Lactoferrin enhanced efficacy of the BCG vaccine to generate host protective responses against challenge with virulent Mycobacterium tuberculosis

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a disease with world wide consequences, affecting nearly a third of the world's population. The established vaccine for TB, an attenuated strain of Mycobacterium bovis Calmette Guerin (BCG), has existed since 1921. Lactoferrin, an iron-binding protein found in mucosal secretions and granules of neutrophils was hypothesized to be an ideal adjuvant to enhance the efficacy of the BCG vaccine, specifically because of previous reports of lactoferrin enhancement of IL-12 production from macrophages infected with BCG. Different vaccination protocols were investigated for generation of host protective responses against MTB infection using lactoferrin admixed to the BCG vaccine. Resulting effects demonstrate that BCG/lactoferrin increased host protection against MTB infection by decreasing organ bacterial load and reducing lung histopathology; significant reduction in tissue CFUs and pathology were observed post-challenge compared to those seen with BCG alone. Addition of lactoferrin to the vaccine led to reduced pathological damage upon subsequent infection with virulent MTB, with positive results demonstrated when admixed in oil-based vehicle (incomplete Freund's adjuvant, IFA) or when given with BCG in saline. The observed post-challenge results paralleled increasing production of IFN-gamma and IL-6, but only limited changes to proinflammatory mediators TNF-alpha or IL-1beta from BCG-stimulated splenocytes. Overall, these studies indicate that lactoferrin is a useful and effective adjuvant to improve efficacy of the BCG vaccine, with potential to reduce related tissue damage and pulmonary histopathology.

Figures

Fig. 1. Lung and splenic bacterial load post-challenge with MTB in mice immunized with BCG and lactoferrin adjuvant: Comparison of IFA and saline formulations

C57BL/6 were immunized once with BCG (1×106 CFU/mouse), or BCG and lactoferrin (100 µg/mouse). At 2 weeks post-immunization, mice were aerosol challenged with Erdman MTB (<100 CFU/mouse). Mice were sacrificed at day 28 post-challenge; lung and spleen were assessed for organ bacterial load. The left panel (A,B) depicts results when the vaccine was formulated in Incomplete Freund’s Adjuvant; the right panel (C,D) shows results of mice immunized with vaccine in a saline formulation. Comparisons were also made to non-immunized control infected mice. At least 6 mice were infected for each group. Average CFU per organ ± standard deviation shown; *p<0.05, **p<0.01, ***p<0.001; indicated comparisons made to BCG-immunized or non-immunized infected mice.

Fig. 2. Lung histopathology in mice immunized with BCG and lactoferrin

Mice immunized as in Figure 1 legend demonstrated reduced histological manifestation of disease in the BCG and lactoferrin immunized groups. Pathology from the unvaccinated control mice (A) is compared with different vaccinated groups. The left panel depicts histopathology from IFA formulated vaccinated mice (B,C); the right panel depicts saline formulation of vaccine (D,E). While BCG formulated in IFA demonstrated reduction in granulomatous response over the saline formulated counterpart, both demonstrated more inflammation than seen in the lactoferrin adjuvant immunized groups (C, E). Marked reduction in granulomatous response was evident in both of the lactoferrin immunized groups. Tissue was embedded, sectioned and stained with H&E (40x). Representative histology from 6 mice per group.

Fig. 3. Cytokine and proinflammatory recall response to BCG antigens in BCG immunized mice with lactoferrin adjuvant

IFN-γ, TNF-α, IL-6, and IL-1β recall responses to BCG antigens were assessed from mice immunized with BCG or BCG/lactoferrin. Results shown indicate splenic responses from mice 2 weeks post single administration of vaccine formulated in IFA (Protocol A, A–D) or formulated in saline (Protocol B, E–H); comparisons in each set are made to non-immunized control mice. Cytokines shown as average ± standard deviation for at least 4 mice per group, done in triplicate; *p<0.05, **p<0.01, ***p<0.001; indicated comparisons made to BCG-immunized or non-immunized infected mice.

Fig. 4. Bacterial load post-challenge with MTB in mice immunized with BCG and lactoferrin adjuvant: Comparison of single vaccination vs. boosting

C57BL/6 were immunized once with BCG, or with BCG and lactoferrin, formulated in saline (Protocol C, left panel), and subsequently aerosol challenged with Erdman MTB 4 weeks later. Alternatively, mice were boosted at 2 weeks post-primary immunization, and then challenged 4 weeks later (Protocol D, right panel). Mice were sacrificed at days 7, 28, and 65 post-challenge, and lung (A,C) and spleen (B,D) tissue were assessed for organ bacterial load. Comparisons were also made to non-immunized control infected mice. At least 6 mice were infected for each group. Average CFU per organ ± standard deviation shown; *p<0.05, **p<0.01, ***p<0.001 compared to BCG immunized or non-immunized control infected mice at indicated times.

Fig. 5. Mice immunized with BCG and lactoferrin demonstrate diminished inflammation and destructive pulmonary histopathology upon challenge with virulent MTB

C57BL/6 mice immunized as in Figure 4 demonstrated reduced histological manifestation of disease in the BCG/lactoferrin immunized groups at day 65 post challenge. The top panel (A) depicts histopathology from a non-immunized control, and is compared to mice singly immunized with vaccine formulated in saline (left panel, B–D), or compared to tissue from mice vaccinated twice (right panel, E–G). Boosting with BCG alone led to a small but detectable reduction in granulomatous response over the saline formulated counterpart, however both demonstrated more inflammation than seen in the lactoferrin adjuvant immunized groups. The lactoferrin adjuvant immunized groups revealed a striking reduction in granulomas with evidence of lymphocytic clusters (white arrowhead) and contained focal pockets of inflamed monocytes (black arrowhead). Tissue was embedded, sectioned and stained with H&E (40x for A–C, E–F; 100x for D,G). Representative histology from 6 mice per group.

Fig. 6. Quantitative analysis of inflammation in immunized mice

Lung weight index (LWI) was calculated at day 7, 28 and 65 post challenge of mice immunized with BCG or BCG/lactoferrin, and compared to non-immunized challenged mice (A). On day 65, histological sections were assessed for changes in lung pathology by quantitative evaluation of inflammatory lesions, represented as percent occlusion of tissue (B). Results are shown as average ± standard deviation for at least 6 mice per group; *p<0.05, **p<0.01; indicated comparisons made to BCG-immunized infected mice.

Fig. 7. Cytokine production to BCG antigens in BCG/lactoferrin immunized mice

IFN-γ, IL-12 and IL-4 recall responses to BCG antigens were assessed from mice immunized with BCG or BCG/lactoferrin. Results shown indicate splenic responses from mice at 4 weeks post single administration of vaccine formulated in saline given once (Protocol C, A–C)) or from mice that were immunized and boosted (Protocol D, D–F)); comparisons in each set are made to non-immunized control mice. Cytokines shown as average ± standard deviation for 4–6 mice per group, done in triplicate; *p<0.05, **p<0.01, ***p<0.001; indicated comparisons made to BCG-immunized or non-immunized infected mice.

Fig. 8. IFN-γ and TNF-α mRNA at 7 days post infection in tissue of BCG/lactoferrin immunized mice

Real time RT-PCR analysis of IFN-γ and TNF-α mRNA from C57BL/6 mice immunized and boosted with BCG alone or with BCG/lactoferrin were evaluated in lung tissue (top) or in spleen (bottom) at 7 days post infection with virulent MTB. Message levels are indicated as average ± standard deviation for at least 4 mice per group, shown as fold change vs. non-immunized controls; *p<0.05, **p<0.01, ***p<0.001; indicated comparisons made to BCG-immunized or non-immunized infected mice.