CHO expressed recombinant human lactoferrin as an adjuvant for BCG

Abstract

Lactoferrin (LF), an iron binding protein with immune modulatory activities, has adjuvant activity to enhance vaccine efficacy. Tuberculosis (TB) is a pulmonary disease caused by the pathogen Mycobacterium tuberculosis (MTB). Progressive TB disease is clinically defined by damaging pulmonary pathology, a result of inflammation due to immune reactivity. The current vaccine for TB, an attenuated strain of Mycobacterium bovis, Bacillus Calmette Guerin (BCG), has only limited efficacy to prevent adult pulmonary TB. This study examines a Chinese hamster ovary (CHO) expressed recombinant human LF (rHLF) to boost efficacy of the BCG vaccine and delay early pathology post infectious challenge. C57BL/6 mice were immunized with BCG, or BCG admixed with either rHLF or bovine LF (bLF; internal control), or remained unvaccinated. Mice were then aerosol challenged with Erdman MTB. All vaccinated mice demonstrated decreased organ bacterial load up to 19 weeks post infection compared with non-vaccinated controls. Furthermore, mice receiving bLF or rHLF supplemented BCG vaccines showed a modest decrease in lung pathology developed over time, compared to the BCG vaccine alone. While mice vaccinated with BCG/rHLF demonstrated increased general lung inflammation at day 7, it occurred without noticeable increase in pro-inflammatory cytokines. At later times, decreased pathology in the rHLF groups correlated with decreased inflammatory cytokines. Splenic recall to BCG antigens showed BCG/rHLF vaccination increased production of IFN-γ, IL-6, and GM-CSF compared to naïve, BCG, and BCG/bLF groups. Analysis of T cell stimulating functions of bone marrow derived macrophages and dendritic cells treated with BCG/bLF or BCG/rHLF showed decreases in IL-10 production when co-cultured with sensitized CD4 and CD8 T cells, compared to those cultured with macrophages/dendritic cells treated with BCG without LF. These results indicate that addition of rHLF to the BCG vaccine can modulate development of host pathology early post infectious challenge, most likely through host immune regulation affecting hypersensitive responses.

Keywords: BCG; CHO; adjuvant; lactoferrin; tuberculosis.

Figure 1

Splenic recall profile to BCG antigens. Splenocytes were isolated from mice immunized and boosted with BCG, BCG/ bLF, or BCG/rHLF, or remained non-vaccinated at 6 weeks post booster. Splenocytes were re-stimulated with heat-killed BCG (MOI 1:1). Supernatants were analyzed for cytokine production by 20-plex multiplex beads at 72 h. Data are represented as box-and-whiskers: average, box min and max identify first and third quartiles, and the error bars define min and max of the dataset. Data are analyzed by one-way ANOVA, and Tukey post-hoc tests were applied. *P <0.05; **P <0.01; ***P <0.001.

Figure 2

Lung weight index of Erdman MTB infected mice. All vaccinated and non-vaccinated mice were infected with Erdman MTB by aerosol at ~100 CFUs/mouse. At set time points, the whole lung was weighed and compared to the weight of the whole mouse to calculate lung weight index (LWI). (a) LWI of groups at day 7 post infection. (b) LWI of groups as a function of time post infection. Data are represented as mean, error bars are standard deviation. Data are analyzed by one-way ANOVA, and Tukey post-hoc tests are applied. *P <0.05; **P <0.01.

Figure 3

Erdman bacterial load in lung, liver, and spleen tissues. All vaccinated and non-vaccinated mice were infected with Erdman MTB by aerosol at ~100 CFUs/mouse. Up to 19 weeks post infection, whole lung collected and homogenized in 1×PBS, serially diluted, and plated onto 7H11 agar plates. Data are represented as Log10 CFU/organ. Data are represented as box-and-whiskers: average, box min and max identify first and third quartiles, and the error bars define min and max of the dataset. Data are analyzed by one-way ANOVA, and Tukey post-hoc tests are applied. *P <0.05; **P <0.01; ***P <0.001.

Figure 4

Pathology of lung tissue during Erdman MTB infection. All vaccinated and non-vaccinated mice were infected with Erdman MTB by aerosol at ~100 CFUs/mouse. The large left lobe lung was collected post infection at times indicated. Formalin fixed tissue was processed, sectioned, and stained with H&E. Images were visualized at 20×. Dark purple clusters represent granulomatous responses of lymphocytes surrounded by light pink designated foamy macrophage clusters; and dark pink monocytic infiltration without foamy cytoplasm.

Figure 5

Percent lung occlusion of mice infected with Erdman MTB. Image from Figure 4 was analyzed by Image J. Percent occlusion was calculated by dividing the area of granulomas (lymphocytes, foamy macrophage, and other infiltrating leukocytes) by the area of the entire lung tissue section and multiplying by 100. Data are a compilation of three lung sections per mouse with five mice per group. No measureable granuloma structures were observed at 1 week post infection. (a) Data are represented as box-and-whiskers: average, box min and max identify first and third quartiles, and the error bars define min and max of the dataset. Data are analyzed by one-way ANOVA, and Tukey post-hoc tests are applied. *P <0.05; **P <0.01; ***P <0.001. (b) Time course of percent lung occlusion of each group up to 19 weeks post infection. Data are represented as mean, with error bar defined by standard deviation.

Figure 6

Lung tissue global cytokine environment post infection. All vaccinated and non-vaccinated mice were infected with Erdman MTB by aerosol at ~100 CFUs/mouse. Up to 19 weeks post infection, lung section was collected and homogenized by pestle in DMEM 10% FBS. The homogenate was incubated at 37°C for 4 h. Supernatants were collected, filtered, and analyzed by ELISA. (a) One week post infection. (b) Four weeks post infection. (c) Ten weeks post infection. (d) Nineteen weeks post infection. Data are represented as box-and-whiskers: average, box min and max identify first and third quartiles, and the error bars define min and max of the dataset. Data are analyzed by one-way ANOVA, and Tukey post-hoc tests are applied. *P <0.05; **P <0.01; ***P <0.001.

Figure 6

Lung tissue global cytokine environment post infection. All vaccinated and non-vaccinated mice were infected with Erdman MTB by aerosol at ~100 CFUs/mouse. Up to 19 weeks post infection, lung section was collected and homogenized by pestle in DMEM 10% FBS. The homogenate was incubated at 37°C for 4 h. Supernatants were collected, filtered, and analyzed by ELISA. (a) One week post infection. (b) Four weeks post infection. (c) Ten weeks post infection. (d) Nineteen weeks post infection. Data are represented as box-and-whiskers: average, box min and max identify first and third quartiles, and the error bars define min and max of the dataset. Data are analyzed by one-way ANOVA, and Tukey post-hoc tests are applied. *P <0.05; **P <0.01; ***P <0.001.

Figure 7

Co-cultures of bone marrow derived dendritic cells and sensitized CD4+ and CD8+ T cells. Bone marrow derived dendritic cells (BM-DCs) were treated with BCG (MOI 1:1) with or without bLF (100 μg/mL) or rHLF (100 μg/mL). At 72 h, treated BM-DCs were washed with 1×PBS and co-cultured with CD4+ or CD8+ T cells isolated from mice immunized with BCG (1×10⁶ CFU/mouse). After 72 h co-culture, supernatants were collected and analyzed by ELISA. Data are represented as box-and-whiskers: average, box min and max identify first and third quartiles, and the error bars define min and max of the dataset. Data are analyzed by one-way ANOVA, and Tukey post-hoc tests are applied. *P <0.05; ***P <0.001.

Figure 8

Co-culture of bone marrow derived macrophages and sensitized CD4+ and CD8+ T cells. Bone marrow derived macrophages (BM-Macs) were treated with BCG (MOI 1:1) with or without bLF (100 μg/mL) or rHLF (100 μg/mL). At 72 h, treated BM-Macs were washed with 1×PBS and co-cultured with CD4+ or CD8+ T cells isolated from mice immunized with BCG (1×10⁶ CFU/mouse). After 72 h co-culture, supernatants were collected and analyzed by ELISA. Data are represented as box-and-whiskers: average, box min and max identify first and third quartiles, and the error bars define min and max of the dataset. Data are analyzed by one-way ANOVA, and Tukey post-hoc tests are applied. *P <0.05; **P <0.01.