Mucosal Immune Response Induced By Chitosan-oligosaccharide (COS)Formulated Poly-epitope-gene Vaccine And Its Protection Against Tuberculosis

Tuberculosis (TB) is one of the oldest contagious diseases in the globe and is coming back since the last20years. Up to one third of the world’s population, that is, over2.2billion individuals are estimated to harbor the causative agent Mycobacterium tuberculosis (MTB) in its dormant form. Globally, there are an estimated9.4million new cases of TB and it caused about1.8million deaths. In China, half the population is currently infected with MTB with1.5million new cases and0.13million deaths yearly. And the threat has been confounded by HIV/AIDS pandemic and multidrug-resistant and extensively drug-resistant TB (MDR-TB and XDR-TB). The current vaccine for TB, BCG, has proven relatively safe and protective against severe forms of childhood TB, but fails to afford protection against adolescent or adult pulmonary TB. With no profound preventive or therapeutic procedures, there would be an estimated30million new TB cases in China in the future10years. Therefore, alternative preventive new TB vaccines are desperately needed.Although our understanding of what constitutes protective immunity in TB is still lacking, Currently CD4+Thl lymphocytes and mononuclear phagocytes are at the center of TB vaccine development for their roles in inhibiting MTB replication. To this aim, in our previous work, genes coding for4predicted T cell epitopes from4well-defined MTB antigens, Ag85A, Ag85B,6-kDa early secretory antigen of T cells (ESAT-6) and10-kDa culture filtrate protein (CFP-10), were respectedly crafted in the scaffold of mycobacterial heat shock protein (HSP)65, using pcDNA3.1as vector, resulting in a multi-epitope DNA vaccine designated as ECANS (epitopes casted in a natural structure), or pHSP65pep in the current work. Intramuscular immunization of this DNA vaccine induced specific Thl immune response and serum IgG, contributing moderate protection against mycobacterial challenge.An increasing number of studies have shown that induction of mucosal immunity is required for effective protection against mucus-infectious pathogens, such as HIV, human papillomavirus and Mycobacterium species. It has been revealed that recruitment and activation of specific T cells in the pulmonic site was more effective to control MTB than that in the spleen. And more evidence is provided showing protective role of pulmonic activated Thl cells. Therefore, how to envoke an immune response at the mucus of MTB transmission is of great intrest.To guarantee the effective immunization of gene vaccine via mucosal route, proper delivery system is needed to circumvent the fast-antigen degradation of the mucus tract. Cationic polysaccharides, one of the non-viral gene delivery systems, have attracted increasing attention. Chitosan, deacetylated derivative of the natural polysaccharide chitin which is soluble in weak acidic environment, easily forms polyelectrolyte complexes with negatively charged DNA by electrostatic interaction, has been well-explored as a mucosal vaccine carrier. It helps to protect DNA from nuclease degradation and promotes mucosal permeability through opening the tight junctions between epithelial cells, therefore enhancing the uptake of DNA by mucus epithelial cells and M-cells. chitosan-DNA compound has been extensively used for eliciting mucosal immunity.The oligomer derivative of chitosan oligosaccharide (COS) is also utilized in this study. It is soluble due to shorter chain length and also ideal for mucosal gene carrier for the solubility in neutral envionment.In the current study, both chitosan and chitosan oligosaccharide (COS) were utilized to encapsulate the multi-epitope gene vaccine-pHSP65pep via intranasal immunization route in order to provoke the mucosal immune response. Pulmonary IFN-y+T cell frequency and specific SIgA level in the lung lavage were followed to evaluate the mucosal immune response; while systemic immune response including splenic IFN-γ+T cell frequency and specific serum IgG was also assessed for systemic immunity. After lethal challenge with lethal BCG, the pathology and bacterial loads in the lung tissue of mice was evaluated.Meanwhile, we observed that some mice receiving pHSP65immunization developed arthritis-like symptoms. The potential mechanism is associated with an autoimmune epitope resident in the H37Rv-originated HSP65180th-188th motif which has over-lapped antigenicity with a glucoprotein of mouse articular cartilage. Therefore, it is necessary to remove the autoimmunogenicity in the180th-188th motif of the gene carrier, HSP65.Altogether, following results have been obtained: 1. Genetic modification of the epitope gene carrier, M.TB-derived HSP65of the TB gene vaccine and confirmation of its immunological characteristics.1) To remove the autoimmunogenicity of180th-188thaa motif in HSP65, three anchor amino acids were site-directed mutated, including the2nd phenylalanine(Phe), the5th glutaminate(Gln) and the8th leucine to alanine (Ala) respectively designated as MT1, MT2, MT3; and plasmid with entire motif deletion was named MT4.2) BALB/c mice were intramuscularly immunized with50μg of DNA vaccine mutants4times biweekly.3) TB specific immunogenicity was first analyzed. We found that the TB specific IFN-y+CD4+Thl cell response induced by MT3/4remained as the immunogenicity of the unmodified gene vaccine pHSP65pep. While that induced by MT1/2was reduced.4) The autoimmune response and pathology was then evaluated3months later. Mice immunized with pHSP65showed weak symptom of arthritis outlookly and their serum anti-dsDNA antibody level also seemed a little increased; HE staining and the immune complex deposition assay of the renal tissue confirmed glomerulonephritis in original pHSP65-immunized mice., while no autoimmune symptoms were observed in MT3and MT4DNA-treated mice.5) Finally,180th-188thaa deletion mutant (MT-4) of pHSP65pep was chosen as the gene vaccine used in the subsequent experiment as it kept its TB-specific immunogenicity while lost its autoimmunogenicity.6) Utilizing of PET-32a(+) system, we expressed and purified HSP65in vitro demonstrated by western blot assay.2. The physical and chemical properties of COS-pHSP65pep mucosal gene vaccine and its ability to induce specific mucosal immune response and its protective effects against TB.In order to induce TB specific mucosal immunity, we adopted two natural polysaccharides originated from chitin, chitosan and COS, to encapsulate pHSP65pep DNA vaccine respectively. Their physical and chemical properties and their immunogenicity and immune protective effect were evaluated.1)0.02%of chitosan solution (PH5.7) and0.4%COS solution were combined with pHSP65pep DNA respectively to form chitosan-DNA(chi-DNA) and COS-DNA particles respectively which turned to be spheroidal particles with uniform size by SEM(scanning electronic microscope) and DLS (dynamic light scattering) assay; the particle size of chi-DNA and COS-DNA were200-400nm and300-500nm respectively.2) Agarose gel electrophoresis confirmed that COS and chitosan could protect DNA from DNase I digestion.3) Using luciferase as reporter gene, the transfection efficiency of chitosan-and COS-DNA was evaluated in HEK293cells. It demonstrated that COS/chitosan can enhance the transfection efficacy of DNA.4) Female BALB/c mice were intranasally immunized with50u g DNA of chi-DNA or COS-DNA4times biweekly. Naked pHSP65pep and pHSP65as well as BCG were used as control.4weeks following the final immunization, mice were challenged with lethal dose of BCG.5) Compared with naked gene vaccines, mucosal gene vaccine maintained their ability to induce systemic immune responses. In terms of specific serum IgG, COS-pHSP65pep and chi-pHSP65pep induced serum IgG titers as15000and12000respectively, similar to that of pHSP65pep group but lower than BCG group (320000). The subclass of IgG was IgG2a. Regarding splenic Thl response evaluated by intracellular staining, the IFN-y+CD4+T(Thl) frequency induced by COS-pHSP65pep was2.88%, similar to that by chi-DNA, but significantly higher than naked DNA(1.22%) and BCG(0.85%). ELISPOT assay again confirmed that COS-pHSP65pep induced specific180SFC/106splenic lymphocytes, being the best one. Therefore, COS-DNA vaccine induced systemic immune response against TB.6) Then pulmonary mucosal immune response was evaluated including pulmonary lavage SIgA and pulmonary Thl response. DNA vaccine alone was poor to induce SIgA, while chitosan or COS formulated DNA vaccine induced high level of SIgA, with titers of1500and1000. The avidity index if SIgA induced by COS-DNA was50, significantly higher than all other groups.7) Regarding pulmonary T cell response, IFN-y+CD4+T(Thl) and IFN-y+CD8+T(CTL) frequency induced by COS-pHSP65pep were4.53%and7.25%, slightly higher than that of chi-pHSP65pep(Thl,3.87%; CTL,5.13%), but significantly higher than that of pHSP65pep(Thl,1.75%; CTL,2.68%) and BCG(Th1,2.05%; CTL,3.41%). ELISPOT assay showed that220and120SFC/106pulmonary lymphocytes were induced by COS-pHSP65pep and chi-pHSP65pep, significantly higher than that by pHSP65pep (50SFC/106). Collectively, intranasally immunization with COS-pHSP65pep significantly enhanced pulmonary mucosal immune response against TB.8) After lethal BCG challenge, it was found that pcDNA3.1-treated mice developed large-area inflammatory lesion in lung tissue. Naked TB gene vaccine provided moderate protection showing small lesion and swollen alveolar cell wall; COS-pHSP65pep immunized mice showed least pathology with only minimal inflammatory lesions. Mock and naked DNA vaccine still left mice with high bacterial loads (105CFU). chitosan-DNA immunization reduced that to101CFU; and COS-DNA-treated mice had only100CFU MTB in tissues. All above data demonstrated that mucosal delivery media like COS could endow DNA vaccine with the ability to induce specific pulmonary SIgA and Thl response in the lung tissue which would confer more protection against TB.3. The BCG prime-COS-DNA boost strategy in inducing specific mucosal immune response and its protective effects against TB.Considering the situation of world-wide BCG inoculation, a BCG prime-chi/COS-DNA mucosal vaccine boost strategy was further adopted.1) BALB/c mice were subcutaneously immunized with1×104CFU BCG, then with3dosed of50μg DNA biweekly. Naked DNA and BCG boost were used as control.4weeks following the final immunization, BCG challenge was performed.2) Regarding systemic immune response, BCG priming strategy significantly elevated the serum IgG level. The IgG titers induced by COS-DNA and chi-DNA were about105, slightly higher than naked DNA (50000). The splenic Thl and CTL frequency induced by COS-DNA boosts were2.15%and1.57%, higher than that of other groups,.3) Pulmonary lavage SIgA titers induced by COS-pHSP65pep or chi-pHSP65pep boosts were1300and700. Again, the COS-DNA boosts enhanced avidity of Ab. Second, in terms of pulmonary Thl and CTL responses, the IFN-y+CD4+T(Thl) and IFN-γ+CD8+T(CTL) cells induced by COS-pHSP65pep boosts were9.32%and9.65%, significantly higher than that of pHSP65pep (Thl,6.43%; CTL,6.78%). ELISPOT results showed that COS-pHSP65pep boost induced260SFC/106pulmonary lymphocytes, significantly higher than that of DNA vaccine. Collectively, BCG prime combined with mucosal vaccine boost strategy significantly enhanced pulmonary T cell immune response. 4) After BCG challenge, pHSP65pep immunization provided moderate protection. While minimal inflammatory lesion was provided by COS-DNA and the bacterial load decreased to the100CFU. It was again demonstrated that strategy of BCG prime-mucosal gene vaccine boosts was good at inducing mucosal immune response which contributed to the improved protective effect against TB.In this study, on the basis of our previously constructed multi-epitope gene vaccine against TB, pHSP65pep, we first modified the HSP65gene to remove the potential auto-immunogenic motif. Then, in order to induce mucosal immune response, two mucosal delivery media, COS and chitosan, were adopted to formulate gene vaccine resulting in COS-DNA and chi-DNA mucosal vaccine. After intranasal immunization, it was demonstrated that COS-DNA and chi-DNA vaccines maintained the ability of gene vaccine to induce systemic immunity manifested by serum IgG and splenic CD4+Th1response; while significantly enhanced specific mucosal immunity manifested by lavage SIgA and pulmonic Thl response. COS-DNA was proved to be more effective to induce mucosal immunity than chi-DNA, and resulted in more effective protection against BCG challenge. This was further confirmed by a BCG prime-mucosal vaccine boost strategy in protection against TB challenge. The significance of this study is to confirm that mucosal delivery system is helpful to elevate mucosal immunogenicity of gene vaccine; and the magnitude of mucosal immunity is closely associated with immunoprotection against TB. Our result could provide two novel potential mucosal TB vaccines as well as two useful mucosal delivery platforms for gene vaccine to enhance mucosal immunity which is critical for early control of mucus-infectious pathogen.