Protective T cell immunity against respiratory syncytial virus is efficiently induced by recombinant BCG

Abstract

Respiratory syncytial virus (RSV) is one of the leading causes of childhood hospitalization and a major health burden worldwide. Unfortunately, because of an inefficient immunological memory, RSV infection provides limited immune protection against reinfection. Furthermore, RSV can induce an inadequate Th2-type immune response that causes severe respiratory tract inflammation and obstruction. It is thought that effective RSV clearance requires the induction of balanced Th1-type immunity, involving the activation of IFN-gamma-secreting cytotoxic T cells. A recognized inducer of Th1 immunity is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which has been used in newborns for decades in several countries as a tuberculosis vaccine. Here, we show that immunization with recombinant BCG strains expressing RSV antigens promotes protective Th1-type immunity against RSV in mice. Activation of RSV-specific T cells producing IFN-gamma and IL-2 was efficiently obtained after immunization with recombinant BCG. This type of T cell immunity was protective against RSV challenge and caused a significant reduction of inflammatory cell infiltration in the airways. Furthermore, mice immunized with recombinant BCG showed no weight loss and reduced lung viral loads. These data strongly support recombinant BCG as an efficient vaccine against RSV because of its capacity to promote protective Th1 immunity.

Fig. 1.

Expression of RSV M2 and N proteins by recombinant BCG strains. BCG was electrotransformed with plasmid pMV361-M2 or pMV361-N and selected on solid 7H10 medium supplemented with 20 μg/mL kanamycin. Expression of recombinant RSV proteins was assessed by Western blot analysis using rabbit polyclonal antisera specific for the M2 or N protein. A total of 25 μg of whole proteins prepared from BCG-M2 or BCG-N was loaded in each gel. As positive controls, recombinant N or M2 proteins and total proteins of RSV-infected HEp-2 cells were loaded. As negative control, whole proteins prepared from WT-BCG (nonrecombinant) were included.

Fig. 2.

Immunization with BCG-N and BCG-M2 protects mice against RSV. Groups of BALB/c mice (5 to 6 weeks of age) received subdermal immunization with 1 × 10⁸ cfu of WT-BCG, BCG-M2, BCG-N, or UV-inactivated RSV and infected with 1 × 10⁷ pfu RSV. Uninfected and unimmunized mice were included as control groups. (A) Body weight loss after RSV infection. Weight loss for BCG-M2-immunized and BCG-N-immunized mice was significantly lower than for mice immunized with WT-BCG or UV-RSV and unimmunized mice (*, P < 0.04, Student's t test between unimmunized and BCG-M2 or BCG-N values). (B) Number of cells in BALs 4 days after RSV infection. BAL cells were obtained as described in Methods and counted with a Neubauer chamber (HBG, Germany). Symbols represent individual mice, and horizontal lines represent means. Cell counts in BALs were significantly lower in mice immunized with BCG-N and BCG-M2 compared with unimmunized mice (***, P < 0.001, Student's t test between unimmunized and BCG-N or BCG-M2 values; NS, nonsignificant, Student's t test between unimmunized and WT-BCG values). (C) Percentage of CD11b-positive cells in BAL 4 days after RSV infection. BAL cells were stained with an FITC-labeled anti-CD11b antibody and analyzed by flow cytometry. Symbols represent individual mice, and horizontal lines represent means. CD11b-positive cell counts were significantly lower in mice vaccinated with BCG-N and BCG-M2 compared with unimmunized mice (***, P < 0.001, Student's t test between unimmunized and BCG-N or BCG-M2 values; NS, nonsignificant, Student's t test between unimmunized and WT-BCG values). Inset shows a CD11b/GR1 dot plot for BAL cells from RSV-infected mice.

Fig. 3.

Immunization with BCG-M2 or BCG-N prevents airway infiltration by neutrophils after RSV infection. (A) Four days after RSV challenge, BALs obtained from unimmunized or immunized mice with BCG-M2, BCG-N, WT-BCG, or UV-RSV were spun on glass slides, stained with May-Grunwald and Giemsa, and observed under a light microscope at 40× magnification. As a control, BALs from uninfected mice were included. (B) Graph shows the amount of total cells (gray) and neutrophils (black) per field, as quantified by light microscopy at 40× magnification in at least 6 random fields per sample. Bars represent means ± SE.

Fig. 4.

Immunization with BCG-M2 and BCG-N reduces inflammatory cell infiltration into the lungs after RSV infection. (A) Four days after RSV infection, mouse lungs were removed and fixed with paraformaldehyde, and 5-μm cuts were stained with hematoxylin/eosin. Significant polymorphonuclear cell infiltration can be observed in unimmunized, UV-RSV, and WT-BCG-immunized mice. Photos are representative of 3 to 6 independent experiments. (B) HPS for each group: 0, no cellular infiltration; 1, minimal cellular infiltration; 2, slight cellular infiltration; 3, moderate cellular infiltration; 4, severe cellular infiltration, as described in Methods (NS indicates nonsignificant; **, P < 0.0036, ***, P < 0.0001, Student's t tests between uninfected and each individual group).

Fig. 5.

Immunization with BCG-M2 and BCG-N reduces virus presence in lung tissues after RSV infection. (A) Four days after infection, lungs were removed, fixed with paraformaldehyde, and stained with an HRP-labeled anti-RSV antibody [first and third columns (arrowheads show positive staining)] or with a biotin-labeled anti-F antibody followed by streptavidin-FITC (second and fourth columns), as described in SI Methods. Fluorescence counterstaining derives from a Cy3-conjugated anti-von Willebrand factor antibody. Positive staining is observed in lungs of unimmunized, UV-RSV, and WT-BCG-immunized mice. Data shown are representative of 3 to 6 independent experiments. (B) Total RNA from lungs of control and infected animals were obtained and reverse transcribed to quantify the number of N-RSV copies by real-time PCR. Data are expressed as the number of N-RSV gene copies per 5,000 copies of β-actin gene (**, P < 0.01, 1-way ANOVA).

Fig. 6.

Immunization with BCG-M2 and BCG-N induces the secretion of Th1-type cytokines and promotes activation of antigen-specific T cells in the spleen. BALB/c mice were immunized with either 100 μL of PBS-Tween 0.02%, 1 × 10⁸ cfu of WT-BCG, BCG-M2, BCG-N, or with 1 × 10⁷ pfu of UV-inactivated RSV. After 21 days of vaccination, spleen cells were recovered and stimulated with 10 μg/mL of either M2 or N proteins for 5 days to evaluate cytokine secretion. (A) IFN-γ, IL-2, and IL-4 secretion was detected in supernatants of cell suspensions, by ELISA. Spleen cells derived from BCG-M2 or BCG-M secreted significant amounts of IFN-γ and IL-2 after stimulation with their cognate proteins (***, P < 0.0002; *, P = 0.02, Student's t test). Secretion of IL-4 was only observed in spleen cells of mice immunized with UV-RSV (*, P = 0.02, Student's t test). (B) CD69 expression on the surface of T cells after stimulation with M2 and N protein. Spleen cell suspensions were stimulated with 10 μg/mL N or M2 proteins or left untreated for 72 h, stained with PE anti-CD69, FITC anti-CD8a, and allophycocyanin (APC) anti-CD4 antibodies, and analyzed by flow cytometry, as described in SI Methods. Representative histograms derived from 3 independent experiments show profiles of CD69 expression by CD4⁺ or CD8⁺ cells. Numbers in each histogram are the percentages of CD4⁺ or CD8⁺ populations positive for CD69. (C) Intracellular IFN-γ production by T cells from BCG-M2-immunized or BCG-N-immunized mice. Representative dot plots of CD4⁺/IFN-γ⁺ cells (CD69 gated) and a graph summarizing percentages of CD4⁺/CD69⁺/IFN-γ⁺ T cells (means ± SE). White bars are cell stimulated with PBS and gray bars are cells stimulated with N or M2 recombinant proteins, respectively. (D) Transfer of T cells from BCG-N-immunized mice protects against RSV. T cells obtained from BALB/c mice 21 days after immunization with BCG-N were stimulated for 3 days with N protein and intravenously injected into naive BALB/c mice. RSV-induced weight loss (Left) and BAL infiltration by CD11b⁺ cells (Right) in BALB/c transferred either with CD4⁺, CD8⁺, or both T cell subsets. Nontransferred (NT) and uninfected (UI) mice were included as controls. Data shown are means ± SE from 3 independent experiments. **, P < 0.01; ***, P < 0.001 between nontransferred and transferred mice, Student's t test. NS indicates nonsignificant.