Enhanced Immune Response and Protective Effects of Nano-chitosan-based DNA Vaccine Encoding T Cell Epitopes of Esat-6 and FL against <i>Mycobacterium Tuberculosis</i> Infection

<div><p>Development of a novel and effective vaccine against <i>Mycobacterium tuberculosis</i> (<i>M.tb</i>) is a challenging for preventing TB infection. In this study, a novel nanoparticle-based recombinant DNA vaccine was developed, which contains Esat-6 three T cell epitopes (Esat-6/3e) and fms-like tyrosine kinase 3 ligand (FL) genes (termed Esat-6/3e-FL), and was enveloped with chitosan (CS) nanoparticles (nano-chitosan). The immunologic and protective efficacy of the nano-chitosan-based DNA vaccine (termed nano-Esat-6/3e-FL) was assessed in C57BL/6 mice after intramuscular prime vaccination with the plasmids DNA and nasal boost with the Esat-6/3e peptides. The results showed that the immunized mice remarkably elicited enhanced T cell responses and protection against <i>M.tb H37Rv</i> challenge. These findings indicate that the nano-chitosan can significantly elevate the immunologic and protective effects of the DNA vaccine, and the nano-Esat-6/3e-FL is a useful vaccine for preventing <i>M.tb</i> infection in mice.</p></div

The protection efficacy against <i>M.tb</i> infection of the mice immunized with different vaccines.

<p>Mice vaccinated with different plasmids and boosted with Esat-6/3e peptides for two times were infected with 1×10⁶ bacilli of <i>M.tb</i> H37Rv for 4 weeks. Bacterial loads in the lungs (<b>A</b>) and spleens (<b>B</b>) of these mice were examined and the sections of the lung tissues from these mice were performed HE staining. Representative histologic changes (100×) depicted the lung tissue of the infected mice (<b>C</b>).</p

Sketch drawing of immunization procedure and challenge by <i>M.tb</i> H37Rv.

<p><b>A.</b> C57BL/6 mice were injected intramuscularly three times at 3-week intervals in both quadriceps muscles with different nano-plasmids, nano-chitosan, Esat-6/3e-FL or BCG. Those mice were then boosted with Esat-6/3e peptides through intranasal administration once a week for 2 weeks, then sacrificed at 1 week after the last peptide boost. <b>B.</b> Mice vaccinated with different nano-plasmids for 3 times and boosted with Esat-6/3e peptides for 2 times as above mentioned time schedules were infected with <i>M.tb</i> H37Rv the airway at 1 week after the last peptide boost, and then sacrificed at 4 weeks post <i>M.tb</i> H37Rv challenge.</p

The CTL activity of the mice elicited by nano-Esat-6/3e-FL or other plasmids.

<p>Splenocytes from naive mice pulsed with (CFSEhigh) or without (CFSElow) peptides were transferred into the immunized mice. The representative histograms (<b>A</b>) and percentages (<b>B</b>) of specific lysis in the immunized mice were compared. Data are one representative results from three performed experiments, and presented as the mean ± SD (<i>n</i> = 6). <i>NS: P</i>>0.05; *<i>P</i><0.05; ***<i>P</i><0.001 versus the mice treated with nano-chitosan, nano-pIRES or nano-FL plasmids; the other differences among nano-Esat-6/3e-FL, nano-Esat-6/3e, Esat-6/3e-FL, nano-Esat-6, nano-Esat-6-FL and BCG treatments were showed on the figure directly.</p

Splenocytes of the mice immunized with different plasmids were cultured and stimulated with Esat-6/3e (10 µg/ml) for 72 h, and the production of IFN-γ, IL-12, IL-4 and IL-10 in the splenocyte supernatants and expression of T-bet and Gata-3 in the splenocytes were measured by ELISA and Western blot, respectively.

<p><b>A.</b> The levels of Th1 type cytokines (IFN-γ and IL-12) and Th2 type cytokines (IL-4 and IL-10). <b>B.</b> The expression levels of T-bet or Gata-3 mRNA and protein. The representative graph and histograms in the immunized mice were showed. The experiments were performed in three times. All data are from one representative of three experiments, and presented as the mean value ± SD (n = 6). The statistics were performed with one-way ANOVA. <i>NS: P</i>>0.05; *<i>P</i><0.05 and ***<i>P</i><0.001 versus the mice with nano-chitosan, nano-pIRES and nano-FL immunization respectively. The other significant differences among the mice vaccinated with nano-Esat-6/3e-FL, nano-Esat-6/3e, Esat-6/3e-FL, nano-Esat-6, nano-Esat-6-FL and BCG were displayed on the figure directly.</p

Construction and identification of pIRES-Esat-6/3e-FL (namely Esat-6/3e-FL).

<p><b>A.</b> Schematic representation of Esat-6/3e-FL. <b>B.</b> The expression of his tag and FL proteins from Esat-6/3e or Esat-6/3e-FL plasmids was determined using Western blot after transfection of the plasmids into rat GMCs for 60 h (1: pIRES-Esat-6/3e. 2: pIRES-Esat-6/3e-FL. 3: pIRES-FL. 4: pIRES). <b>C.</b> The irregular solid spheres of nano-chitosan and nano-Esat-6/3e-FL plasmids under EM. <b>D.</b> The expression of his or FL proteins from the Esat-6/3e-FL plasmids enclosed with chitosan nanoparticle (nano-Esat-6/3e-FL) was confirmed using Western blot with anti-his or anti-FL antibody at 60 h after transfection into rat GMCs (1: MEM. 2: nano-pIRES. 3: nano-Esat-6/3e-FL).</p

The proliferation of the splenocytes and number of IFN-γ⁺ T cells of the mice immunized by nano-Esat-6/3e-FL.

<p><b>A.</b> Splenocytes were cultured and stimulated with the Esat-6/3e (10 µg/ml) for 72 h. The stimulation index (SI) of splenocytes in the mice treated with different vaccines was calculated to determine the proliferation activity. <b>B.</b> The numbers of IFN-γ⁺ T cells from the splenocytes were quantified by ELISPOT assays. Data are one representative of three experiments and presented as mean value ± SD (n = 6). The statistics were performed with one-way ANOVA. <i>NS: P</i>>0.05; ***<i>P</i><0.001 versus the mice treated with nano-chitosan, nano-pIRES and nano-FL plasmids. The differences among other treatments were shown directly on the figure.</p

The groups of mice given with different treatments.

<p>The groups of mice given with different treatments.</p

Proposed model of granzyme A-mediated suppression of intracellular mycobacterial growth by γ₉δ₂ T cells.

<p>Mycobacteria-infected macrophages present antigens to γ₉δ₂ T cells (1) which secrete granzyme A upon activation (2). Granzyme A in turn induces TNF-α production by infected and/or bystander macrophages (3) apparently independent of perforin. TNF-α activates intracellular mechanisms which alone or in concert with other unknown granzyme A-induced intracellular mechanisms suppress mycobacterial growth (4).</p

Granzyme A secretion by γ₉δ₂ T cells mediates inhibition of intracellular mycobacteria by induction of inflammatory responses in mycobacteria-infected macrophages.

<p><i>A</i>, The levels of granzyme A produced by γ₉δ₂ T cells are directly and highly correlated with inhibition of intracellular mycobacterial growth (r = 0.7564; p<0.0001). Supernatant levels of granzyme A, measured by CBA, were correlated with the level of mycobacterial growth inhibition observed in γ₉δ₂ T cell co-cultures with BCG-infected macrophages. <i>B</i>, Purified granzyme A induces the production of pro-inflammatory cytokines TNF-α and IL-1β by BCG-infected macrophages (*p<0.05, n = 4; statistical comparisons performed using Friedman's test). The indicated concentrations of purified granzyme A were added to BCG-infected monocytes for 3 days and the levels of TNF-α and IL-1β in the culture supernatants determined by CBA. <i>C-D</i>, Purified granzyme A alone can induce inhibition of intracellular mycobacterial growth in the absence of perforin. The indicated concentrations of granzyme A were added to BCG-infected (<i>C</i>) or <i>M. tuberculosis</i> H37Rv-infected (<i>D</i>) co-cultures for 3 days and the surviving bacteria quantified by H³-uridine incorporation and CFU counting. (*p<0.03; n = 6–12 by Wilcoxon matched pairs test comparing levels of mycobacterial intracellular growth in cultures with and without added purified granzyme A). Purified granzyme A had no direct effects on extracellular mycobacterial growth (data not shown). <i>E</i>, siRNA knockdown of granzyme A in γ₉δ₂ T cells reduces the inhibitory activity of γ₉δ₂ T cells for mycobacterial growth (*p<0.05; n = 3 by one-way ANOVA comparing levels of intracellular mycobacterial growth inhibition in cultures with or without γ₉δ₂ T cells producing granzyme A). γ₉δ₂ T cells transduced with the indicated siRNA-lentivirus targeting GzmA or a negative control (NC) were co-cultured with BCG-infected macrophages for 3 days and surviving bacteria quantified by H³-uridine incorporation.</p