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-γ and IL-6, but only limited changes to proinflammatory mediators TNF-α or IL-1β 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.

Introduction

The Mycobacterium bovis bacillus Calmette Guerin (BCG), an attenuated strain of M. bovis, is the current vaccine for tuberculosis (TB), a disease caused by the intracellular pathogen Mycobacterium tuberculosis (MTB). BCG is the most widely used vaccine in the world and has remained relatively unchanged since 1921 [1], [2]. The World Health Organization (WHO) estimates that roughly one third of the world's population is currently infected with MTB, and the incidence rate for new infections is approximately 0.6% per year [3]. While BCG is effective in protecting children against TB disease manifestations [4], the response wanes in adulthood, potentially leading to disease development upon infection [5]. The observed diminished efficacy of BCG vaccination has resulted in intensive research into developing novel TB vaccines that could surpass the efficacy of the current BCG vaccine [6], [7], [8]. Despite investigations into developing alternatives, BCG remains the gold standard for TB vaccines [9], [10], [11].

The BCG vaccine generates host protective responses against MTB infection by promoting development of a mycobacterial antigenic specific delayed type hypersensitivity (DTH) response, characterized as a T-cell helper type-1 (T_H1) immunity with antigen specific production of IFN-γ [12]. A proven experimental method for enhancing the efficacy of the existing BCG vaccine is through addition of adjuvant components that lead to host protection against MTB infection, resulting in decreased organ bacterial load, reduced lung inflammation and increased mycobacterial antigen specific production of IFN-γ [13], [14], [15].

A novel adjuvant candidate that could potentially enhance efficacy of the BCG vaccine is lactoferrin, an iron-binding protein found primarily in mucosal secretions and neutrophilic granules [16]. Lactoferrin possesses a wide range of immunomodulatory activities [17], [18], [19], [20], [21] including promotion of the T-cell dominated DTH response towards BCG antigens [22], [23]. The ability of lactoferrin to enhance the generation of antigen specific DTH responses suggests that lactoferrin could promote development of specific T-cell responses against a complex antigen, such as BCG [24], [25].

The development of T-cell helper type 1 (T_H1) immunity is, in part, regulated by production of IL-12 [26], [27]. A variety of in vivo studies have shown lactoferrin capable of increasing production of IL-12 [28], [29], [30]. IL-12 is clearly a critical component involved in directing T_H1 development effective in promoting protective host responses during MTB infection [31], [32]. More so, addition of lactoferrin to BCG infected macrophages increased the IL-12:IL-10 ratio, setting up an environment that could potentially promote BCG specific T_H1 development in vivo [25], [33]. It was therefore hypothesized that lactoferrin possesses adjuvant activity that could be useful to enhance the efficacy of the BCG vaccine, leading to an increase in host protection against subsequent infection with MTB.

These studies were designed to investigate various vaccine formulations of BCG admixed with lactoferrin and the effect of different vaccine delivery schedules to increase host protection against subsequent infection with virulent Erdman MTB and to generate DTH responses to BCG antigens. The overall goal was to maximize the lactoferrin-mediated augmentation of the BCG vaccine, to result in decreased organ bacterial load and reduced lung histopathology following challenge with virulent mycobacterium. In addition, splenic recall to BCG antigens were examined to monitor for changes in production of IFN-γ to assess the adjuvant activity of lactoferrin to stimulate generation of cell-mediated (T_H1) immunity [34], [35].