An Alveoli-targeting Poly-epitope TB Vaccine

Tuberculosis (TB) is a potentially fatal contagious disease caused by infection of Mycobacterium tuberculosis (M.tb) that can affect almost any part of the body but mainly the lungs. Following inhalation of contaminated aerosols, M.Tuberculosis moves to the lower respiratory tract where it is recognized and engulfed by alveolar macrophages. Various lymphocytes are recruited to control the infection but finally lead to formation and maturation of the hallmark of the disease:tuberculous granuloma and then pulmonary tuberculosis. MTB can establish a long-term latent TB infection (LTBI) for decades without overt disease in 95% infected population; with only 5-10% will progress to overt, active tuberculous disease depending on host and environmental factors. As one of the most lethal infectious disease in nineteen and twentieth century, it was not controlled until 1919 when French bacteriologists Calmette and Guerin developd an attenuated strain of Mycobacterium bovis, Bacillus Calmette-Guerin (BCG), after 230 generation’s culture.which was proved to be safe and effective for prevention of human tuberculosis, With the isolation of streptomycin (1944) as the first antibiotic agent against M. tb and the development of mycobactericidal drugs such as isoniazid (1952) and rifampin (1970), TB cases are significantly reduced in the 1980s. However, with the rise of drug-resistant strains and the emergency of HIV, there is a great resurgence of TB in the late 1980s. It is estimated that one third of the world’s population is latently infected with Mtb. With 1.5 million deaths (including 0.36 million AIDS) and 9.0 million (India and China accounte for 24% and 11%) new cases in 2013 (WHO tuberculosis report 2014), TB remains a global health care problem representing one of the three infectious diseases world-wide (HIV and Malaria). Control of the global TB epidemic has been impaired by the lack of effective vaccines, the emergency of drug-resistant forms of Mtb and the lack of sensitive and rapid diagnostics. Neonatal immunization with BCG, the only licensed TB vaccine, reduces TB incidence in children but its protective efficacy against pulmonary TB varies with no apparent protection in aldult population, and mechanisms of its immune protection is poorly known, highlighting the challenges of developing novel vaccines against M.tb that requires cellular immunity to contain an established infection.Successful rational design of effective vaccines against M. tb has been hampered by the lack of definitive immunological correlates of protection. Unlike the available human vaccines which protect people mainly by induction of neutralizing Abs, evidence supports an important role for T cell-mediated responses in eliciting protective immunity against TB. CD4+ T cells and CD4+ T-derived gamma interferon (IFN-γ) has long been considered required for host resistance to TB. IFN-y activates macrophages to kill intracellular bacteria by activating downstream antimicrobial effector pathways including iNOS, IFN-γ inducible GTPases, phagosomal maturation and acidification, autophagy, and Vitamin D receptor signaling. However, most people with active TB have no obvious defects in T cell compartment and generate vivid M. tuberculosis-specific IFN-γ responses; Patients whose T cells produce greater amounts of IFN-γ are more likely to progress to active disease than patients with weaker responses. And IFN-γ KO mice would generate an automatic autoimmune disease after repeated infections indicating the regulatory role of IFN-γ. Therefore T cell functions other than IFN-y production can mediate protection and the best quantity of IFN-γ in local environment might be important. Apart from IFN-γ cytokines TNF-α, GM-CSF, IL-1β, IL-2 and vitamins C/D are all implicated as mediators that activate macrophages to control M. tb growth. TNF-γ up-regulation is important to stimulate apoptosis of infected macrophage and the successful elimination of the invading mycobacteria. So it has been proposed that Mtb-specific multifunctional/polyfunctional CD4+T cells producing a combination of IFN-γ, IL-2, and/or TNF-α should be used in vaccine studies as a measurable immune parameter, reflecting activity of a vaccine and potentially predicting protection. Although the role of CD8+T cells in TB is less clear, they are generally considered to contribute to optimal immunity and protection by lysing infected macrophages and releasing TB antigens. Although serum IgG might be ineffective to control TB directly, B cells can modulate immune responses to M. tb through restriction of free bacterium, antigen-presentation, ADCC, production of cytokines at the site of infection and by regulating T immune response.In recent years, it has become clear that a large proportion of immune cells reside outside lymphoid organs such as lung and intestine and such tissue-resident mucosal immunity with specialized phenotypic and functional properties of T/B cells as well as innate cells have an important role in protection against infection. The lung is the portal of entry for Mtb and animal experimental evidence indicates that local immune defense mechanisms might be crucial for protective immunity. TB antigen-specific T cells are found in both lung interstitial tissue and in BAL. Both CD4+ and CD8+ T cells can mediate early inhibition of Mtb growth after challenge. It indicates that activated lung T cells located at the site of entry of inhaled mycobacteria are able to inhibit growth of mycobacteria early after Mtb infection. Meanwhile, the early activation of local macrophages and production of IFN-γ/TNF-α, the early infiltration of neutrophils, the activated Th1, Th17 and CTL response in the acute phase, the late regulatory response, all these locally initiated cellular immune responses have an important role in the early control of Mtb infection after exposure, although definitive evidence for their role in mediating protection in humans has yet to be obtained.Secretory IgA (SIgA) is the predominant Ig isotype in mucosal tissue, and is believed to be involved in defense against viral and bacterial infections at these sites. Owing to highly efficient neutralization activity of dimeric IgA compared to the monomeric form of IgG, SIgA not only control extracellular pathogens in body fluids but also can neutralize intracellular pathogen after translocation into epithelial cells by pIgR receptor. Recent study revealed that IgA is carried inside cells during infection of adenovirus and recognized by the newly identified cytosolic Fc receptor, tripartite motifcontaining protein 21 (TRIM21), leading to destruction of virus by the proteasome and immune activation suggesting and enlarging the role of IgA in immune protection against mucosal infection. To better control of TB, it has been suggested that parenteral priming (BCG) and mucosal boosting might have the advantage that local lung immunity would control pathogen growth in the lungs, whereas systemic immunity might control growth of organisms that escape to other tissues. Delivery of appropriate innate signals to the lung innate immune system is also critical for induction of effective local immunity.Taken together, to develop a novel TB vaccine, we highlight the induction of poly-antigen-specifc T immune response, poly-functional T response and mucosal immune response. To facilitate muti-functional T immune response, strategy of poly-epitope vaccine is adopted by our group to couple multiple CD4+ and CD8+ T cell epitopes into one vaccine. To induce mucosal response, ideal mucosal delivery system should be utilized to help local immunization of poly-epitope vaccine, and a targeting adjuvant is further adjusted to this mucosal TB vaccine to achieve the best immunogenic and protective effect..For the molecular designing of multi-epitope vaccine, an "poly epitopes-grafted in protein scaffold, PES" strategy is used by our group by genetically grafting 5 various T cell epitopes elaborately elicited from Ag85B, ESAT-6, CFP-10,PPE19 and PE25 antigen of M.tb into various sites of H37Rv-heat shock protein 65 (HSP65) scaffold. The innovations of this designing lies in:1) multiple TB epitopes and protein are coupled into one vaccine; 2) HSP65 has potent immunogenicity; 3) The optimal spatial conformation of epitopes makes more accurately and efficiently recognition and digestion of protesomes after they are uptaken within APCs; 4) new B cell epitopes and T cell epitopes may be generated by this poly epitope-protein fusion. HSP65 is selected as protein vector because it combines the ideal properties of antigen carriage (chaperoning) and APC targeting as well as activation. HSP functions as chaperones by binding intracellular polypeptide chains and misfolded proteins, preventing aggregation and supporting folding and transport. More importantly, HSP modulate innate immune system by binding to TLRs on surface of APCs, triggerring maturation of DC and secretion of pro-inflammatory cytokines and chemokines. HSP is so called ’chaperokine’ meaning both chaperone and cytokine activity of HSP. Resent study suggests that HSP65 derived from M.tb interacts with TLR2/4 expressed on DC and promotes DC release of RANTES indicating its adjuvant potential. Meanwhile, HSP65 itself has a potent immunogenicity to protect against Mtb infection. Taken together, M.tb derived HSP65 is used as the scaffold protein of our vaccine, poly-T cell epitopes were then grafted into non-functional domains of HSP65, generating a novel poly-T epitope TB DNA vaccine, designated as pPES vaccine.Chitosan is adopted as mucosal delivery carrier for pPES vaccine. As a cationic polysaccharides, chitosan is famous for and long used as non-viral gene delivery system. By encapsulation DNA into nanoparticle componds, chitosan helps to enhance the integrity of DNA vaccine on the mucosal surface and the uptake of vaccines by mucosal APCs. Our group have previously developed chitosan-DNA mucosal vaccine against Mtb and CVB3, and independently improved anti-infection protection via significant elevation of mucosal T and SIgA responses.Taken together, strong evidence supports an important role for mucosal T cell-responses as well as SIgA in protection against M.tb. Thus gives us inspiration to integrate both mucosal and systemic immune activation into our rational design of novel TB vaccines. To this aim, in the current study, a poly-T-epitope DNA vaccine in HSP65 scaffold was designed and further packaged into a mucosal vaccine by chitosan (CS) delivery. Following intranasal immunization of this mucosal TB vaccine for 4 times, induction of both systemic and mucosal immune response is evidenced. The protection is evaluated after lethal BCG challenge, To further improve the mucosal targeting and antigen uptake efficienty by APCs in the lung, chitosan is mannosylated (MCS) to facilitate more efficient targeting of vaccine to mannose R-rich macrophages and DCs in the lung. It is found that the immune responses as well as immune-protection are greatly enhanced. Our study highlights a novel mucosal vaccine designing platform to coupling multi-immunodominant TB epitopes and TB proteins together and to facilitate its efficient respiratory tract delivery and induction of mucosal immunity in the respiratory tract and lung, thus making local control of TB infection more potent and sufficient.Part 1 Designing, construction, expression, and immunogenicity characterization of poly-epitope DNA vaccine1. Molecular designing of poly-epitope DNA vaccineFive T cell epitopes were selected from H37Rv strain of M.tb, including four CD4+Thl epitopes (ESAT-61-20, Ag85B241-255, PPE25241-255, PE194-18) and one CD8+CTL epitope (MTB 10.43-11). According to the principle of homogeneous structure compatible with each-other, ESAT-61-20、Ag85B241-255、MTB10.43-11、PPE25241-255-AAY- PE194-18 were genetically grafted into 146aa,151aa,157aa,395aa sites of H37Rv-derived HSP65 protein individually.The Eukaryotic expression plasmid of pHSP65 was constructed. Through primer overlap extension PCR, HSP65 gene containing five T cell epitopes (PES) were obtained and then cloned into pcDNA3.1 vector, getting pPES plasmid. Enzyme digestion and DNA sequencing confirmed the construction. STAR analysis if secondary structure demonstrated that the secondary structure of PES was very similar to HSP65.As the control of pPES, plasmids with only 5-epitopes (p5pep), HSP65-series connected to 5 epitopes (pHSP65-pep) were also constructed.2. Purification of HSP65 protein.pET32a-HSP65 plasmid was constructed and then transformed to BL21. After IPTG induction, HSP65 protein within inclusion body was purified by freezing method. 90% purity of HSP65 was confirmed and it could be recognized by mouse anti-HSP65 antibody as confirmed by Western Blot assay.3. The eukaryotic expression efficiency of pPES plasmid.To verify the transcriptional efficiency of pPES plasmid in vitro,293T cells were transfected with pPES or pHSP65, electrophoresed, bloted to PVDF membrane and detected by anti-HSP65 mAb. It is confirmed that HSP65 protein (65KD) and PES protein (75KD) could be expressed in eukaryotic cells.4. Specific T cell response induced by pPES vaccine.To confirm the immunogenicity of pPES vaccine, and its priority over pHSP65-pep and pPEP vaccine, we immunized female C57BL/6 mice intramuscularly with pcDNA3.1, pHSP65, pPES, p5pep, pHSP65-pep for four times in 10days interval,50μg DNA each time, total 200μg DNA dose.10 days after last immunization, splenocytes were isolated and stimulated with single peptides, mixed peptides, HSP65 protein and H37Rv antigen, then ELISPOT assay was performed to investigate specific IFN-γ+T cell response. p5pep vaccine could hardly induce specific T cell responses; pHSP65 vaccine could induce HSP65-specific IFN-γ+T cells; pHSP65-pep construction induced moderate T cell response; while only pPES injection generated significantly increased numbers of IFN-γ+T cells to 5 epitope and mixed epitope, and to ESAT-61-20 which was absent in BCG. These data suggested that pPES vaccine designing is indeed superior than other construction to induce IFN-γ+T response.5. Protective efficacy of pPES vaccine.To determine the protective potential of the pPES vaccination,4 weeks after lethal challenge with 1×107 CFU BCG, tissue pathology was examined. Extensive infiltration and tissue injury was found in lung tissues of mice treated with pcDNA3.1 and p5pep, indicating that epitopes connected in series did not provide protection. Reduced thickening degree of alveolar septum could. Be seen in pHSP65-immunized mice; Less inflammation and improved alveolar morphology was observed in pHSP65-pep-treated mice compared to the above 3 vaccines. The best protection was found in pPES-immunized mice, with least pulmonary inflammation. In accordance with that, a significant reduction of bacteria loads in lung (1065 to 1054) and spleen (105.5 to 104.6) was confirmed in pPES-treated mice. It suggested that the poly-epitope DNA vaccine, pPES is more protective than other constructions.Part 2 Mucosal immune response induced by chitosan formulated DNA vaccine and its protective effects1. Specific Ab response elicited by intranasal immunization with chitosan-pPES (CS-pPES)To assess if intranasal immunization with chitosan-pPES would induce HSP65-specific antibody, C57BL/6 mice were i.n. immunized with chitosan-formulated, DNA vaccine (CS-vector, CS-pHSP65, CS-pPES) 4 times biweekly, totally 200μg DNA. One dose(1×106CFU) of BCG i.d immunization was used as positive control. No detectable serum IgG was detected in mice receiving CS-vector. In other groups, serum IgG gradually increased from week 6, reaching peak at week 8. Level of serum IgG induced by CS-pPES was significantly higher than that by CS-vector and CS-pHSP65, but less than BCG indicating that epitope grafing did not affect the immunogenicity of the HSP65 carrier protein..To detect whether CS-pPES would induce HSP65-specific mucosal SIgA, bronchoalveolar lavage (BAL) was collected two weeks after final immunization. A significant higher level of BAL SIgA was seen in mice receiving CS-pPES than other vaccines, except BCG. These results indicated CS-pPES intranasal immunization induced both HSP65 specific serum IgG and mucosal SIgA.2. Specific systemic (spleen) T cell response elicited by intranasal immunization with CS-pPESTo investigate specific T immune responses by intranasal immunization with CS-pPES, spleen lymphocytes were collected and stimulated with selected peptides, mixed peptides, HSP65 protein and H37Rv then subjected to IFN-y+ELISPOT assay. Results showed that CS-pPES induced higher IFN-γ+T response to all TB antigens than other vaccine did (p<0.05), which was a little lower than those of BCG. Only CS-pPES immunized splenocytes responded to ESAT-61-2o.FACS assay was used to access multifunctional CD4+ and CD8+T cell response. CS-pPES induced H37Rv-specific IFN-γ+, IL-2+ and TNF-α+CD4+ and CD8+T cell response, which were significantly higher than CS-pHSP65 and CS-vector (p<0.05).but not higher than BCG did, suggesting i.n. immunization with CS-pPES effectively induced multifunctional CD4+ and CD8+T response in spleen.3. Specific mucosal (lung) T response elicited by CS-pPESPulmonary monocytes were isolated by Collagenase digestion two weeks after final immunization, IFN-γ+ ELISPOT assay showed that CS-pPES group effectively induced specific IFN-γ+T cell response to almost all TB antigen (except PPE25241-255 and PE194-18), which was significantly higher than CS-pHSP65 and CS-vector did but less than BCG did. FACS assay revealed that IFN-γ+, IL-2+ and TNF-α+ multifunctional pulmonary CD4+ and CD8+T frequency induced by CS-pPES was significantly higher than CS-pHSP65, CS-vector and BCG did (p<0.05)4. Protection conferred by CS-pPES.Four weeks after leathal BCG challenge, the histopathological assay indicated that: BCG vaccination showed best protection; while the lung pathology condition of CS-pPES-treated mice was significantly better than that of other groups, although there was still alveolar septum thickening and moderated immune-inflammation. A significant reduction of bacteria loads in the lung (105.8to 104.6) and spleen (105.1 to 1044) also suggested that CS-pPES could effectively protect against Mtb local challenge, although not as good as BCG did.In conclusion, we constructed a poly-epitope DNA vaccine (pPES) containing 5 T cell epitopes and a protein from M.tb. Formulation of this DNA vaccine with chitosan significantly enhanced pulmonary IFN-γ+, IL-2+, TNF-α+T cell response and BAL SIgA response of naked DNA vaccine; although the splenic T cell response and protection effects seemed lower than thosed induced by BCG. Next, we would use a novel adjuvant to further optimize and improve the effect of this mucosal vaccine.Part 3 Increased mucosal response and protection of DNA vaccine by mannosylated chitosanTo further enhance the mucosal immunogenicity of pPES vaccine, we modified chitosan with mannose aiming to targeting mannose R-rich alveolar macrophages and DC in the lung; then made MCS-pPES particles. The characterization, immunogenicity and protection effects of MCS-pPES were detected in mice.1. Characterization of MCS-DNA and CS-DNA particleUniform MCS-DNA and CS-DNA complex were obtained by co-acervation of chitosan or mannosylated chitosan (MCS) with pPES. Transmission electron microscopy confirmed that freshly synthesized MCS-DNA complexes were nearly spherical and was approximately 250-400 nm in size which was a little larger than CS-DNA particle. Their encapsulation rate was approximately 100% and could protect plasmid DNA from DNase I degradation.2. Specific Ab response elicited by intranasal immunization with MCS-pPESMice were i.n. immunized with MCS-vector, CS-vector, pPES, MCS-pPES and CS-pPES comprising 50μg DNA for 4 times at 2 week intervals, BCG as positive control. The HSP65-specific serum IgG titer in MCS-DNA -treated mice was nearly 1:4000. significantly higher than that in mice immunized with CS-DNA (1:2800) and DNA. The BAL SIgA titer of MCS-DNA group reached 1:1100, significantly higher than that of CS-DNA and BCG (1: 600, p<0.05) group. These results indicated that chitosan mannosylation significantly increased SIgA response of pPES vaccine.3. Systemic (spleen) T response elicited by intranasal immunization with MCS-pPES.ELISPOT assay showed that MCS-pPES induced significantly higher level of IFN-γ+T response than that of CS-pPES. and similar to that of BCG. More importantly. MCS-pPES induced IFN-γ+ T response to ESAT-61-20.which could not be induced by BCG.FACS assay showed that MCS-pPES could significantly enhance H37Rv specific multifunctional T cell response (IFN-γ+CD4+/CD8+T, IL-2+CD8+T. TNFα+CD4+T) compared to CS-pPES vaccine, which was slightly lower than those of BCG. The level of IFN-γ+CD4+/CD8+T, IL-2+CD4+T. TNFα+CD4+/CD8+T response of MCS-DNA to 5 peptide mixture was significantly higher than that CS-pPES (p<0.05):and TNFα+T response even significantly higher than BCG (p<0.05), suggesting i.n. immunization with MCS-pPES significantly enhanced H37Rv and mixed-peptide-specific multifunctional CD4+ and CD8+T response in spleen.4. Mucosal (lung) T response elicited by MCS-pPES.ELISPOT assay showed that MCS-pPES significantly enhanced IFN-γ+T cell response than CS-pPES and naked DNA. And the T response to 5 peptides and HSP65 protein was even significantly higher than that of BCG (p<0.05)FACS assay showed the pulmonary multifunctional H37Rv specific T cell response (IFN-γ+CD4+/CD8+T、TNF-α+CD4+T) induced by MCS-pPES was significantly higher than CS-pPES and control (p<0.05); approximate to BCG’s effect.5 epitopes-specific pulmonary IFN-γ+CD4+/CD8+T, IL-2+CD4+T. TNFα+CD4+/CD8+T responses of MCS-DNA were all significantly higher than CS-pPES, and than BCG group (p<0.05). These data indicated that mannosylated chitosan encapsulation of pPES could significantly increase pulomonary multifunctional T response, better than not only CS-pPES but also BCG vaccine.5. Protection conferred by MCS-pPES vaccineNaked DNA provided moderate protection. MCS-pPES i.n. immunization resulted in significantly reduced inflammation, improved alveolar morphology and lower bacterial burden in the lung tissue; which was similar to effect of BCG immunization and significantly better than effect of CS-pPES vaccine.Above all, to improve immunogenicity and protective efficacy of chitosan-pPES vaccine, mannosylated chitosan was used as mucosal delivery system and adjuvant. It was confirmed that MCS could significantly enhance serum IgG, BAL SIgA, splenic and pulmonary IFN-y+T cell response of DNA vaccine formulated with chitosan, Among those, BAL SIgA level, pulmonary 5 peptides specific IFN-y+T, IFN-γ+CD4+/CD8+T, IL-2+CD4+T and TNFa+CD4+/CD8+T response were all significantly higher than CS-pPES and BCG, indicating enhanced mucosal response was associated improved protection.Part 4 Mechanism of mannosylated chitosan for targeting alveolar macrophageTo investigate the mechanism of mannosylated chitosan to enhance mucosal response, we used HSP65 and LacZ as reporter antigen to detecte the delivery and expression efficiency of pPES vaccine formulated with CS and MCS in the lung tissue. Confocal assay was used to assess the alveolar targeting characteristics of MCS.1. The expression efficiency of MCS-DNA in lung.β-galactosidase as reporter antigen:2 days after after i.n. immunization of mice with 50μg CS-pLacZ and MCS-pLacZ, tissue expression of LacZ in pulmonary alveoli was evaluated with X-Gal staining. Naked DNA could hardly express in the alveoli, CS-pLacZ immunization led to moderate β-galactosidase expression in alveolar interstitial. While MCS-pLacZ significantly enhanced protein expression in alveoli and alveolar interstitial, indicating both chitosan and mannosylated chitosan could enhance local expression of DNA, while mannosylated chitosan was the best one.HSP65 as reporter antigen:The frozen section of lung tissue was stained with HSP65 specific antibody and found that both CS and MCS could increase HSP65 expression in alveolar interstitial and alveoli, while MCS-pHSP65 was much better indicating again among chitosan and mannosylated chitosan which enhance local antigen expression MCS was the best one.2. FITC tracing the targeting effect of MCS to alveolar macrophageTo detect whether mannosylated chitosan could efficiently target to alveolar macrophage, we labeled mannosylated chitosan and chitosan with FITC respectively, then delivery labeled CS/MCS-pPES to lung by intratracheal instillation.24hrs later, frozen sections were stained with MOMA-2 Ab (monocyte and macrophage) and DAPI. Confocal laser scanning microscopy revealed that beyond weak fluorescence of FITC-CS-DNA in alveolar macrophages, MCS significantly enhanced the fluorescence reaching alveolar macrophages indicating mannosylated chitosan displayed better targeting property than chitosan to alveolar macrophages.3. FITC tracing the macrophages uptaking of MCS-DNATo further exploring the vaccine-uptake ability of macrophages, the subcellular distribution of FITC-MCS or CS-DNA was observed with a confocal laser scanning microscope after incubation nanoparticles with Raw264.7 for 0.5 h. Results showed that the uptaking of MCS-DNA was significantly enhanced compared to CS-DNA. Overall, mannosylation of chitosan carrier could significantly enhance the uptaking of DNA by macrophages.In conclusion, we designed and constructed a novel poly T-epitope TB DNA vaccine grafted in HSP65 scaffold, named pPES. We confirmed that this kind of poly epitope-protein coupling and vaccine construction was better than epitopes connected in series or HSP65 connection with epitopes in series. To enhance TB antigen-specific mucosal immunity (BAL SIgA pulmonary T resonse), chitosan were formulated with pPES for intranasal immunization. CS-DNA significantly enhanced BAL SIgA and pulmonary IFN-y+T response, which could not be induced by naked DNA. However the overall level of CS-DNA-induced mucosal immunity was still not better than BCG. To improve this, chitosan was mannosylated to generated MCS-pPES mucosal vaccine. MCS-pPES vaccine significantly enhanced both systemic and mucosal immune response of CS-DNA.; and the level of BAL SIgA,5 peptides-specific IFN-γ+T, IL-2+CD4+T and TNFα+CD4+/CD8+T responses were significantly higher than BCG positive control, which resulted in significantly improved protection. Finally we demonstrated that mannosylated chitosan enhanced mucosal immunogenicity of DNA vaccine by increasing local Ag expression, alveolar macrophage targeting and macrophage uptaking efficiency. This novel mucosal delivery carrier and adjuvant harnesses both alveoli-targeting and APC function stimulation, making it an ideal mucosal delivery system for DNA vaccine.This study provides a novel designing platform for poly-epitope TB vaccine and a novel mucosal delivery system for TB vaccine; meanwhile, it adds new data to support the association of immunoprotection against TB with mucosal immune response., More potent mucosal immunity and protection efficiency is hoped to be achieved if more immunodominant TB epitopes as well as proteins would be identified and grafted into pPES vaccine designing platform in the future.