG) in C57BL/6 mice. Our results demonstrated that A1D4/MTO could provide more significant protection against M. tuberculosis infection than the PBS control or MTO adjuvant alone judging from the A1D4-specific Th1-type immune response; however, its efficacy was inferior to BCG as demonstrated by the bacterial load in the lung and spleen, and by the pathological changes in the lung. Antigen-specific single IL-2-secreting cells and different combinations with IL-2-secreting CD4+ T cells were beneficial and correlated with BCG vaccine-induced protection against TB. Antigen-specific IFN-γ+IL-2+ CD4+ T cells were the only effective biomarker significantly induced by A1D4/MTO. Among all groups, A1D4/MTO immunization also conferred the highest number of antigen-specific single IFN-γ+ and IFN-γ+TNF-α+ CD4+ T cells, which might be related to the antigen load in vivo, and single IFN-γ+ CD8+ T cells by mimicking the immune patterns of LTBIs or curable TB patients. Our strategy seems promising for the development of a TB vaccine based on multistage antigens, and subunit antigen A1D4 suspended in MTO adjuvant warrants preclinical evaluation in animal models of latent infection and may boost BCG vaccination.

Highlights

Protein subunit vaccines, consisting of defined immunodominant antigens of Mycobacterium tuberculosis (M. tuberculosis), are sufficiently safe as a stand-alone prophylactic vaccine in a population where Bacillus Calmette-Guerin (BCG) immunization is not recommended, or as a boost to the currently used BCG vaccine
The recombinant fusion protein A1D4 was constructed by tandem-linking the five M. tuberculosis antigens Rv1813, Rv2660c, Ag85B, Rv2623, and HspX, and expression in the plasmid pET30b-A1D4 (Fig 1), and the successful plasmid generation was verified by enzyme digestion (Fig 1)
The recombinant protein A1D4 with a C-terminal His-tag was efficiently expressed in E. coli BL21 (DE3) as inclusion bodies, and purification was performed under denaturing conditions (Fig 1)

Summary

Introduction

Protein subunit vaccines, consisting of defined immunodominant antigens of Mycobacterium tuberculosis (M. tuberculosis), are sufficiently safe as a stand-alone prophylactic vaccine in a population where Bacillus Calmette-Guerin (BCG) immunization is not recommended, or as a boost to the currently used BCG vaccine. T cells, especially CD4+ Th1-type cells that were sensitized in the local lymph node and homing the infected lesion via the pulmonary circulation, can secrete Th1-typed cytokines such as IFN-γ, which activates macrophage phagocytosis, thereby killing intracellular bacteria, and TNF-α, which attracts more infiltrating macrophages and lymphocytes and promotes granuloma formation at the site of infection, leading to latent TB infection (LTBI) During this chronic phase, the metabolic status of M. tuberculosis aggregating in the granuloma transfers from growing replication to dormancy in vivo with limited antigen presentation [18]. These studies partially confirm the vaccine potential of stage-specific antigens expressed by M. tuberculosis

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