c)

Tuberculosis 89 (2009) S1, S49–S54

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Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube

Lactoferrin enhances efficacy of the BCG vaccine: comparison between two inbred mice strains (C57BL/6 and BALB/c) Shen-An Hwanga , Ranjana Aroraa , Marian L. Kruzelb , Jeffrey K. Actora,c, * a Department of Pathology, Medical School, b Department of Integrative Biology and Pharmacology, c Program in Molecular Pathology, University of Texas-Houston Medical School, Houston, TX, USA

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Keywords: Lactoferrin BCG Vaccine Adjuvant Tuberculosis

The current vaccine for tuberculosis (TB), an attenuated strain of Mycobacterium bovis Bacillus Calmette Guerin (BCG), is effective to prevent childhood onset of the disease, but its efficacy is reduced in adults. One strategy to improve the existing vaccine is to develop more effective adjuvants. Lactoferrin, an iron-binding glycoprotein possessing immune modulatory activities, is a promising adjuvant candidate. The studies presented here examine the effect of lactoferrin to enhance efficacy of the BCG vaccine using a vaccination/challenge protocol (8 weeks boost and challenge at 12 weeks post-boost) that focuses on reduction in development of pathological changes to lung tissue. C57BL/6 and BALB/c mice vaccinated with BCG/lactoferrin exhibited protection upon Mycobacterium tuberculosis (MTB) challenge, showing reduced pulmonary disease pathology and decreased organ bacterial load. In addition, BCG/lactoferrin-treated macrophages isolated from BALB/c mice, which express a relative reduced TH 1 phenotypic response to MTB antigens compared to the C57BL/6 mouse, were able to activate a higher percentage of IFN-g-producing CD4+ splenocytes. Overall, lactoferrin stands as an adjuvant capable of enhancing efficacy of the BCG vaccine through induction of TH 1 immune responses, even in hosts typically demonstrative of reduced TH 1 responsiveness to BCG antigens. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction The current vaccine for tuberculosis (TB) is an attenuated strain of Mycobacterium bovis Bacillus Calmette Guerin (BCG), which is often effective in preventing childhood onset TB, but demonstrates waning efficacy in adulthood.1–3 Multiple efforts are underway to develop a vaccine capable of surpassing BCG, however, the most promising candidates are not yet approved for human use.4 What is required is a vaccine that would limit pathology, and thus limit spread of disease between individuals by establishing a “firebreak” to slow transmission.5 A promising avenue to increase BCG vaccine efficacy is through the incorporation of adjuvant components. Lactoferrin, an iron-binding glycoprotein found in mucosal secretions and granules of neutrophils, is just such a potential adjuvant. Lactoferrin has demonstrated multiple immune-modulatory functions, including promoting B-cell, T-cell, and dendritic cell maturation,6–8 stimulating cytokine production from naive or suboptimally activated macrophages,9–12 regulating cytokine production from highly activated macrophages,13,14 and up-regulating * Corresponding author. Jeffrey K. Actor, Ph.D. Professor, Department of Pathology and Laboratory Medicine, MSB 2.214, University of TexasHouston Medical School, 6431 Fannin, Houston, TX 77030, USA. Tel: +1 (713) 500 5344; fax: +1 (713) 500 0730. E-mail address: [email protected] (J.K. Actor). 1472-9792 /$ – see front matter © 2009 Elsevier Ltd. All rights reserved.

surface expression of presentation and co-stimulatory molecules on macrophages and dendritic cells.15 Indirectly, lactoferrin promotes macrophage and dendritic cells to stimulate IFN-g production from antigen-specific T-cells.15 Additionally, lactoferrin increases the delayed type hypersensitivity (DTH) response to multiple diverse antigens.9,16–18 These data suggest that lactoferrin would function as an ideal adjuvant to enhance efficacy of the BCG vaccine to promote generation of host protection against subsequent MTB infection. In the murine challenge model of pulmonary TB, addition of lactoferrin to the BCG vaccine significantly reduced manifestation of disease pathology, described as decreased total lung area occupied by granulomas and maintenance of normal parenchyma surrounding granulomatous structures.19,20 These changes correlated with increased T-cell antigen-specific recall responses, as measured by production of IFN-g. This implies that vaccination with BCG/lactoferrin led to generation of antigen-specific T-cell helper type 1 (TH 1) response, crucial for limitation of pathological damage during MTB infection.21 Further evaluation discovered that macrophages and dendritic cells are the target phenotypic cells responsive to lactoferrin, allowing for promotion of stimulation and activation of antigen-specific T-cells. It is well established that IFN-g production defines the presence of a strong TH 1 phenotypic response, whereas production of IL-4, IL-5, and/or IL-13 indicates the activation of TH 2 cells.22 Evidence suggests that lactoferrin acts as an adjuvant for the BCG vaccine to

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favor development of a TH 1 recall response,20 while also suppressing early IL-4 production during establishment of infection.23 To test this hypothesis in vivo, two strains of mice (C57BL/6 and BALB/c), with native differences in generation of TH 1 immune response to mycobacterial antigens, were vaccinated with BCG/lactoferrin and examined for protection upon subsequent MTB infection. Protection after MTB challenge was then monitored by changes in pulmonary disease pathology and organ bacterial loads. The effect of lactoferrin to promote macrophage stimulation of antigenspecific T-cells was also examined, by measuring the production of IFN-g from BCG-sensitized T-cells co-cultured with bone marrowderived macrophages (C57BL/6 or BALB/c) which were pre-treated with BCG in the presence or absence of lactoferrin. In this way, we were able to address how lactoferrin might effectively contribute as an adjuvant to poorly responsive populations in generation of pathological protective responses. 2. Materials and methods 2.1. Animals Female C57BL/6 mice (6 weeks, Jackson Laboratories, Bar Harbor, ME) or BALB/c mice (6 weeks, Harlan Laboratories, Indianapolis, IN) of 20–25 g initial body weight were utilized for experimentation. All in vivo studies were conducted under approved guidelines of the animal ethics committee at the University of Texas, Health Science Center at Houston (HSC-AWC-06-100 and HSC-AWC-07-016). 2.2. Lactoferrin, BCG and MTB Low endotoxin bovine milk lactoferrin (<0.2 E.U./mg, <20% iron saturated, >95% purity) was provided by PharmaReview Corporation (Houston, TX). Mycobacterium bovis Calmette Guerin (BCG) Pasteur strain (TMC 1011, ATCC, Manassas, VA) was grown in Dubos base (without addition of glycerol) in 10% supplement (5% BSA and 7.5% dextrose in saline) on an orbital shaker at 37°C. BCG was diluted with 1× Dulbecco’s phosphate-buffered saline (PBS) (Cellgro, Herndon, VA) to 3 × 108 organisms/mL, estimated using McFarland standards (Sigma), with confirmed dose measured by plating dilutions on 7H11 agar (Remel, Lenesa, KS). Colony growth was measured after incubation at 37°C with 5% CO2 for 3–4 weeks. Mycobacterium tuberculosis (MTB; TMC 107, ATCC) was grown in Dubos base with 5.6% glycerol and 10% supplement for 3–4 weeks before use. Bacteria were taken during log phase growth, resuspended in 1× PBS, sonicated for 10 seconds to dislodge any clumping that might have occurred. Erdman was diluted in 1× PBS to 3 × 108 bacteria/mL, and concentration was estimated using McFarland standards and confirmed by plating dilutions onto 7H11 agar plates and enumerating after 3–4 weeks incubation at 37°C. 2.3. Vaccination and challenge Mice (at least 5 mice/group) were immunized with BCG (1 × 106 colony-forming units [CFU]/mouse), with or without lactoferrin (100 mg/mouse) in 1× PBS. Comparisons were made to mice that remained non-immunized. At 8 weeks, all mice were boosted and 12 weeks later mice were aerosol challenged with Erdman strain of MTB. MTB (1 × 108 CFU/mL) was suspended in 5 mL 1× PBS and aerosolized using an inhalation exposure system (IES) (GLASCOL Model #A4212 099c Serial #377782). Total inoculation dose (approximately 50–100 CFU/mouse) was calculated by sacrificing 4 mice at day 1 post-infection and assessing for lung bacterial load after plating lung homogenates on 7H11 media. Groups of mice were sacrificed at 65 days post-challenge. Lung, liver, and spleen tissue were sectioned for CFU counts and histological analysis.

2.4. Histological techniques Histological analysis of the lung was performed on day 65 following aerosol infection. Lungs were fixed in 10% formalin and embedded in paraffin using standard techniques.24,25 Sections, 5 mm thick, were stained with hematoxilin and eosin (H&E) and subsequently reviewed histologically; the pathologist viewing and interpreting the slides was blind to the type of experiment and treatment. Multiple sections of each lung from at least 5 mice per group were digitized with a 2× objective and analyzed using software (NIH Image J). Relative area of occluding lesions was obtained and calculated as percent of total (100%) lung section area as described.20 2.5. Intracellular IFN-g production Bone marrow-derived macrophages were differentiated from C57BL/6 or BALB/c bone marrow cells by culturing at 37°C with 5% CO2 for 7 days in GM-CSF (10 ng/mL) containing McCoy’s 5A Medium (Sigma) supplemented with 10% FBS (Sigma). As described, adherent populations (approximately 5 × 105 cells/mL) were stimulated with lactoferrin (100 mg/mL), BCG (MOI 5:1), or BCG/lactoferrin, or remained unstimulated.15 After 3 days treatment, BM macrophages were washed and co-cultured (1:1 ratio) with BCG-sensitized CD3+ CD4+ splenocytes isolated using magnetic beads (Miltenyi) from mice immunized and boosted with BCG after 6 weeks. At 72 h, co-cultured cells were restimulated with ConA (2 mg/mL), in the presence of 1 mL of GolgiPlug (BD Biosciences); cells were further incubated for 6 additional hours. Non-adherent cells were isolated and stained for CD4+ FITC and intracellular IFN-g-PE following manufacturer’s directions (BD Biosciences). Antibodies were diluted to working concentration of 1 mg/106 cells in staining buffer (1% BSA in 1× PBS). Nonspecific staining sites were blocked with Fc Block™ (CD16/32, BD Biosciences Pharmingen, San Diego, CA). Stained cells were washed with staining buffer (1% BSA in 1× PBS) and fixed with 4% w/v paraformaldehyde. Analysis was performed using Coulter FlowCentre™ (EPICS XL-MCL). 2.6. ELISA (Enzyme-linked immunosorbent assay) Supernatants were assayed for cytokine production using the DuoSet ELISA kits (R&D Systems, Minneapolis, MN), according to manufacturer’s instructions as described.26,27 Supernatants were assayed for production of IFN-g, IL-2, and IL-4. Lower limit of detection for assessment was between 15 and 32 pg/mL. 2.7. Statistics In vivo experiments were repeated twice using 5 mice/group. BMmacrophage and T-cell co-cultures were repeated at least 6 times and data reported as mean ± standard deviation for flow cytometric analysis. Likewise, results are shown as mean ± standard deviation for all other analyses, and statistical analysis was performed using OneWay ANOVA or T-test. 3. Results 3.1. Immunization with BCG/lactoferrin limits manifestation of disease-related pathology C57BL/6 and BALB/c mice were vaccinated with BCG or BCG/ lactoferrin and challenged at 12 weeks post-final immunization. The time chosen for challenge was sufficient to allow development of memory protective responses, while limiting non-specific activation due to the vaccination procedures. After 65 days of infection, lungs from C57BL/6 and BALB/c mice were collected and examined histologically. One of the most striking changes observed in BCG/lactoferrin-immunized mice was the decreased development

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(A)

(A)

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% Lung Occlusion

25

25

(B)

*

20

***

15 10 5

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(B)

**

20

p=0.279

15 10 5 0

0 Non-immunized

BCG

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BCG/Lf

Fig. 1. Vaccination with lactoferrin adjuvant protects MTB-challenged C57BL/6 mice from disease-induced pathology. C57BL/6 mice vaccinated with BCG or with BCG/lactoferrin were boosted at 8 weeks, and challenged with Erdman MTB at 12 weeks post-boost. Mice were sacrificed at day 65 post-challenge, lung sections collected, embedded in paraffin, sectioned, and stained with H&E (20×). Comparisons are made to non-immunized controls. (A) Representative histopathology from one mouse per immunization group. (B) Quantitation of lung granulomatous response was performed; results are shown as average ± standard deviation for at least 5 mice per group. *p < 0.05, ***p < 0.001.

Fig. 2. MTB-challenged BALB/c mice vaccinated with BCG/lactoferrin exhibit reduced disease pathology. BALB/c mice were vaccinated with BCG or with BCG/lactoferrin, boosted at 8 weeks, and challenged with Erdman MTB at 12 weeks post-boost. Mice were sacrificed at day 65 post-challenge, lung sections collected, embedded in paraffin, sectioned, and stained with H&E. Comparisons are made to non-immunized or BCG-alone immunized controls. (A) Representative histopathology from one mouse per immunization group (H&E, 20×). (B) Quantitation of lung granulomatous response was performed; results are shown as average ± standard deviation for at least 5 mice per group. **p < 0.01.

of destructive lung pathology compared to other immunized groups or non-immunized mice. Specifically, C57BL/6 mice immunized with BCG/lactoferrin exhibited a decrease in lung pathology, as previously observed,20 with evidence of smaller and more compact granulomas. In addition, there was little to no inflammation in the parenchyma adjacent to the granulomas, as compared to both the non-immunized and the BCG-immunized groups (Fig 1A). Quantification of the total area occupied by granuloma structures showed significant decrease in total lung area occluded in mice immunized with BCG/lactoferrin (8.51±3.12%) as compared to both the BCG alone (12.33±6.79%, p < 0.05) or non-immunized groups (14.24±6.04%, p < 0.001) (Fig 1B). Pathological responses in BALB/c mice responding to infectious challenge with MTB are similar to those of the C57BL/6 strain, in spite of reported cytokine differences elicited to BCG antigens.28–31 Examination of the control non-immunized BALB/c mice at 65 days post challenge revealed disease pathology in lungs similar to that of the C57BL/6 mice, again defined by presence of large granulomas comprised of increased accumulation of lymphocytes with associated clusters of foamy macrophages. Vaccination with BCG led to reduced inflammation in the surrounding tissue, although the granuloma structures remained similar in nature with lymphocytes and foamy macrophages. However, the lung pathology exhibited by BALB/c mice vaccinated with BCG/lactoferrin was characterized by granuloma structures that appear smaller and more compact, with marked reduction in foamy macrophages (Fig. 2A). Quantification of the histological observation revealed

that mice immunized with BCG/lactoferrin significantly decreased total lung area occluded by granuloma structures (7.17%±5.22%, p < 0.01) compared to the non-immunized group (15.56%±6.37%), and showed a strong reductive trend compared to the BCG-only group (10.18%±5.39%, p = 0.28) (Fig 2B). 3.2. Reduced bacterial loads in lactoferrin-adjuvant vaccinated mice Mice vaccinated and challenged as described typically demonstrate greatest reduction in organ CFU at 30 days post challenge, with number of organisms rising to levels seen in non-vaccinated mice by 60 days after challenge.32,33 Therefore, the BCG/lactoferrinimmunized mice were investigated for presence of CFU in tissue at the later time point (day 65 post-challenge). Lung, liver, and spleen were collected, and CFU were enumerated. C57BL/6 mice vaccinated with BCG or BCG/lactoferrin demonstrated significantly decreased number of organisms in the liver and spleen, and slightly lowered organisms in the lung (p = 0.089) compared to controls (Fig. 3). In comparison, the BCG/lactoferrin-vaccinated BALB/c mice showed significantly decreased organ bacterial load in all tissues examined (lung, liver, and spleen) compared to both the BCG- and non-immunized groups (Fig 3). Overall, the lactoferrin adjuvant extended the protection in the immunized group to time periods not typically associated with reduction in CFUs in similar mouse model systems.

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BALB/c

C57BL/6 p=0.089

7

6

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Log CFU/Lung

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5 4 3 2

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0.5

0.0

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*

6

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*

6

**

5

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4

0

0

Spleen

5

1

1

Liver

*

7

6

5 4 3 2 1 0 Non-immunized

BCG

BCG/lactoferrin

Fig. 3. Mice vaccinated with BCG/lactoferrin demonstrate decreased organ bacterial loads. CFU from C57BL/6 and BALB/c mice vaccinated with BCG or with BCG/lactoferrin were enumerated at 65 days post challenge with Erdman MTB. Comparisons are made to non-immunized mice. Numbers of organisms in lung, liver and spleen are indicated in log CFU values, showing average ± standard deviation for at least 5 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001.

3.3. BCG-infected macrophages cultured with lactoferrin increase IFN-g production from sensitized CD4+ splenocytes Historically, BALB/c T cells respond in a weaker manner to BCG antigens than their C57BL/6 counterparts, even after immunization. A study was therefore designed to examine if lactoferrin could boost BALB/c presenting cells in a way to augment IFN-g production from antigen-specific CD4+ splenocytes.34 Stimulation of CD4+ T-cells was compared between the C57BL/6 and BALB/c populations. Bone marrow-derived macrophages (BMM) were infected with BCG or remained uninfected, and cultured with or without lactoferrin. BCG-sensitized CD3+ CD4+ splenocytes (obtained from mice immunized only with BCG) were added, and T cells were stained for production of intracellular IFN-g. As expected, the BCG-infected C57BL/6 macrophages cultured with or without lactoferrin stimulated comparable IFN-g production from antigen-specific CD4+ splenocytes, both in percent cells positive for IFN-g and mean fluorescent intensity (MFI). There was a slight increase in IFN-g production from the CD4+ T-cells from C57BL/6 mice that were overlaid onto macrophages treated with BCG/lactoferrin (MFI 716±111), compared to T-cells overlaid onto macrophages treated with BCG alone (MFI 675±89). A similar trend, albeit lower in magnitude, was observed for the effect seen on BALB/c macrophages. However, BCG-infected BALB/c macrophages cultured with lactoferrin stimulated an overall increase in percent of CD4+ splenocytes producing IFN-g (2.53±0.44%) compared to CD4+ splenocytes overlaid onto only BCG infected macrophages alone (2.09±0.24%). In this group, more cells were responsive within the population of CD3+CD4+ cells, with no differences in amount of IFN-g producted per cell as indicated by analysis in the MFI.

4. Discussion The ultimate goal of vaccination to protect against development of tuberculosis should encompass the ability to limit transmission between infected individuals and those non-infected that are at risk from contact.5 Any TB vaccine that limits development of caseous pathology would have the outcome of also limiting aerosol spread of organisms. Lactoferrin is proving to be an ideal adjuvant candidate to enhance efficacy of the BCG vaccine to improve host protection against infection with virulent MTB by limiting development of destructive pulmonary pathology. These vaccinated and challenged mice demonstrated granulomatous lesions that were considerably smaller and more compact when compared to BCG-alone immunized mice. Additionally, BALB/c mice vaccinated with BCG/lactoferrin exhibited decreased organ bacterial load for substantially longer periods than previously defined, perhaps also delaying organism expansion within the infected host. Examination of splenic recall responses is routinely used to predict vaccine efficacy. Yet evidence is accumulating that suggests that increased IFN-g production in mice does not necessarily correlate with subsequent host protection.35 Thus, it may be more appropriate to develop vaccines by measuring outcomes where pathological damage to tissue is limited, which may more accurately represent subsequently slowing of disease spread between individuals. Even so, it remains critical for vaccines to retain strong development of specific adaptive function that includes IFN-g production to BCG antigens which share common epitopes with MTB. A proposed action for lactoferrin during vaccination is that it potentially promotes macrophage stimulation of antigen-specific T-cells, increasing environmental (local) cytokines that promote TH 1-like development which is critical for containment of MTB infection.21 The experiments presented here reaffirm the previously published adjuvant activity of lactoferrin19,20 and suggest that

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lactoferrin may be an ideal candidate to include as an adjuvant for vaccines targeting generation of cell-mediated memory response, specifically those that require a TH 1-mediated response. Previous in vivo studies examining the potential adjuvant activity of lactoferrin to increase efficacy of the BCG vaccine utilized a limited immunization protocol comprised of a short-term boost at 2 weeks and challenge with virulent MTB soon thereafter (4 weeks post final boost).20 In the experiments outlined here, an immunization protocol was adopted to exclude effects due to residual activation of cellular responses relative to the act of vaccination. Here, a boost was done at 8 weeks followed by MTB challenge at 12 weeks post final boost. This allowed a more stringent vaccination/challenge protocol, and it remained clear that mice vaccinated with the lactoferrin adjuvant displayed augmented protection under these conditions. The potential of lactoferrin to serve as an adjuvant to influence efficacy of the BCG vaccine has previously been studied exclusively using the C57BL/6 mouse, which along with the BALB/c strain, has the susceptible Bcg allele.36 Yet these two strains demonstrate a dichotomy of response to other intracellular pathogens, and are loosely characterized with innate shifts towards TH 1-like phenotypes (C57BL/6) or TH 2-like responses (BALB/c) against defined pathogens.37–40 The experiments presented were designed to investigate the effectiveness of lactoferrin to promote IFN-g response in a host that does not possess a strong TH 1 immune response upon MTB infection.41 Of interest, a main difference was observed between the two strains in which the BCG/lactoferrinvaccinated C57BL/6 mice demonstrated significantly reduced pulmonary disease pathology, but only marginal effects on reduction in long-term control of organ bacterial loads. In contrast, BCG/lactoferrin-vaccinated BALB/c mice significantly reduced organ bacteria loads through day 65, as well as demonstrated a reduced pulmonary disease pathology. These differences could certainly be attributed to various factors, including differences in MHC haplotype, T-cell recognition of presented epitopes, participation of total numbers of CD4+ and CD8+ populations, as well as differences in induction of IFN-g production.31,42,43 More effort will be needed to fully define the responses identified in the BALB/c mice. One of the main reported differences in the development of the TH 1 response between C57BL/6 and BALB/c mice is that the latter requires IL-1a and TNF-a to maximize IL-12 production.44 Indeed, the immune response to MTB during infection of BALB/c mice can be enhanced by exogenous IL-12.34 It is speculated that the underlying mechanisms of enhanced adjuvant activity of lactoferrin may be attributed to increased TNF-a and IL-12 production.11,16,19 In support of this hypothesis, BCG-infected BALB/c macrophages cultured in the presence of lactoferrin displayed a strong trend of increased IFN-g production from antigen-specific CD3+ CD4+ splenocytes. It is likely that a more complicated scenario exists because although addition of exogenous IL-12 can boost immune response in MTB-infected BALB/c mice, supplementation of IL-12 during BCG vaccination does not enhance vaccine efficacy to protect the BALB/c host against infection with MTB.34 The potential for lactoferrin to function as an effective adjuvant to enhance efficacy of the BCG vaccine has now been demonstrated under a long-term vaccination/challenge protocol that focuses on development of pathological protective immunity. The adjuvant effect of lactoferrin was observed in two strains of inbred mice that show natural divergent immune responses to intracellular pathogenic infections. In the current environment, where developing novel TB vaccines remains years away from approval for human use, there is a distinct advantage for incorporation of an effective adjuvant that would enhance pathological protection, and thus limit or delay spread of organisms between individuals.

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Acknowledgements: This work was supported in part by NIH grants 1R41GM079810-01 and R42-AI051050-03. This work was presented in part at the Texas Tuberculosis Research Symposium (TTRS) 2009, Houston, TX, co-sponsored by the University of Texas Health Sciences Center-Houston and the Methodist Hospital Research Institute. Competing interests: M.L. Kruzel is a founder of PharmaReview Inc., that supplied lactoferrin for the study; J.K. Actor is a member of the scientific advisory board for PharmaReview, Corp.

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