Lactoferrin as an Adjuvant for the Generation of Delayed Type Hypersensitivity to Orally Administered Antigen.

OBJECTIVE: The aim of this investigation was to evaluate the property of bovine lactoferrin (LF) in the generation of delayed type hypersensitivity (DTH) as an oral adjuvant during immunization with ovalbumin (OVA) and BCG. METHODS: LF admixed with OVA or BCG was used for immunization of CBA or C57BL/6 mice when given via oral or subcutaneous routes. Elicited DTH response was measured post immunization. Inhibition studies using mannose or galactose were accomplished by gavage prior to oral administration of antigens. LF was also examined for effects on BCG uptake by bone marrow derived macrophages (BMM). RESULTS: LF at doses of 1.0 mg and 10.0 mg, admixed with OVA (10.0 mg), significantly enhanced the antigen-specific DTH reaction. The stimulatory effects of LF were inhibited by the oral pretreatment of mice with 50.0 mg of mannose but not galactose. LF also enhanced the DTH reaction to orally administered BCG. LF enhanced uptake of BCG by BMM in a dose-dependent manner. CONCLUSION: LF was able to augment development of DTH when orally administered with OVA or BCG antigens. Inhibition studies suggest the involvement of the receptor with an affinity to mannose in mediation of the adjuvant effect. LF augmentation of the DTH response was partially effective when given in advance of oral delivery of the antigen; this effect could also be saturated by mannose. BCG studies provide preliminary evidence for LF in the potential augmentation of oral vaccination to prevent mycobacterial infection. In vitro experiments provide evidence that LF plays a role in modulation of antigen presenting cell activation.

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

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.

Lactoferrin modulation of antigen-presenting-cell response to BCG infection.

INTRODUCTION: Lactoferrin, an 80-kDa basic N-glycoprotein, has been identified as a potent immune modulator. When used experimentally as an adjuvant in mice, lactoferrin can boost the efficacy of the BCG vaccine to increase delayed type hypersensitive responses and to limit subsequent pathology upon infection with virulent mycobacterium. These studies outline preliminary findings to examine the multiple mechanisms of action of lactoferrin on antigen-presenting cells which would enhance vaccination and bridge the innate and adaptive responses. MATERIAL/METHODS: Bone marrow-derived macrophages (BMMs) and human and murine cells lines were infected with BCG in the presence or absence of bovine lactoferrin. The cells were examined for changes in uptake of BCG post infection and increases in the surface expressions of Class I or Class II molecules by flow cytometric analysis. Infected cultures were collected to examine cytokine production by ELISA. RESULTS: Lactoferrin was found to significantly increase the uptake of BCG organisms during infection of BMMs and human monocyte cell lines. Lactoferrin added to BCG-infected BMMs demonstrated significantly increased surface expression of Class II (I-Ab), but no change in Class I (H-2kb) molecules. In addition, BCG-infected cells incubated in the presence of lactoferrin demonstrated a significant increase in relative IL-12 to IL-10 ratios in a dose-dependent manner. CONCLUSIONS: Overall, lactoferrin was able to alter BCG-infected antigen-presenting cells (APCs) in vitroin a manner consistent with the induction of responses required for successful presentation of antigen to the adaptive arm of the immune response, which would lead to the generation of strong T-helper 1 type immunity.

Lactoferrin modulation of IL-12 and IL-10 response from activated murine leukocytes.

Lactoferrin possesses a wide range of immunomodulatory activities, including promotion of the delayed type hypersensitivity response (DTH) towards BCG (Bacillus Calmette Guerin) antigens. Addition of Lactoferrin as an adjuvant to the BCG vaccine was previously demonstrated to augment protection against subsequent mycobacterial challenge, with concomitant development of a strong T cell helper type 1 (TH1) immunity. Because generation of TH1 immunity is in large part dependent on the balance of monocytic pro- and anti-inflammatory cytokines, the effect of Lactoferrin on leukocytes was investigated. Lactoferrin enhanced proinflammatory responses in a dose-dependant manner from splenocyte and adherent (F4/80+) splenocyte populations, bone marrow derived monocytes (BMM), and J774A.1 cultured cells. In all scenarios tested, Lactoferrin induced a strong increase in the ratio of IL-12:IL-10 production from LPS stimulated cells. Examination of Lactoferrin effects on BCG infected J774A.1 cells and on BMM revealed similar immunomodulatory effects, with particularly strong increase in IL-12 production. Furthermore, immunization of mice with BCG admixed with Lactoferrin led to increased generation of CD4+ cells expressing IFN-gamma upon restimulation with BCG antigens. These results provide molecular evidence to support the role of Lactoferrin as an adjuvant candidate to augment development of DTH response to vaccine antigens.