WO2006063329A2 - Enhanced protection against mycobacterium tuberculosis - Google Patents

Abstract

The method of the present invention provides a novel use of Lactoferrin as an adjuvant composition suitable to be used in vaccines. More specifically, the present invention is directed to the use of Lactoferrin to augment a vaccine efficacy by generation of T helper response and subsequent protection against challenge with a virulent pathogen. Also provided by the present invention is a method of manufacture of the adjuvant and it use as medicament.

Description

ENHANCEDPROTECTIONAGAINSTMYCOBACTERIUMTUBERCULOSIS

Field of the Invention

The present invention relates to lactoferrin as an adjuvant composition suitable to be used in vaccines or in conjunction with administered vaccines. In particular, lactoferrin is used to augment a vaccine efficacy by generation of T lymphocyte response and subsequent protection against challenge with a virulent pathogen. Also provided by the present invention is a method of manufacture of the adjuvant and it use as medicament.

Background of the Invention

Tuberculosis (TB) is the leading cause of morbidity due to an infectious disease and is a serious, unresolved burden upon the world's population despite aggressive vaccine implementation and progressive antibiotic treatment. The causative agent is Mycobacterium tuberculosis (MTB), an intracellular bacterium whose primary host cell is the macrophage. It is estimated that over a third of the world's population is infected with MTB, with incidence of infection continuously on the rise (World-Health-Organization. 2003. Global Tuberculosis Control. Surveillance, Planning, Financing. The World Health Report 2003. World Health Organization, Geneva, Switzerland). The 2003 World Health Organization (WHO) report on tuberculosis (TB) incidence rates around the world shows dense epidemic areas where TB vaccine is widely applied clearly illustrating the limitations of the BCG vaccine towards spread of disease (Lietman, T., and S. M. Blower. 2000. Potential impact of tuberculosis vaccines as epidemic control agents. Clin Infect Dis 30 Suppl 3:S316). Delining BCG efficacy will continue to be an obstacle in eradicating tuberculosis; a modeling study predicted that a MTB vaccine with only 50% efficacy would save thousands of lives in the next 10 years (Murray, C. J., and J. A. Salomon. 1998. Modeling the impact of global tuberculosis control strategies. Proc Natl Acad Sci U S A 95:13881 ). Accompanying strategies for the eradication of TB have included aggressive managed treatment, such as the directly observed treatment short course program (DOTS), however, dramatic strides in improving TB incidences will only occur with the development of improved vaccines and adjuvants. Protection against MTB infection requires host generation of a strong cell mediated immunity (CMI); specifically, a T-cell mediated delayed type hypersensitivity (DTH) response, involving the activation of both CD4+ and CD8+ lymphocytes and development of a strong T-cell helper type-1 (TH1) response as indicated by production of interferon gamma (IFN-κ) and macrophage-, dendritic cell- , or NK cell-derived interleukin-12 (IL-12), activating host macrophages and leading to bacterial clearance (Flynn, J. L. 2004. Immunology of tuberculosis and implications in vaccine development. Tuberculosis (Edinb) 84:93 and Bloom, B. R., J. Flynn, K. McDonough, Y. Kress, and J. Chan. 1994. Experimental approaches to mechanisms of protection and pathogenesis in M. tuberculosis infection, lmmunobiology 191 :526). DTH contributes to the development of protective granulomatous response, limiting organism dissemination. The widely used TB vaccine is a live attenuated strain of Mycobacterium bovis Bacillus Calmette-Guerin (BCG). The global efficacy of BCG in generating a protective host response against MTB has fallen, especially in regards to preventing adult onset disease. BCG as a live vaccine has historically been more efficacious than killed or subunit vaccines by being able to induce CMl required for protection. BCG remains the gold standard by which other vaccines are judged. Advantages of the BCG vaccine lie in its potential ability to persist in vivo for longer periods of time and to induce prolonged immunological memory, thus generating CMI and immune responses at mucosal surfaces at relatively low cost. However, efficacy of BCG has fallen during the past two decades, especially in protection against adult pulmonary tuberculosis. Clinical studies indicate a range of efficacy from 80% effective in the United Kingdom to almost 0% efficacy in India, Japan, and Malawi (Brennan, M. 2004. A new generation of tuberculosis vaccine. In Vaccines: Preventing Disease & Preventing Health. C. A. de Quadras, ed. Pan American Health Organization, Washington, DC, p. 177 and Colditz, G. A., T. F. Brewer, C. S. Berkey, M. E. Wilson, E. Burdick, H. V. Fineberg, and F. Mosteller. 1994. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. Jama 271 :698). The reasons for this are unclear at this time. The most important problem with the current BCG vaccine is its inability to generate and sustain the necessary protective DTH and CMI responses needed for control of MTB infection. It is also clear that there is a great need to develop novel vaccines or adjuvants that can overcome the failure of BCG, offering protection against infection and limiting dissemination of organisms. To date, no research vaccine against tuberculosis has been able to consistently surpass BCG in reduction of lung organisms following challenge with virulent MTB (McMurray, D. N. 2003. Recent progress in the development and testing of vaccines against human tuberculosis, lnt J Parasitol 33:547). Multiple alternative vaccines failed to protect against MTB. Indeed, surpassing BCG seemed an unrealistic goal that may never be accomplished. The need for new MTB vaccines is paramount, as evident by the number of new vaccine formulations currently in the process of Phase I clinical testing. These include, but are not limited to, vaccines utilizing live attenuated MTB, recombinant BCG with MTB Ag85 antigens, inactived M. vaccae organisms, DNA vaccines utilizing Heat Shock protein 65 DNA, dendritic vaccines incorporating Ag85-ESAT6 proteins, and modified (auxotrophic) organisms (Wang, J., and Z. Xing. 2002. Tuberculosis vaccines: the past, present and future. Expert Rev Vaccines 1 :341 and Kumar, H., D. Malhotra, S. Goswami, and R. N. Bamezai. 2003. How far have we reached in tuberculosis vaccine development? Crit Rev Microbiol 29:297).

Employing adjuvants as a strategy to improve BCG vaccine efficacy is a major world-wide research focus. Most adjunct adjuvants, while effective in enhancing humoral immunity, fail to increase T-cell responses considered protective during subsequent MTB challenge. Presently, the model adjuvant capable of promoting generation of CMI and DTH responses is complete Freund's adjuvant (CFA); a water-in-oil emulsion containing 50% mineral oil, emulsifying agent Arlacel A, and heat-killed avirulent MTB strain, H37Ra. CFA is highly toxic; it is not suitable for human use and becoming more undesirable for use in animals. Recent advances in adjuvant development are directed towards generating vaccines that approach the efficacy of CFA while possessing none of its toxic properties (Brennan, M. 2004. A new generation of tuberculosis vaccine. In Vaccines: Preventing Disease & Preventing Health. C. A. de Quadras, ed. Pan American Health Organization, Washington, DC, p. 177).

A promising natural adjuvant candidate is Lactoferrin, an 8OkDa iron binding protein commonly found in secretory fluids and present in secondary granules of neutrophils (Zimecki, M., J. Artym, G. Chodaczek, M. Kocieba, and M. L. Kruzel. 2004. Protective effects of lactoferrin in Escherichia coli-induced bacteremia in mice: Relationship to reduced serum TNF alpha level and increased turnover of neutrophils, lnflamm Res 53:292 and Legrand, D., E. Elass, A. Pierce, and J. Mazurier. 2004. Lactoferrin and host defense: an overview of its immuno-modulating and anti-inflammatory properties. Biometals 17:225). Lactoferrin is non-toxic, and has been examined in depth for its ability to modulate excessive proinflammatory responses during sepsis (Kruzel, M. L., Y. Harari, D. Mailman, J. K. Actor, and M. Zimecki. 2002. Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice. Clin Exp Immunol 130:25 and Adamik, B., M. Zimecki, A. Wlaszczyk, P. Berezowicz, and A. Kubler. 1998. Lactoferrin effects on the in vitro immune response in critically ill patients. Arch Immunol Ther Exp (Warsz) 46:169). Cell surface receptors for Lactoferrin have been identified on multiple leukocyte populations, including macrophages and lymphocytes (Suzuki, Y. A., and B. Lonnerdal. 2002. Characterization of mammalian receptors for lactoferrin. Biochem Cell Biol 80:75). Lactoferrin stimulates T cells to proliferate, promotes lymphocyte maturation, increases surface expression of leukocyte functional adhesion molecule LFA, and enhances natural killer cell activity (Kruzel, M. L., and M. Zimecki. 2002. Lactoferrin and immunologic dissonance: clinical implications. Arch Immunol Ther Exp (Warsz) 50:399).

Lactoferrin exhibited adjuvant properties through generation of lymphocytic responses with suboptimal antigen doses (Zimecki, M., and M. L. Kruzel. 2000. Systemic or local co-administration of lactoferrin with sensitizing dose of antigen enhances delayed type hypersensitivity in mice. Immunol Lett 74:183). Preliminary studies also demonstrated efficacy of Lactoferrin to boost DTH responses against multiple antigens, including BCG. Lactoferrin stimulated naϊve macrophages to increase production of IL-12 (Actor, J. K., S. A. Hwang, M. Olsen, M. Zimecki, R. L. Hunter, Jr., and M. L. Kruzel. 2002. Lactoferrin immunomodulation of DTH response in mice, lnt lmmunopharmacol 2:475), an essential cytokine produced by macrophages and dendritic cells to promote TH1 immunity; thereby identifying a potential molecular mechanism by which Lactoferrin promotes antigen specific lymphocytic responses. According to present invention, Lactoferrin is a useful adjunct adjuvant to boost efforts to BCG based vaccines and perhaps to assist through augmentation of other vaccine strategies as well. Summary of the Invention

The method of the present invention provides a novel use of lactoferrin to modulate the molecular events during development of pathogen-induced immune responses in humans. More specifically, the present invention is directed to the use of lactoferrin as an adjuvant to enhance effectiveness of vaccine, and/or reduce disease related pathology manifested after subsequent infection with infectious organisms.

Brief Description of the Drawings

The present invention will be further understood from the following detailed description and Examples with reference to the accompanying drawings, in which:

Figure 1 shows that Lactoferrin increases IL-12 production from stimulated macrophages. J774A.1 (top) or RAW 264.7 cells (bottom) were stimulated with LPS (200 ng/ml) and increasing concentrations of Lactoferrin (1 to 1000 μg/ml). Supematants were assessed for IL-12 (closed bar) and IL-10 (open bar). Average values (pg/ml) with standard errors are shown. *, p<0.05 compared to LPS alone.

Figure 2 shows Stimulation (Proliferation) Index of splenocytes from mice immunized with BCG. Splenocytes from mice immunized one time with BCG in IFA ( ), BCG in IFA with Lactoferrin (100 μg/mouse) (D), or BCG in CFA (D) were assessed for proliferative response to Heat-Killed BCG. Average Stimulation Index values with standard errors are shown. Responses are compared to non-immunized control mice ( ). *, p<0.05; **, p<0.05 as measured by two-tailed unpaired Student's t-test for indicated comparisons.

Figure 3 shows that BCG Immunization with adjunct Lactoferrin adjuvant augments proinflammatory mediators from splenocytes. Splenocytes from mice immunized one time with BCG in IFA ( ), BCG in IFA with Lactoferrin (100 μg/mouse) (D), or BCG in CFA (D) were assessed for response to Heat-Killed BCG. Supematants were assessed by ELISA for TNF-cr, IL-1/?, and IL-6. Average values (pg/ml) with standard errors are shown. Responses are compared to non-immunized control mice ( ). *, p<0.05 vs non-immunized; **, p<0.05 vs. BCG alone; ***, p<0.05 CFA vs. Lactoferrin group. Figure 4 shows that adjunct Lactoferrin adjuvant increases TH1 cytokine IFN- D from splenocytes, as well as TNF-a and IL-6 production. Splenocytes from mice immunized one time with BCG in IFA ( ), BCG in IFA with Lactoferrin (100 μg/mouse) (D)₁ or BCG in CFA (D) were assessed for production of IFN-K, TNF-a, or IL-6 in response to Heat-Killed BCG. Average values (pg/ml) with standard errors are shown. Responses are compared to non-immunized control mice ( ).^*, p<0.05 vs. non-immunized; **, p<0.05 vs. BCG alone; ***, p<0.05 CFA vs. Lactoferrin group.

Figure 5 shows that BCG vaccination with adjunct Lactoferrin adjuvant limits mycobacterial dissemination following aerosol challenge with MTB. Mice immunized one time with BCG in IFA ( ), BCG in IFA with Lactoferrin (100 μg/mouse) ( ), or BCG in CFA ( ) were aerosol challenged with approximately 100 CFU virulent MTB, strain Erdman. Non-immunized controls are indicated (D). 28 days post infection lung tissue (left) was assessed for bacterial load and spleen tissue (right) was examined for dissemination of organisms to peripheral tissue. Individual CFU values for 4 mice per group are shown; bars indicate average for group. Responses are compared between groups. *, p<0.05 vs. non-immunized. 3 of 4 mice in the Lactoferrin group exhibited sterilizing immunity in the spleen; values were placed at organism limit of detection (Log10 of 500 CFU/organ = 2.69). Data representative of two experiments with similar results and reduction of CFU.

Figure 6 shows that BCG admixed with Lactoferrin increased antigen specific IFN-/ production during recall response. Splenocytes were isolated from mice immunized with 10⁶ BCG/mouse with or without 100//g/mL of Lactoferrin (boosted at 2 weeks) at 6 weeks post-boost and stimulated with Heat Killed-BCG (10:1). Supernatants were collected at 72 hours and analyzed by ELISA.

Figure 7 shows that BCG vaccination with adjunct Lactoferrin adjuvant limits MTB induced pathological damage. Mice were immunized twice with BCG, or with BCG and Lactoferrin. 4 weeks after final immunization, mice were aerosol challenged with approximately 100 CFU virulent Erdman MTB. Mice were sacrificed 65 days post challenge, lungs were isolated and sectioned for histology (H&E, 4Ox and 100x). Control (non-immunized) mice also shown. Detailed Description of the Invention

The present invention provides novel techniques which can be employed for enhancing effectiveness of vaccination protocols. Lactoferrin effects are demonstrated by the ability to elicit in vitro and in vivo responses to BCG. Stringent conditions were applied to demonstrate Lactoferrin's ability to enhance BCG vaccine efficacy for protection against subsequent aerosol challenge. For example, for these studies, mice were administered vaccine, thus allowing for identification of differences in response generation. All three vaccines (BCG alone, BCG in CFA, BCG with lactoferrin) were productive in limiting mycobacterial growth in the organ of implantation, with lung CFUs reduced nearly one log after only 28 days. However, the Lactoferrin group was superior in limiting bacterial dissemination, with 3 of 4 mice exhibiting sterilizing immunity in the spleen. Although the other two vaccinated groups had reduction in splenic CFUs, neither was able to achieve sterilizing immunity and completely inhibit or control spread of organisms to that tissue.

A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for the purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Methods of immunology and biochemistry used but not explicitly described in this disclosure and these Examples are amply reported in the scientific literature and are well within the ability of those skilled in the art.

Cell lines: Two murine macrophage cell lines, J774A.1 and RAW 264.7, purchased from American Type Culture Collection (ATTC), were used for stimulation experiments. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma) and 0.01% HEPES (Sigma) and L-Arginine (Sigma). Macrophages were plated into 24 well plates at 1x10⁶ cells/mL/well using the media as outlined in 3.1. Triplicate cells of macrophages were stimulated with 200ng/mL of lipopolysacrridθ (E. coli 0111 :B4 LPS; approximately 3x10⁶ E.U./mg) (Sigma), with increasing concentrations of bovine Lactoferrin (1 Dg, 10Dg, 100Dg, 500Dg, and 1 mg/ml_), with Lactoferrin and LPS. Low endotoxin bovine Lactoferrin utilized was characterized as <1 E.U./mg, less than 25% iron saturated, and >95% purity. Cells were incubated at 37⁰C with 5% CO₂. Supernatants were collected after 72hrs and frozen at -20oC until analyzed by ELISA for cytokine production.

Microorganisms: The BCG Pasteur strain (TMC 1011 , ATCC, Manassas, VA) was grown in Dubos base (without addition of glycerol) with 10% supplement (5% BSA and 7.5% dextrose in saline) on an orbital shaker at 37°C for two weeks before use. The Erdman strain of Mycobacterium tuberculosis (TMC 107, ATCC) used for challenge was grown in Dubos base (with 5.6% glycerol) with 10% supplement for three weeks before use. Cultures were taken during log growth period. Organisms were washed with 1x PBS (Dulbecco's Phosphate buffered saline 10X, Cellgro, Herndon, VA) and resuspended in 1x PBS. Suspensions were sonicated for 5 seconds prior to use. Bacterial concentration was determined using McFarland standards, and confirmed by plating dilutions onto 7H11 agar plates (Remel, Lenesa, KS). Plates were incubated at 37⁰C with 5% CO2 for 3 to 4 weeks, and colonies were enumerated. Heat-killed BCG (HK-BCG) was produced by autoclaving the BCG suspension in 1x PBS at 121°C for 20min. Death of BCG was verified by plating of autoclaved BCG on 7H11 plates.

Immunizations: Immunizations were performed using standard NIH protocols for evaluation of BCG vaccines, modified as follows. Ten to twelve female C57BL/6 mice (4-5 weeks, Jackson Laboratories, Bar Harbor, ME) were immunized per group with 10O/vI of the formulation, subcutaneously (s.c.) at the base of the tail. All formulations of BCG with or without Lactoferrin utilized BCG at 10⁶ CFU/mouse. Lactoferrin was administered at 100 //g/mouse. BCG and Lactoferrin were emulsified with Freund's adjuvant in a 1 :1 ratio. IFA and CFA were used as described previously.

Antigenic Responses: 17 days post-immunization, spleens were harvested from each immunization group, homongenized, and red-blood cells lysed by ACK buffer (Cambrex Bio Science, East Rutherford, NJ). Splenocytes were washed 2x with 1x PBS and resuspended in DMEM, supplemented with 10% heat-inactivated FBS, 0.005% 2-mercaptoethanol (2Me, Gibco, Carlsbad, CA), 0.01 % penicillin G (Sigma) and Gentamycin (Sigma), 0.01 % HEPES (Sigma) and L-Arginine (Sigma). Cells were plated at 10⁶ cells/mL and stimulated with HK-BCG at 5:1 ratio. 4 to 6 mice were used per group. Assay was done in duplicate or triplicate. Supernatants were collected at 24 and 72 hrs and frozen at -2O⁰C until evaluation by ELISA. Recall response experiments were repeated a total of 3 times with similar results.

Proliferation Assay: Splenocytes at 5x10⁵ cells/1 OOμL/well were plated in DMEM without phenol red (Sigma) and stimulated with HK-BCG at 5:1 ratio. After 48 hours, proliferation was measured using the MTT assay (Sigma) following manufacture protocols. MTT was dissolved in 1x PBS at 5mg/mL and filtered through a 0.2μm filter. 10//L of MTT solution was added to each well, and plates were incubated at 37oC for 4 hours. Supernatants were removed and 100μl_ of 0.1 N of hydrochloric acid (HCI) in anhydrous isopropanol was added. 4 to 6 mice were used per group; assay was performed in triplicate. Absorbance was read at 570nm subtracting background at 690nm. Proliferation Index was calculated relative to naϊve control splenocytes in the absence of HK-BCG stimulation.

ELISA: Supernatants were assayed in triplicate for cytokine production using enzyme-linked immunosorbent assay (DuoSet ELISA kit, R&D Systems, Minneapolis, MN), according to manufacture's instructions. Supernatants were analyzed for T-cell cytokine \FN-y, cellular mediators of TH1 response (IL-12p40, IL- 10), and proinflammatory mediators (IL-1/?, IL-6, TNF-σ). IL-1/?and TNF-α were analyzed at 24 hrs, all others at 72 hrs. Values (pg/ml) were by regression analysis of data to standard curves generated.

Erdman challenge: 14 days post-immunization, 4 mice from each group were aerosol challenged with Erdman MTB (13). Each mouse was infected via aerosol with <100CFU/mouse. Aerosol infection was achieved using an inhalation exposure system (IES) (GLAS-COL Model #A4212 099c Serial # 377782). Verification of infectious dose was accomplished at 1 day post-infection; 4 mice were sacrificed, lungs collected and homogenized, and dilutions plated onto 7H11 agar plates for CFU counts. 28 days after Erdman challenge, mice were sacrificed. Lung and spleen tissues were isolated, homogenized, and plated on 7H11 agar plates for CFU determination. Plates were incubated at 370C for 3 to 4 weeks before enumeration. Statistics: One way ANOVA comparison or Student's t-test were used for data analysis. Statistical significance was assigned for values of p<0.05 or as indicated, comparing average values and standard errors between groups.

EXAMPLE 1

Lactoferrin enhances IL-12 production from stimulated macrophages.

Lactoferrin was examined for the ability to enhance IL-12 production in vitro from LPS stimulated macrophage cell lines. J774A.1 or RAW 264.7 cultured macrophages were stimulated with LPS (200ng/mL) in the presence of increasing Lactoferrin concentrations (Figure 1 ). Stimulation of both cell lines with LPS led to production of IL-12(p40), with J774A.1 cells slightly more responsive than the RAW 264.7 line. Addition of Lactoferrin led to significant (p<0.05) increase in IL-12 from both cell lines. Increased IL-12 was apparent at 100 μg/ml of Lactoferrin and above. The RAW 264.7 cells were more sensitive to Lactoferrin treatment with increased IL- 12 production at all Lactoferrin concentrations tested from 1μg/ml through 1000 μg/ml. J774A.1 or RAW 264.7 macrophages did not produce any significant levels of IL-12p40 without stimulation with LPS or when stimulated with Lactoferrin alone (not shown).

In a converse relationship, Lactoferrin reduced the production of IL-10 from LPS stimulated macrophages (Figure 1). The J774A.1 cell line produced approximately 81 μg/ml IL-10; addition of 100 μg/ml Lactoferrin and above led to significant (p<0.05) decrease. Although the levels of IL-10 produced upon LPS stimulation from the RAW 264.7 cell line was lower, Lactoferrin was able to reduce IL-10 production at higher concentrations. J774A.1 or RAW 264.7 macrophages did not produce any significant levels of IL-10 without stimulation with LPS or when stimulated with Lactoferrin alone (not shown).

Overall, Lactoferrin was able to affect LPS stimulation of macrophage cell lines, resulting in an increased ratio of IL-12:IL-10 production, leading to increased production of mediators necessary for driving TH1 mediated functions.

EXAMPLE 2

Lactoferrin enhances in vivo development of TH1 mediators to BCG antigen. Lactoferrin was examined for ability to effectively increase BCG immunization for promotion of TH1 immunity. Immunization conditions were stringent to investigate early events in generation of response. Mice were immunized only once s.c. with 10⁶ BCG emulsified in IFA, in IFA and Lactoferrin, or in CFA. Splenocytes were harvested 17 days post-immunization and stimulated in vitro with Heat-Killed- BCG (HK-BCG) at a ratio of 5:1. Supernatants were collected after 72 hrs and analyzed for IL-12(p40) and IL-10 (Table I).

Table I. BCG Immunization with adjunct Lactoferrin adjuvant increases IL- 12(p40):lL-10 ratios (with standard deviation).

A single administration of BCG in IFA and Lactoferrin resulted in significantly increased production of IL-12 in the splenic recall assay (p<0.05), with 86 pg/ml produced. Likewise, the CFA positive control immunization group also demonstrated significant increase in IL-12 production, relative to non-treated mice. In comparison, with only one immunization and short time to recall, the group receiving BCG emulsified in IFA alone did not generate significantly increased IL-12 production (50 pg/ml). Evaluation of IL-10 was also performed; immunization with Lactoferrin did not significantly increase production of this cytokine. Splenic recall to HK-BCG demonstrated only modest increases in IL-10 production for both the IFA alone and IFA with Lactoferrin groups. In contrast, BCG administered in CFA demonstrated significant increase in IL-10.

A comparison of IL-12 to IL-10 produced revealed strong and significant shift in response generated between groups. Immunization in IFA and Lactoferrin adjuvant demonstrated a significantly higher ratio of IL-12:IL-10 for the IFA and Lactoferrin group (ratio of 1.43) compared to either IFA or CFA alone (ratios of 0.70 and 0.78 respectively).

EXAMPLE 3

Lactoferrin enhances in vivo development of proinflammatory mediators and IFN-K.

Splenocytes from Lactoferrin immunized mice demonstrated strong proliferative response to HK-BCG (Figure 2). A comparison of stimulation index (Sl) revealed specific proliferation to antigen was significantly enhanced for the IFA with Lactoferrin and for the CFA immunized groups (2.81 ±0.43 and 4.24 ± 0.42) versus the non-immunized group (1.31 ±0.23) or BCG emulsified in IFA group (0.58 ±0.25).

The assay was extended to further examine generation of proinflammatory response (TNF-α, IL-/? and IL-6) in antigenic recall to HK-BCG. Splenocytes from individual mice were stimulated with HK-BCG (5:1 ). Supernatants were collected and assayed (Figure 3). In concert with the increased stimulation index, there was significant production of all three proinflammatory mediators in the Lactoferrin immunization group compared to both the non-immunized and IFA immunized groups (p<0.05). Likewise, the CFA immunization group remained statistically higher for TNF-α, IL-/? and IL-6, with TNF-α and IL-6 markedly elevated compared to all other groups.

IFN-K response was measured as a direct indicator of generated TH1 response, and as a marker for required function of vaccine efficacy to protect against virulent mycobacterial infection (Figure 4). Increased IFN-κ was found upon in vitro stimulation with HK-BCG. Specifically, the IFA/Lactoferrin group produced 556.2 ±63.8 pg/ml IFN-K compared with 149.5 ±7.6 pg/ml for the IFA alone group. As expected, the positive control CFA adjuvant group generated the highest level of IFN-K with 2668.0 ± 136.6 pg/ml.

EXAMPLE 4

Vaccination with Lactoferrin adjuvant increases protection against challenge with virulent M. tuberculosis. Immunized mice were aerosol challenged with virulent Mycobacterium tuberculosis, strain Erdman (<100 CFU per lung). Tissue was obtained four weeks following implantation and CFU were enumerated. All immunization groups showed significant reduction in lung organism load (p<0.05) compared to the non-immunized control, with nearly 1 log less organisms present indicating local growth control within the tissue of implantation (Figure 5, left). Of major importance is the ability to restrict organism dissemination to peripheral organs following aerosol challenge. In this regard, CFUs enumerated from spleen tissue detailed differences between immunization groups (Figure 5, right). In the spleen, the non-immunized mice showed 4.49 ± 0.25 log CFU, indicating spread of organisms to that tissue. In contrast, all BCG immunized groups limited dissemination. The IFA and Lactoferrin adjuvant group demonstrated the largest and most consistent reduction (p<0.065) in bacterial load within the spleen (2.760 ± 0.32 log CFU); three of 4 mice demonstrated sterilizing immunity in that tissue with CFU levels below the limit of detection (500 CFU per organ). Indeed, the Lactoferrin immunized group further reduced bacterial loads compared to the IFA alone group (3.278 ±0.23 log CFU) and the CFA group (3.243 ±0.27 log CFU). At this day 28 time point, no detectable organisms were found in the liver for any group, including the non-vaccinated controls (data not shown).

EXAMPLE 5

Lactoferrin Adjuvant Admixed with BCG vaccine increases antigen specific IFN- y production and limits tuberculosis related pathology.

Mice were immunized subcutaneously (s.c.) at the base of the tail and boosted at 2 weeks. The immunization groups were: 1 ) non-immunized, 2) 10⁶ BCG (ATCC)₁ and 3) 100ug of Lactoferrin with 10⁶ BCG. Figure 6 shows that BCG admixed with Lactoferrin increased antigen specific IFN-K production during recall response. 10⁶ Splenocytes were isolated from mice immunized with 10⁶ BCG/mouse with or without 100μg/mL of Lactoferrin (boosted at 2 weeks) at 6 weeks post-boost and stimulated with Heat Killed-BCG (10:1). Comparisons were made to non-immunized controls. Supernatants were collected at 72 hours and analyzed by ELISA for IFN-K, TNF-a and IL-6. For all three cytokines tested, the immunization with BCG and lactoferrin generated significant recall responses from splenocytes which was significantly greater in magnitude to responses generated by splenocytes isolated from BCG alone immunized mice, or from splenocytes isolated from non- immunized controls. This confirms the ability to generate specific IFN-K producing cells in mice immunized in the presence of lactoferrin adjuvant.

Lactoferrin adjuvant also increased the protection against subsequent challenge with virulent organisms. Mice were immunized subcutaneously (s.c.) at the base of the tail and boosted at 2 weeks. There were 20 mice per immunization group: 1) non-immunized, 2) 10⁶ BCG (ATCC), and 3) 100ug of Lactoferrin with 10⁶ BCG. Four weeks after boosting, mice were infected with approximately 100 CFU aerosolized Erdman Mycobacterium tuberculosis (MTB) (ATCC). Each mouse was aerosol infected with approximately 100 organisms. Mice were sacrificed at day 7, 28 and 65 post-challenge. Lung and spleen were isolated and plated on 7H11 plates (Remel) for colony forming units (CFU) assessments. Sections were taken for histological study (H&E staining). Lungs from non-immunized control mice (Figure 7A, 7D) and lungs from mice immunized with BCG alone (Figure 7B, 7E) demonstrate granulomatous responses with marked destructive pathology. The group immunized with BCG and lactoferrin (Figure 7C, 7F) demonstrated reduced manifestation of pathological damage and controlled responses to infection within lung tissue.

Specific Lactoferrin in the present invention are illustrated in the following Examples:

EXAMPLE 6

Bovine milk lactoferrin (BLF): Bovine milk lactoferrin is a highly purified Lactoferrin derived from cow's milk. The commercially available BLF (Glanbia Nutritionals, Twin Falls, ID, USA) is further purified by metal ion affinity chromatography (IMAC). An imminodiacetic acid-epoxy activated gel (Pharmacia Fine Chemicals, Uppsala, Sweden, CHELATING SEPHAROSE™ 6B) is washed with water and equilibrated with 0.1 M sodium acetate buffer (pH 4.0) containing 1 M sodium chloride. The gel is then packed into a chromatographic column (1.2 cm x 10 cm) and saturated with 4 bed volumes of the same sodium acetate buffer further containing 5 mg/ml of nickel chloride. Excess metal is washed from the column with the sodium acetate buffer, and the gel is equilibrated with 20 mM HEPES buffer (pH 7.0) containing 1 M sodium chloride and 2 mM imidazole. The commercially available BLF is mixed with HEPES, sodium chloride, and imidazole to obtain a pH of 7.0, 20 mM HEPES, 1 M sodium chloride, and 2 mM imidazole. The mixture is applied onto the column at a flow rate of about 1 ml/min followed by, washing the gel with 2 bed volumes of 20 mM HEPES buffer (pH 7.0) containing 1 M sodium chloride and 2 mM imidazole. The non- adsorbed fraction is discarded, and the adsorbed fraction containing lactoferrin is eluted using 2 bed volumes of 20 mM HEPES buffer (pH 7.0) containing 1 M sodium chloride and 20 mM imidazole. The final material is dialyzed against water and lyophilized. BLF obtained in this example is at least 95% pure and is free of Coliform bacteria, Salmonella and pathogenic Staphylococcus. For oral administration BLF is reconstituted with water at concentration 0.5% (w/v) and stored at 4⁰C.

EXAMPLE 7

Human Milk Lactoferrin (HML): Human Milk Lactoferrin is a highly purified Lactoferrin derived from human milk. The commercially available Lactoferrin (Sigma Chemical Company; St Louis, MO; USA) is further purified by metal ion affinity chromatography (IMAC) as described in Example 6. Alternatively, the gel is then packed into a chromatographic column (1.2 cm x 10 cm) and saturated with 4 bed volumes of the same sodium acetate buffer further containing 5 mg/ml of copper sulfate. Excess metal is washed from the column with the sodium acetate buffer, and the gel is equilibrated with 50 mM TRIS HCL buffer (pH 8.0) containing 1 M sodium chloride. Lactoferrin is equilibrated to 50 mM TRIS HCL pH 8.0, 1 M NaCI and applied onto the column at a flow rate of about 1 ml/min followed by washing the gel with 2 bed volumes of 50 mM TRIS HCL buffer (pH 7.0) containing 1 M sodium chloride. The non-adsorbed fraction is discarded, and the adsorbed fraction containing lactoferrin is eluted using 2 bed volumes of 200 mM sodium acetate buffer pH 3.0. The eluate is neutralized with sodium hydroxide (final pH 7.2), sterilized by filtration and lyophilized. The final material is at least 95% pure. For the systemic administration the HML is reconstituted with sterile saline to obtain the final concentration of 10 mg/ml and is stored at 4⁰C. EXAMPLE 8

Recombinant human Lactoferrin (RHL): Highly purified recombinant Lactoferrin described in the U.S. Patent 6,066,469, U.S. Patent 6,277,817 B1 and U.S. Patent 6,455,687 B1 all of which are incorporated herein by reference.

According to the present invention Lactoferrin used as an adjuvant may be HML, BLF or recombinant human Lactoferrin alone or in combination from the stock solutions described in example 6 and 7.

Lactoferrin is administered in accordance with the present invention in either enteraly, preferably orally, in the form of a powder, aqueous or non-aqueous solution or gel, or parenterally, preferably intramuscularlly, in the form of an injectable solution, as an adjuvant to augment a vaccine efficacy by generation of T helper response and subsequent protection against challenge with a virulent pathogen. Preferable formulations or medicaments of the present invention comprise lactoferrin alone or in combination with pharmaceutical or nutritional carriers such as, water, saline, starch, maltodextrin, pullulan, silica, talcum, stearic acid, its magnesium or calcium salt, polyethyleneglycol, arabic, xanthan or locoust bean gums and fatty emulsions and suspensions that will be readily apparent to the skilled artisan. The lactoferrin is preferably present in the formulation at a level of 0.01 milligram to 10 milligram, more preferably between 0.1 to 5 milligram, based on 1 milliliter or 1 gram of the carrier. An effective amount of lactoferrin varies depending on the individual treated and the form of administration. Preferable in treating individual, a single dose of 0.01 milligram to 20 milligrams, more preferable 0.1 milligram to 1 milligram of lactoferrin per kilogram of body weight is administrated. Lactoferrin can also be delivered as a liposomal formulation, including transdermal patches.

According to the present invention, lactoferrin can be incorporated in formulation with any drug adjuvant therapy and delivered alone or simultaneously per os, intravenously, intraperitonealy, intraarterialy, intramascularly, subcutanoeusly, transdermal^, or as an intranasal spray, or intrabroncheal inhalation mist, at the effective concentration ranges set forth herein above. Preferred formulations or medicaments of the present invention comprise incorporating the lactoferrin into a steril aqueous solution as exemplified in Example 7.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

What is claimed is:

1. A method for augmenting TB vaccine efficacy by generation of T helper response and subsequent protection against challenge with a virulent TB pathogen in mammals comprising the steps of: a) administering an effective amount of pharmaceutically acceptable Lactoferrin; and b) administering an effective immunizing amount of said TB vaccine.

2. The method according to claim 1 wherein said Lactoferrin is administered prior to TB vaccine.

3. The method according to claim 1 wherein said Lactoferrin is administered concomitantly with the TB vaccine.

4. The method according to claim 1 wherein said mammal is human.

5. The method according to claim 1 wherein said Lactoferrin is bovine Lactoferrin.

6. The method according to claim 1 wherein said Lactoferrin is human recombinant Lactoferrin.

7. The method according to claim 1 wherein said Lactoferrin is administered enterally or parenterally.

8. A method for enhancing a cell mediated immune response to pathogenic microorganism in mammals comprising the steps of: a) administering an effective amount of pharmaceutically acceptable Lactoferrin; and b) administering a vaccine composition which comprises an effective immunizing amount of an antigen.

9. The method according to claim 8 wherein said Lactoferrin is administered prior to immunizing antigen.

10. The method according to claim 8 wherein said Lactoferrin is administered concomitantly with immunizing antigen

11. The method according to claim 8 wherein said mammal is human.

12. The method according to claim 8 wherein said Lactoferrin is bovine Lactoferrin.

13. The method according to claim 8 wherein said Lactoferrin is human recombinant Lactoferrin.

14. The method according to claim 8 wherein said Lactoferrin is administered enterally or parenterally.

15. A method for protection against pathological manifestation when challenged with a virulent pathogen in mammals comprising the step of administering an effective amount of pharmaceutically acceptable Lactoferrin and a vaccine composition which comprises an effective immunizing amount of an antigen.

16. The method according to claim 15 wherein said Lactoferrin is administered prior to immunizing antigen.

17. The method according to claim 15 wherein said Lactoferrin is administered concomitantly with immunizing antigen

18. The method according to claim 15 wherein said mammal is human.

19. The method according to claim 15 wherein said Lactoferrin is bovine Lactoferrin.

20. The method according to claim 15 wherein said Lactoferrin is human recombinant Lactoferrin.

21. The method according to claim 15 wherein said Lactoferrin is administered enterally or parenterally.

22. The use of Lactoferrin in the manufacture of a medicament use as an adjuvant in vaccine.