A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector Cells with Higher Protective Efficacy against Tuberculosis

<div><p>Tuberculosis (TB) is an infectious disease caused by <i>Mycobacterium tuberculosis</i> (Mtb) that is a major public health problem. The vaccine used for TB prevention is <i>Mycobacterium bovis</i> bacillus Calmette-Guérin (BCG), which provides variable efficacy in protecting against pulmonary TB among adults. Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG. Here we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein (CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and evaluated its immunogenicity and protection in a murine model of infection. The stability of the vaccine <i>in vivo</i> was maintained for up to 20 days post-vaccination. rBCG-CMX was efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO) production. Following mouse immunization, this vaccine induced a specific immune response in cells from lungs and spleen to the fusion protein and to each of the component recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination. This study describes the creation of a new promising vaccine for TB that we hope will be used in further studies to address its safety before proceeding to clinical trials.</p></div

Representative lung pathology of Balb/c mice after challenge.

<p>Vaccinated mice were challenged <i>i.v</i>. with 10⁵ CFU of virulent <i>M. tuberculosis</i> H37Rv strain. Forty-five days after infection, lung tissue sections from different vaccine groups were harvested. Images are representative of two distinct experiments. HE staining is shown with 20X magnification. (A) Unvaccinated group. Black arrowheads: Foamy macrophages. (B) BCG-vaccinated group. (C) rBCG-CMX vaccinated group. (D) Histological score of the lesion area from three representative fields obtained by AxioVision 4.9.1 software, through ratio of lesioned and total field area. Data are presented as percentages (%).</p

Plasmid construction and CMX expression for three different rBCG-CMX vaccines.

<p>(A) PCR products corresponding to the CMX fusion gene, CMX (∼860 bp), from all three plasmid constructions and their respective empty controls: pLA71, pLA73 and pMIP12. M: molecular weight marker; NC: negative control reaction, using water; BCG: DNA from BCG-Moreau; rBCG transformed with pLA71, pLA73 and pMIP12, with (CMX) or without (empty) the fusion gene; PC: positive control reaction. (B) Analysis of CMX expression in rBCG-pLA71/CMX. Western blot of BCG transformants containing pLA71/CMX or empty vector using polyclonal antibody produced against rCMX. M: molecular mass marker; CMX: purified recombinant CMX; pLA71/CMX: rBCG with plasmid pLA71/CMX; pLA71: rBCG with plasmid pLA71.</p

Levels of CD4⁺IL-17⁺ T cells induced by <i>ex vivo</i> stimulation with recombinant Ag85, MPT51, and HspX.

<p>Thirty days after vaccination, lung and spleen suspensions were stimulated <i>ex</i><i>vivo</i> with Ag85, MPT51, HspX, or medium alone. The number of cells positive for CD4 and IL-17 was determined by flow cytometry. Lymphocytes were selected based on size and granularity. Gates were set to analyze CD4⁺ T cells, and then the fluorescence of antibodies detecting IL-17⁺ cells was recorded. (A–B) Spleen cells from mice vaccinated with (A) rBCG-CMX or (B) BCG. (C–D) Lung cells from mice vaccinated with (C) rBCG-CMX or (D) BCG. In A and C, all results were different from the medium stimulation. These data are representative of two independent experiments (N = 6, *p<0.05).</p

Levels of CD4⁺IFN-γ⁺ T cells induced by <i>ex vivo</i> stimulation with recombinant Ag85, MPT51, and HspX.

<p>Thirty days after vaccination, lung and spleen suspensions were stimulated <i>ex</i><i>vivo</i> with Ag85, MPT51, HspX, or medium alone. The number of cells positive for CD4 and IFN-γ was determined by flow cytometry. Lymphocytes were selected based on size and granularity. Gates were set to analyze CD4⁺ T cells, and then the fluorescence of antibodies detecting IFN-γ⁺ cells was recorded. (A–B) Spleen cells from mice vaccinated with (A) rBCG-CMX or (B) BCG. (C–D) Lung cells from mice vaccinated with (C) rBCG-CMX or (D) BCG. In A and C, all results were different from the medium stimulation. These data are representative of two independent experiments (N = 6, *p<0.05).</p

Levels of phagocytosis by peritoneal macrophages of BCG and rBCG-CMX after infection (MOI = 10).

<p>(A) Macrophages were infected with BCG or rBCG-CMX and the bacterial load in both the supernatant (sup) and inside the macrophages (Mφ) were determined. The amount of viable bacteria was determined by plating supernatant or cell lysates onto 7H11 agar supplemented with OADC and counting the CFU 28 days after incubation at 37°C. *(p<0.01) significant difference between the compared groups (log10 scale). (B) Nitric oxide (NO) production by macrophages infected with BCG or rBCG-CMX was determined. Uninfected media (Control) and LPS-stimulated (LPS) macrophages were included as controls. (C) Microscopic evaluation of peritoneal macrophages, 3 hours after infection with BCG or rBCG-CMX stained with Instant Prov or Ziehl Neelsen. Uninfected macrophages (Control) were included as a negative control. The results shown are representative of three different experiments.</p

Stability of rBCG-CMX <i>in vivo</i>.

<p>(A) Images of plates showing the mycobacterial growth of rBCG-CMX recovered from the dorsal region of mice 5, 10 and 15 days after subcutaneous immunization, and plated on media with kanamycin (kan) or without (W/o). (B) CFU counts recovered at different time points from the dorsal region of mice after immunization. (C) CMX gene detection by PCR for three isolated colonies from plates W/o kanamycin (Lanes 3–5). Lanes 1: M: molecular weight marker; 2: Negative control: NC: water.</p

Levels of polyfunctional CD4⁺ T cells induced by BCG and rBCG-CMX vaccines.

<p>Spleen (A and C) and lung (B and D) cell suspensions from vaccinated and control mice stimulated with rCMX. (A–B) CD4⁺IL-2⁺IFN-γ⁺ cells or (C–D) CD4⁺TNF-α⁺IFN-γ⁺ cells were analyzed by flow cytometry. Lymphocytes were selected based on size and granularity. Gates were set to analyze CD4⁺ T cells, and then the fluorescence of antibodies detecting IL-2⁺ and IFN-γ⁺ or TNF-α⁺ and IFN-γ⁺ cells was recorded. These data are representative of two independent experiments (N = 6, *p<0.05).</p

Bacterial load in the lungs of BALB/c mice 45 days after <i>Mycobacterium tuberculosis</i> challenge.

<p>Ninety days after immunization, three mice from each group (control, BCG and rBCG-CMX) were challenged with 10⁵ CFU of <i>Mycobacterium tuberculosis</i> H37Rv intravenously into the orbital sinus plexus. One additional group of animals received a booster of rCMX/CPG DNA, 30 days after rBCG-CMX vaccination and challenged with Mtb 30 days post the immunization (rBCG-CMX+CMX). Forty-five days after challenge, mice were euthanized and the anterior and mediastinal right lung lobes were collected, homogenized, and plated on Middlebrook 7H11agar supplemented with OADC to determine the bacterial load by counting the number of CFU. * Significant differences between infected (control) and vaccinated groups. # Significant differences between rBCG-CMX and rBCG-CMX+CMX groups. | Significant differences between rBCG-CMX and BCG groups analyzed by <i>t</i> test (p<0.05).</p

Immunogenicity of rBCG-CMX in BALB/c mice.

<p>(A) Experimental time line. BALB/c mice were immunized with rBCG-CMX or BCG Moreau. Thirty days later, 6 mice per group were euthanized for evaluation of vaccine-induced immunogenicity. Ninety days after immunization, mice were intravenously (<i>i.v.</i>) challenged with 10⁵ CFU of H37Rv. Forty-five days after <i>i.v.</i> challenge, the lung bacterial load (CFU) and lesions (H&E) were assessed. (B–E) Specific cellular immune responses induced with rCMX stimulation <i>ex</i><i>vivo</i>. Spleen (B and D) and lung (C and E) cell suspensions from vaccinated and unvaccinated (Control) mice were stimulated with rCMX. Cells positive for both CD4 and IFN-γ (B and C) or CD4 and IL-17 (D and E) were determined by flow cytometry. Lymphocytes were selected based on size and granularity. Flow cytometry gates were set to analyze CD4⁺ T cells, and then the fluorescence of antibodies detecting IFN-γ⁺ or IL-17⁺ cells was recorded. These data are representative of two independent experiments (N = 6, *p<0.05).</p