Lactoferrin: An adjuvant to enhance the efficacy of the BCG vaccine

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 virtually unchanged since 1921. Intensive research is focused on developing a TB vaccine that can surpass and improve the existing BCG vaccine. 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, Lactoferrin enhanced the ratio of IL-12:IL-10 production from macrophages stimulated with LFS or infected with BCG, indicating the potential to affect T-cell development in vivo. Five different vaccination protocols were investigated for generation of host protective responses against MTB infection using Lactoferrin admixed to the BCG vaccine. Mice immunized and boosted at 2 weeks with BCG/Lactofefrin increased host protection against MTB infection by decreasing organ bacterial load and reducing lung histopathology. The observed postchallenge results paralleled with increasing production of IFN-γ, IL-2, TNF-α, and IL-12 from BCG stimulated splenocytes. In vitro studies examined possible mechanisms of Lactoferrin action on BCG infected macrophages and dendritic cells. Addition of Lactoferrin to BCG infected macrophages and dendritic cells increased stimulation of presensitized CD3+ and CD4+ T-cells. Analysis by fluorescent activated cell sorting (FACS) revealed an increase in surface expression of MHC I and decreased ratio of CD80/86 from BCG infected macrophages cultured with Lactoferrin. In contrast, Lactoferrin decreased surface expression of MHC I, MHC II, CD80, CD86, and CD40, but increased CD 11c, from BCG infected dendritic cells, indicating involvement of adhesion molecules. Overall, these studies indicate that Lactoferrin is a useful and effective adjuvant to improve efficacy of the BCG vaccine by enhancing generation of mycobacterial antigen specific T-cell responses through promotion of antigen presentation and T-cell stimulation.^

NOVEL MECHANISMS OF ANTIGEN PROCESSING THAT ENHANCE BCG VACCINE EFFICACY

Mycobacterium tuberculosis, the causative agent of tuberculosis, is the most lethal single infectious agent afflicting man today causing 2 million deaths per year. The World Health Organization recommends a vaccine as the best option to prevent this disease. The current vaccine, BCG, has a variable efficacy and does not protect adults. It is known that BCG vaccine becomes sequestered in special phagosome compartments of macrophages that do not fuse with lysosomes. Since lysosome fusion is necessary for peptide production and T cell priming leading to protective TH1 immunity, we hypothesized that vaccine efficacy is reduced and occurs perhaps due to non-lysosome dependent mechanisms. We therefore proposed an in depth analysis of phagosome environment, and its proteome to unravel mechanisms of antigen processing and presentation. We initially discovered that three mechanisms of pH regulation including vacuolar proton ATPase, phagocyte oxidase and superoxide dismutase (SOD) secretion from BCG vaccine affect antigen processing within phagosomes. These studies led to the discovery that a mutant of BCG vaccine which lacked SOD was a better vaccine. Subsequently, the proteomic analysis of vaccine phagosomes led to the discovery of novel protease (γ-secretase) enriched on BCG vaccine phagosomes. We then demonstrated that these proteases generated a peptide from the BCG vaccine which was presented through the MHC-II pathway to T cells and induced a TH1 response. The specificity of antigen production from γ-secretase was confirmed through siRNA knockdown of the components of the protease namely, nicastrin, presenilin and APH, which led to a decrease in antigen presentation. We therefore conclude that, even though BCG phagosomes are sequestered and do not fuse with lysosomes to generate peptide antigens, there are complex and novel in situ mechanisms within phagosomes that are capable of generating an immune response. We conclude that TH1 immunity to BCG vaccine arises mostly due to non-lysosome dependent immune mechanisms of macrophages and dendritic cells.