Construction And Evaluation Of A Multistage Subunit Vaccine And The Role Of Interleukin-17in Tuberculosis

Tuberculosis (TB) is a chronic infectious disease, caused by Mycobacteriumtuberculosis (M. tuberculosis, Mtb), from which human, animal and birds aresuffering. It is one of the most severe communicable diseases on Earth. With theincreasing drug-resistant strains especially multidrug-resistant and humanimmunodeficiency virus (HIV) infection, TB remains one of the major diseases withhigh morbidity and mortality worldwide. Central to the success of M. tuberculosis as apathogen is its ability to persist for long periods of time in latent state in human cellsto escape from specific immunity and may develop into active disease oncehypoimmunity. The latent infection is a quiz to control and even eliminate TB. Thecurrent vaccine, Bacilli Calmette-Guerin (BCG)，is widely used in many areas of theworld. It protects children from severe TB, but can not prevent reactivation of latentTB and is unreliable against the pulmonary TB in adults. Therefore, novel vaccinesand vaccination strategy which target adult TB, especially latent infection are urgentlyneeded. In addition, during Mycobacterium tuberculosis (Mtb) infection, cells of theimmune system rely on cytokines to regulate the activity of other immune andstructural cells. It suggested that cytokines are critical for evaluation of novel TBvaccine such as IFN-γ, major cytokine from Th1cells, provides protection againstMycobacterium tuberculosis (Mtb), while IL-4weakens Th1type immune responseand benefits to Mtb surviving in body. Recent studies suggest that Th17cellssecreting IL-17might play a potential role on tuberculosis (TB) infection. Someinvestigation has assessed the production of IL-17, IFN-γ and IL-4in the peripheralblood of patients with TB.In this study, a novel fusion protein consisting of Hsp16.3, the190to198peptideof Mpt64and Ag85B, which were confirmed to be the effective protective antigens mainly expressed in the dormancy and exponential phase of growth, was constructedand its immunogenicity was investigated; The protective efficacy of the vaccine wasdetected by BCG-primed and protein subunit vaccine-boost strategy; To assess IL-17,the production of IL-17and IFN-γ from lymphocytes of novel subunit vaccine，MHand EAMM，boost mice was detected. Meanwhile, IL-17and IFN-γ production inperipheral blood is reviewed and analyzed following BCG vaccination andMycobacterium tuberculosis infection in human for approach to the potential role ofIL-17and evaluation index。1. Construction and analysis of a multistage subunit vaccine for tuberculosisObjective：To construct protective immunity to Mycobacterium tuberculosis latentand replication stages，a novel fusion protein consisting of Hsp16.3, Ag85B and the190to198peptide of Mpt64, which were confirmed to be the effective protectiveantigens mainly expressed in the dormancy and exponential phase of growth, wasconstructed and its immunogenicity was investigated.Methods：the sequences, Ag85B and Mpt64190-198-Hsp16.3, were amplified bypolymerase chain reaction, subsequently, cloned in order into plasmids pET-28a.Ag85B-Mpt64190-198-Hsp16.3(AMH), a fusion protein, was induced and expressed inE. coli BL21, and then purified with Ni-NTA Resins. C57BL/6mice were immunizedthree times at2-week intervals subcutaneously with AMH formulated with theadjuvant composed of dimethyl-dioctyldecyl ammonium bromide (DDA) and BCGpolysaccharide nucleic acid（BCG-PSN）. Cell-mediated and humoral immunityresponse were checked at the11th week.Results：The fusion protein AMH was not only expressed stably in E. Coli but alsopurified well by Ni–NTA affinity chromatography; Splenic lymphocyte of C57BL/6mice immunized with AMH subunit vaccine delivered more IFN-γ to tuberculosisantigens such as Ag85B, Mpt64190-198, Hsp16.3and PPD. And there were high IgG1、IgG2a，IgG2a/IgG1in serum of AMH-vaccinated mice.Conclusion: AMH subunit vaccine induced intensively specific cellullar andhumoral immunologic response to tuberculosis antigens. It is necessary to furtherresearch for AMH as a promising candidate antigen of TB subunit vaccine. 2. Protective efficacy of multistage subunit vaccine as booster to enhance BCGprimed immunityObjective: The protective efficacy of the immunization and the direction wereobserved for subunit vaccines, consisting of multiple antigens from replication anddormant stages of Mycobacterium tuberculosis, to boost BCG.Ag85B-Mpt64190-198-Hsp16.3(AMH) with a latent antigen, Ag85B-Mpt64190-198-Mtb8.4（AMM） without latent antigen and a mixture of the both were used asboosters.Methods: BCG-immunized C57BL/6mice were boosted twice at the8th and10thweeks with Ag85B, AMH, AMM and AMM+AMH in the adjuvant composed ofdimethyl-dioctyldecyl ammonium bromide (DDA) and BCG polysaccharide nucleicacid （BCG-PSN） respectively. The level of antibody of serum and spleniclymphocyte-produced IFN-γ were detected at the14th week. Six weeks later aftermice were challenged with H37Rv at the22th week, the immunity reaction andprotective efficacy were determined.Results: Compare with BCG group, the Ag85B-specific IgG1and IgG2a inducedby subunit vaccines boosting were higher; the spleen cells from C57BL/6miceimmunized with fusion protein vaccines could generate higher IFN-γ with thestimulation of Ag85B and purified protein derivation (PPD) in vitro. After virulent M.tuberculosis H37Rv strain challenging, the percentage of gramulomas of all fusionprotein vaccines were not more than BCG group; colony forming units (CFUs) andthe positive degree of acid-fast were lower in lung of AMM+AMH group.Conclusion: AMM+AMH vaccine boosting induced specific cell-mediated andhumoral immune responses and improved the BCG-primed protective efficacy againstcaudal vein injection M. tuberculosis in mice. Fusion protein vaccines as boostersprovided protective efficacy but single protein. 3. The role of Interleukin-17for tuberculosis in human and miceObjective： To assess the effect and potential mechanisms of IL-17on TBdevelopment, the production of IL-17and IFN-γ from lymphocytes of BCG-primedand subunit vaccine-boost mice. Meanwhile, we provide a systematic review aboutthe production of IL-17and IFN-γ as well as IL-4in the infants or children vaccinatedwith BCG and in the patients with TB.Methods: The mice primed with BCG and boosted by MH and EAMM subunitvaccine two times at the12th and14th week were used to analyze its cell-mediatedimmunity boosting BCG at6weeks after the last immunization. The literaturerelevant with IL-17and IFN-γ production with or without IL-4on human TB wasretrieved from PubMed, EMBASE, Cochrane Library, BIOSIS Previews and theChina Biomedicine Literature Databases (CBM) using the search terms"Interleukin-17or Th17cells" and "Tuberculosis". The information of includedstudies, the production of IL-17and IFN-γ responding to antigens in the peripheralblood in vitro, was independently extracted by the first two researchers andsubsequently qualitatively analyzed.Results: The frequencies of IFN-γ and IL-17secreting lymphocytes from the miceprimed by BCG and boosted by subunit vaccines were higher than that immunizedwith BCG without boosting or PBS. Nine studies from a total of226retrievedpublications met the criteria. These included studies showed that BCG vaccinationinduced dramatically high level of IL-17similar to IFN-γ; in patients with active TB,the level of IL-17and IFN-γ were low while the level of IL-4was high; IL-17andIFN-γ had a similar trend of increase during the conversion from active TB to latentTB while IL-4inclined to decrease in this process.Conclusion: There are similar responses of IL-17and IFN-γ to antigens of Mtb inanimal model. In systematic review, IL-17acts as an effector molecule analogous toIFN-γ after BCG vaccination and Mtb infection to protect human beings against TB. The current findings do not support IL-17as an inducer of tissue damage intuberculosis.