Bronchus-associated lymphoid tissue (BALT) and survival in a vaccine mouse model of tularemia

Abstract

Background: Francisella tularensis causes severe pulmonary disease, and nasal vaccination could be the ideal measure to effectively prevent it. Nevertheless, the efficacy of this type of vaccine is influenced by the lack of an effective mucosal adjuvant.

Methodology/principal findings: Mice were immunized via the nasal route with lipopolysaccharide isolated from F. tularensis and neisserial recombinant PorB as an adjuvant candidate. Then, mice were challenged via the same route with the F. tularensis attenuated live vaccine strain (LVS). Mouse survival and analysis of a number of immune parameters were conducted following intranasal challenge. Vaccination induced a systemic antibody response and 70% of mice were protected from challenge as showed by their improved survival and weight regain. Lungs from mice recovering from infection presented prominent lymphoid aggregates in peribronchial and perivascular areas, consistent with the location of bronchus-associated lymphoid tissue (BALT). BALT areas contained proliferating B and T cells, germinal centers, T cell infiltrates, dendritic cells (DCs). We also observed local production of antibody generating cells and homeostatic chemokines in BALT areas.

Conclusions: These data indicate that PorB might be an optimal adjuvant candidate for improving the protective effect of F. tularensis antigens. The presence of BALT induced after intranasal challenge in vaccinated mice might play a role in regulation of local immunity and long-term protection, but more work is needed to elucidate mechanisms that lead to its formation.

Figure 1. Recombinant PorB and evaluation of antibody response against F.tularensis LPS.

(A) The band of rPorB detected by Coomassie gel stain is indicated by the arrow. (B) Serum was collected four weeks after vaccination, just before i.n. challenge. Antibody production was analyzed by ELISA and results are presented as nanograms (ng) of LPS-specific IgG and IgM per milliliter (ml) of serum. Black dots indicate individual mice, while bars represent mean values. Lower antibody concentrations include values between 286 and 436 ng/ml as opposed to those that were not detected (ND) in control groups. Significant differences were calculated by Mann Whitney test and are indicated by *** (P<0.001) for LPS+rPorB compared with PBS.

Figure 2. Survival, morbidity and bacterial dissemination following intranasal challenge.

(A) Protection was monitored for 21 days in challenged groups after inoculation with 10⁶ and 10⁵ CFU of LVS. (B) Morbidity associated with bacterial infection was determined by analyzing changes in body weight. The weight regain curve for the LPS-vaccinated group includes the only mouse that survived intranasal challenge (filled grey squares). (C) Bacterial loads in blood of challenged mice. Black dots represent individual mice, while bars represent mean values. ** (P<0.01); *** (P<0.001).

Figure 3. Lymphoid aggregates in lungs of vaccinated mice were detected at 30 days post-challenge (arrows).

(A) Representative lung from naïve mouse was used as a negative control; (B) Lung from LPS vaccinated/challenged; (C and D) Lung from LPS+rPorB vaccinated/challenged mouse and (E) Lung from LPS+rPorB vaccinated/unchallenged mouse. Formalin fixed, paraffin embedded sections were stained with H&E and representative pictures were taken with a 10× objective.

Figure 4. Detection of B and T cell infiltrates in lungs of vaccinated mice 30 days post-challenge.

(A) Representative lung from naïve mouse as a negative control; (B) Mediastinal lymph node from LPS+rPorB/challenged mouse as an internal positive control; (C) Lung from LPS vaccinated/challenged mouse; (D, E) Lung from LPS/rPorB vaccinated/challenged mouse and (F) lung from LPS+PorB vaccinated/unchallenged mouse. Cell surface markers used were B220 for B cells (green- Alexa 488) and CD3 epsilon for visualizing T cells (red- Alexa 549). Cell nuclei were stained with 4′-6-Diamino-2-phenylindole (DAPI-blue). Representative pictures were taken with a 20× objective.

Figure 5. Detection of proliferating B cells within BALT of vaccinated mice 30 days post-challenge.

(A) Lung from naïve mouse as a negative control; (B) mediastinal lymph node from LPS+rPorB/challenged mouse as an internal positive control; (C) Lung from LPS vaccinated/challenged mouse; (D, E) Lung from LPS+rPorB vaccinated/challenged mouse and (F) lung from LPS+PorB vaccinated/unchallenged mice. B cells were detected with B220 (green- Alexa 488) and PCNA was used to identify proliferating cells (red- Alexa 549). Areas of mixed color overlap indicate proliferation of B cells (see arrows). Cell nuclei were stained with 4′-6-Diamino-2-phenylindole (DAPI-blue). Representative pictures were taken with a 20× objective.

Figure 6. Germinal center B cells are exclusively found within BALT from LPS+rPorB vaccinated/LVS challenged mice.

(A) Representative lung from naïve negative control; (B) mediastinal lymph node from LPS+rPorB/challenged mouse as an internal positive control; (C) Lung from LPS vaccinated/challenged; (D, E) Lung from LPS+rPorB vaccinated/challenged mouse and (F) lung from LPS+PorB vaccinated/unchallenged mouse. Cells markers are B220 for B cells (red- Alexa 549) and PNA to detect germinal center B cells (green- Alexa 488). Areas of color overlap indicate germinal centers (see arrows). Cell nuclei were stained with 4′-6-Diamino-2-phenylindole (DAPI-blue). Representative pictures were taken with a 20× objective.

Figure 7. Morphometric analysis of follicles and pulmonary dendritic cells at 30 days post-challenge.

Data are expressed as average size of (A) and area occupied by (B) lymphoid and B cell follicles (µm²). (C) Dendritic cells (DCs) are shown from one representative mouse immunized with LPS+PorB and challenged with LVS (the two panels show different fields of the same lung where cells were detected). DCs were detected within follicles by CD11b (green), CD11c (red) or both (color overlap) and characterized by cytoplasmic prolongations. DCs are indicated by the yellow arrows. The white arrow on the left panel indicates a macrophage, characterized by a rounded shape. ** (P<0.01). Sections were viewed with a 20× objective.

Figure 8. Antibody producing cells and homeostatic chemokines in BALT areas at 30 days post-challenge.

(A) Representative lungs from mouse vaccinated with LPS+rPorB and challenged with LVS. The PCNA-CD3 combined stain was conducted to differentiate between nuclear (PCNA+) and surface (CD3+) staining. The upper left panel shows GC (cluster is outlined by the white dashed line) shown by the presence of cells with large PCNA+ nuclei (red nucleus staining). IgG producing proliferating plasma cells (indicated by the yellow arrows-main panel and insert) are detected with an anti-mouse IgG antibody (green cytoplasm) and PCNA (red nuclei), and few CD3+ cells (red surface) were identified in the same location. The upper middle panel shows IgM producing proliferating plasma cells (indicated by the yellow arrows-main panel and insert), identified by PCNA+ stain (red nuclei) and a dense IgM+ antibody signal (green cytoplasm). IgM+ B cells had a distinctive signal localized exclusively on the surface (green surface), and some CD3+ cells (red surface) were also detected. In the upper right panel, few IgA producing plasma cells (green cytoplasm) are present, but no proliferation is evident. Several CD3+ cells (red surface) were identified in the same area. In the lower left panel, some CXCL13 signal (red surface) co-localizes with the FDC network (combination of CD21, CD35 and FDCM1 markers-green) in the BALT area. The lower middle panel shows B cell clusters (green surface) and a CCL21+ high endothelial venule like structure (HEV) (red surface) as indicated by the yellow arrowhead. In the lower right panel, CCL21+ cells (red surface), resembling lymphatics or endothelial cells are shown by the white arrows. All sections were viewed with a 20× objective, and a 40× objective for the inserts in the first two upper panels. (B) Levels of CCL21 in lung lysate and serum are expressed as picograms (pg) per milliliter (ml) of lung lysate or serum. * (P<0.05).