accines represent one of the most powerful and cost-effective mechanisms for prevention of infectious disease, with many successful efforts leading to significant reduction in morbidity and mortality due to microbial assault [41
]. Substantial progress has also been made during the past 15 years towards the development of improved vaccine for tuberculosis [7
], in large part due to the availability of genome sequences and advances in common antigenic characterization among multiple strains common to larger communities. The ultimate goal of vaccination to prevent tuberculosis disease encompasses not only long term protection to the individual, but also long term protection of the greater society [9
]. There are enormous obstacles to the effective control of tuberculosis [43
]. However, while the causative agent, Mycobacterium tuberculosis
(MTB), can infect many animals, there is no animal reservoir. Man is the only species from which the organism can escape to infect new subjects. The transmissibility of infection, and thus the organism’s continued existence, depends on its ability to escape from immune hosts to infect new ones. Therefore, the goal of a protective tuberculosis vaccine should include the ability to limit cavitary formation leading to subsequent spread of disease [10
The results presented are designed to provide a logical framework for development of a recombinant human lactoferrin based BCG vaccine that functions to limit immune related pathologies. It is well accepted that a cell-mediated immune (CMI) response of the TH
1 type, characterized by elevated production of gamma interferon (IFN-γ) and interleukin-12 (IL-12), is critical in development of protective immunity against M. tuberculosis
. Experiments reported here extend the understanding of immunomodulatory effects of lactoferrin to assist the BCG vaccine in augmentation of CMI resulting in protection of alveolar integrity upon subsequent infection. In this case, alveolar integrity is defined as limited acute destruction of lung parenchyma that culminates in a destructive pulmonary pathology. Indeed, these studies give strong support for the potential of lactoferrin, especially the fully humanized recombinant molecule, to function as an adjuvant component to augment primary interactions that lead to elevated production of TH
1 cellular responses upon vaccination. The mice that were immunized with recombinant human lactoferrin of the sialylated form demonstrated antigenic recall responses with significantly increased IFN-γ, and a shift towards greater IL-12 production. Overall, this suggests that treatment with recombinant human lactoferrin adjuvant augments a local environment that would potentiate development of T cells towards a recall TH
1 phenotype to BCG antigens. The mechanisms for augmentation of response are not fully understood at this time, but bovine lactoferrin-induced immune modulation of T cell responses have been presented elsewhere [30
]. Specific description of modulation of activated macrophages in cytokine secretion and antigen presentation that would lead to enhancement of antigenic presentation and direction of focused T helper responses has been reported [29
]. It is hypothesized that similar mechanisms would allow human lactoferrin to function in this same manner.
Addition of lactoferrin to the BCG vaccine generated protective responses upon subsequent infection with virulent mycobacterium, with decreased immune related pathology due to infection. The effects of lactoferrin adjuvant based vaccination protocols also appeared to be long lasting; protection was maintained in the sialylated form of the recombinant human lactoferrin group through 150 days post challenge. The use of human recombinant lactoferrin in the vaccine allowed an augmented level of lymphocyte recall response to mycobacterial antigen that correlated well with observed cytokines in lung tissue soon after infection in immunized mice. Of major interest was that the group immunized with the sialylated form of the recombinant lactoferrin demonstrated a marked early increase in IL-6 with concurrent reduction in IL-4 (indicating indirectly an effective push to TH
1 response) rather than a mixed format as seen in mice immunized with BCG alone. Specifically, increased level of IFN-γ was generated, which has been shown to correlate well with a reduction in disease progression [24
]. IL-6, although not essential for development of protective immunity, is involved in stimulating early IFN-γ production [48
]. The uniqueness with the novel recombinant is the drop in IL-4, indicating a functional shift away from early activated TH
2 response, which also correlates well with disease progression in vaccinated humans [49
], theoretically undermining TH
1-mediated immunity to drive inappropriate alternative activation of macrophages and development of pathological damage in the host.
Of surprise to expected findings, the bacterial load with lung and spleen tissue was not greatly decreased in the lactoferrin vaccinated groups, compared to mice vaccinated with BCG alone. A modest reduction in liver CFU was apparent, perhaps indicative of delayed dissemination of organisms. However, the overall difference between vaccination groups was manifested primarily in reduced development of pathological damage and lung occlusion. Studies in the guinea pig suggest that survival following challenge may occur in vaccinated animals in the absence of decreased early bacillary loads [8
]. Dascher, et al., speculate that use of defined adjuvants may alter development of T cell subsets which function more effectively over longer periods of infection. [20
]. The data described here is in agreement with assessment that prolonged survival may be predicated on changes in the pathological manifestation of disease within lung tissue [51
], with improvement seen in the absence of decreased early bacillary loads. In this scenario, increase in lymphocytic content is seen in vaccinated mice in the absence of help to macrophages to increase bactericidal effector function. This could limit the magnitude of pathological damage, even in the absence of markedly reduced CFU.
Multiple mechanisms of action have been described for bovine lactoferrin to function as an immune modulator [28
], While these studies are scientifically informative, bovine lactoferrin is only 70% homologous to human lactoferrin in amino acid sequence [54
], and contains markedly different glycosylation patterns [55
]. Furthermore, the bovine form is not amenable for parenteral utility formulated with a human vaccine. While many options are available to produce recombinant human lactoferrin, full functionality as an immune modulator appears dependent upon both glycosylation and sialylation of the molecule [38
]. There are two primary forms of human lactoferrin, one contained in exocrine secretions including milk, tears, saliva, bronchial and intestinal secretions. The other form is present in the secondary granules of neutrophils. While the two forms of human lactoferrin are identical in their amino acid sequence, they differ in sugar moieties. The granulocytic lactoferrin is not fucosylated thereby allowing transduction of signals that do not require fucose-specific receptors such as the mannose receptor [58
]. In addition, while the secreted form is thought to be involved in the host defense against microbial infection at mucosal sites, the granulocytic lactoferrin has notable immunomodulatory function [31
]. The comparative immunizations investigated in this study extend these observations, with the milk-derived human lactoferrin determined as less effective in adjuvant function. The different glycosylation pattern for milk-derived lactoferrin and neutrophilic form (replicated as rhLF) may be responsible for the difference in adjuvant activity. One possibility is that fucosylated lactoferrin (milk-derived) has lower affinity to the receptor that is responsible for signal transduction mechanism in those cells. While intuitive in hindsight, it would be an evolutionary advantage for neutrophils recruited to sites of inflammation to release a glycosylated-modified molecule which could both function locally to inhibit bacterial growth as well as exert effects on macrophage responses to modulate their ability to present novel antigens to subsequent interacting lymphocytes.
Overall, it is prudent to continue to search for alternative mechanisms to boost the current BCG vaccine, especially in light that no research vaccine against tuberculosis that can consistently surpass BCG in protecting the host is near FDA approval. The studies here suggest that fully humanized recombinant lactoferrin produced in Pichia pastoris may have utility to augment the BCG vaccine and confer protection against formation of cavitary disease after infection with mycobacteria. This new knowledge of lactoferrin immunomodulation paves the way to more general design of T cell-dependent vaccines that incorporate naturally occurring granulocytic components.