(BCG) AND METHODS OF USE

I. CROSS-REFERENCE TO RELATED APPLICATIONS

1. This application claims the benefit of U.S Provisional Application No. 62/517,269, filed on June 9, 2017 and U.S. Provisional Application No. 62/544,077, filed on August 11,

2017, which are incorporated herein by reference in their entirety.

II. BACKGROUND

2. Mycobacterium tuberculosis (M.tb) the causative agent of tuberculosis (TB) continues to cause significant morbidity and mortality around the world, and the rise of extensive-, extreme-, and total-drug resistant M.tb endangers eradication efforts (World Health Organization, 2016). The only currently licensed vaccine against TB, Mycobacterium bovis Bacille Calmette et Guerin (BCG), is ineffective against pulmonary TB (PTB) despite it being efficacious against other forms of mycobacterial disease such as TB meningitis and military TB. What are needed are new vaccines that are effective against pulmonary TB.

III. SUMMARY

3. Disclosed are methods and compositions related to attenuated Mycobacterium bovis Bacille Calmette et Guerin (BCG).

4. Disclosed herein are attenuated Mycobacterium bovis Bacille Calmette et Guerin (BCG) wherein the amount of one or more of trehalose dimycolate (TDM), phenolic glycolipid (PGL), Mycoside B (MycB), tri-acyl glycerol (TAG), and phthiocerol dimycocerosate (PDMI) on the cell wall of the BCG has been reduced.

5. Also disclosed herein are attenuated BCGs of any preceding aspect, wherein the reduction of one or more of TDM, PGL, MycB, TAG, and PDMI on the cell wall of the BCG occurs due to exposure of a delipidating agent (such as, for example, petroleum ether).

6. In one aspect, disclosed herein are vaccines against Mycobacterium tuberculosis comprising the attenuated BCG of any of preceding aspect.

7. Also disclosed herein are methods of inhibiting a Mycobacterium tuberculosis infection in a subject comprising administering to the subject the vaccine of any preceding aspect.

8. In one aspect, disclosed herein are methods of attenuating a BCG, comprising treating the BCG with a delipidating agent (such as, for example, petroleum ether) and reducing the amount of on ore more of trehalose dimycolate (TDM), phenolic glycolipid (PGL), Mycoside B (MycB), tri-acylglycerol (TAG), and phthiocerol dimycocerosate (PDMI) on the cell wall of the BCG.

9. Also disclosed herein are methods of reducing the amount of inflammatory cytokines (such as, for example, T Fa, Π.1β, IL-6, and/or IL-10) released at the site of a BCG infection in a subject comprising treating the BCG with a delipidating agent (such as, for example, petroleum ether) thereby reducing the amount of one or more of trehalose dimycolate (TDM), phenolic glycolipid (PGL), Mycoside B (MycB), tri-acylglycerol (TAG), and phthiocerol dimycocerosate (PDMI) on the cell wall of the BCG prior to administration of the BCG and administering to the subject the delipidated BCG.

10. In one aspect, disclosed herein are methods of increasing the amount of effector memory (CD62L-CD44+) and central memory (CD62L+CD44+) T cells that arise in a subject following a BCG vaccination, comprising treating the BCG with a delipidating agent (such as, for example, petroleum ether) thereby reducing the amount of one or more of trehalose dimycolate (TDM), phenolic glycolipid (PGL), Mycoside B (MycB), tri-acylglycerol (TAG), and phthiocerol dimycocerosate (PDMI) on the cell wall of the BCG and administering to the subject the delipidated BCG.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

11. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

12. Figures 1A, IB, 1C, ID, and IE show that petroleum ether extracts TDM, PGL, MycB, PDEVIs, and TAGs without affecting viability of BCG. To assess nature and percentage of the lipids remaining on the BCG cell wall following extraction with PE (PE BCG extract), PE treated BCG was subjected to C:M (2: 1, v/v) extraction for 12 h at 37°C (C:M BCG extract) (n=3; TLC, C:M, 95:5 v/v). Representative TLC images of PE BCG extracts in triplicate. BCG total lipid (BCG TL) extracts were included as a reference. Figure 1 A shows TDM, MycB, and PGL are highly extractable by PE. Figure IB shows that some PDFMs and TAGs are extracted from BCG by PE (n=3; TLC, petroleum ether/acetone, 96:4 v/v). Figure 1C shows that PFMs are not extractable by PE (n=3, TLC, chloroform/acetic acid/methanol/water, 40:25:3 :6 v/v/v/v). Fiure ID shows densitometry analysis of all lipids plotted as percent extracted following treatment with the corresponding solvents, (PE BCG extracts, grey bars), (C:M BCG extracts, black bars), (n=3). Figure IE shows the viability of BCG treated with PBS, PE or C:M (2: 1, v/v) assessed by plating serial dilutions (n=6). Representative experiment shown, each experiment performed at least three times, mean ± SEM; Student's t-test, ***p<0.001; ns: not significant, nd: no data.

13. Figures 2 A, 2B, 2C, 2D, 2E, and 2F show that delipidation of BCG significantly reduces its survival and attenuates inflammatory responses in human macrophages. MDMs monolayers (2.5xl0 ⁵ cells) were infected with viable BCG (grey bars) or dBCG (black bars) bacilli. Colony forming unit (CFU) assays were used to assess growth of BCG or dBCG in vitro. Figure 2A shows Inoculum used to infect MDMs showed no significant differences between the two groups. A representative experiment of n=4 in triplicate is shown, mean ± SEM; Student's t- test; ns: not significant. Figure 2B shows that human macrophages were infected with BCG or dBCG at an MOI 1 : 1 and bacterial growth was determined at the indicated intervals (2-120 h). A representative experiment of n=4 in triplicate is shown, mean ± SEM; Student's t-test, *p<0.05, **p<0.01, ***p<0.001. Figure 2C shows that MDMs were infected with BCG or dBCG at an MOI 10: 1 and supernatants were probed for the inflammatory cytokines T Fa, IL-6, and IL-Ιβ. A representative experiment of n=4 in triplicate is shown, mean ± SEM; Student's t-test, *p<0.05, **p<0.01, ***p<0.001. Figure 2D shows IL-10 levels in the supernatant of dBCG infected macrophages were also significantly reduced. No significant differences were observed for IL-12p40. A representative experiment of n=4 in triplicate is shown, mean ± SEM; Student's t-test, *p<0.05. Figure 2E shows the levels of LDH as a measure of cytotoxicity in BCG or dBCG MOI 10: 1 infected macrophages. A representative experiment of n=2 in triplicate is shown, mean ± SEM; Student's t-test, **p<0.01, ***p<0.001. Figure 2F shows representative pictures of MDM monolayers at 120 h not infected or infected with BCG or dBCG at MOI 10: 1; final magnification: lOOx.

14. Figures 3 A, 3B, 3C, and 3D show that delipidated BCG is quickly eliminated from the lung and is associated with little pathology of the lung. C57BL/6J mice were intranasally inoculated with 5xl0 ⁵ viable BCG (grey bar or grey circle) or dBCG (black bar or black circle) bacilli. Mice were sacrificed at 2, 7, 21, 50, and 150 DPV to assess BCG bacterial burden in the lung. Figure 3 A shows the inoculum used to vaccinate mice showed no significant differences between the two groups. Representative experiment of n=2, mean ± SEM; Student's t-test; ns: not significant. Figure 3B shows BCG CFUs in the lung was assessed at 2, 7, 21, 50, and 150 DPV by plating serial dilutions onto OADC supplemented 7H11. Pooled results from n=2 with 4-5 mice/group per time-point, mean ± SEM; Student' s t test, **p<0.01, ***p<0.001. Figure 3C shows that at 7, 21, 50, and 150 DPV, mice were sacrificed and lungs fixed in 10% NBF, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to visualize tissue morphology. Representative images are shown. Figure 3D shows areas of cell aggregation and infiltration (inflammation) in BCG (grey bars) or dBCG (black bars) vaccinated mice were quantified using Aperio Imagescope by calculating the area of inflammatory foci (i.e.

involvement) divided by the total area of the lung. Representative images at a final magnification of 20X. Representative experiment from n=2 with 4-5 mice/group, mean ± SEM; Student's t- test, *p<0.05, **p<0.01, ***p<0.001.

15. Figures 4 A, 4B, 4C, and 4D show that pulmonary vaccination with delipidated BCG increases memory and effector T cell responses in the lung. C57BL/6J mice were intranasally inoculated with 5xl0 ⁵ viable BCG (grey squares) or dBCG (black circles) bacilli. A group of naive mice (white circles) was included as a control to assess changes in the immune cell population due to vaccination. Mice were sacrificed at 7, 21, and 50 DPV to characterize memory T cell responses in the lung. Figure 4A shows the total number of CD4+ and CD8+ T cells in the lung across time. Figure 4B shows the proportion of CD4+ or CD8+ T cells displaying a naive phenotype (CD62L+CD44-). Figure 4C shows the proportion of CD4+ or CD8+ effector T cells (CD62L-CD44+) increased across time, and reached statistical significance in dBCG vaccinated mice at 50 DPV. Figure 4D shows the proportion of CD4+ or CD8+ memory T cells (CD62L+CD44+) also increased across time and peaked at 50 DPV in dBCG vaccinated mice. Pooled experiment from n=2 with 4-5 mice/group, mean ± SEM;

Student's t-test (for comparisons between groups), *p<0.05; One way ANOVA with Tukey's post-hoc test (for comparisons across groups), §p<0.05, §§p<0.01, §§§p<0.001. DPV: day post vaccination.

16. Figures 5 A, 5B, 5C, and 5D show that pulmonary vaccination with dBCG reduces M.tb bacterial burden in the lung and peripheral organs of infected mice. C57BL/6J mice were intranasally inoculated with PBS (vehicle; white circles) or 5xl0 ⁵ viable BCG (grey squares) or dBCG (black circles) bacilli. Fifty days later, mice were infected with a low dose aerosol of M.tb. At 21, 60, and 150 DPI mice were euthanized to assess bacterial burden in the (5A) lung, (5B) spleen, (5C) liver, and (5D) MLN. Pooled results from n=2 with 4-5 mice/group per time- point, mean ± SEM; One way ANOVA with Tukey's post-hoc test, *p<0.05, **p<0.01, ***p<0.001; ns: not significant; MLN: mediastinal lymph node.

17. Figures 6A and 6B show that pulmonary vaccination with dBCG is associated with decreased M.tb lung pathology across time. C57BL/6J mice were intranasally inoculated with PBS (vehicle) or 5xl0 ⁵ viable BCG or dBCG bacilli. Fifty days later, mice were infected with a low dose aerosol oiM.tb. Figure 6A shows that at 21, 60, and 150 DPI mice were sacrificed and lungs fixed in 10% NBF, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to visualize tissue morphology. Figure 6B shows the areas of cell aggregation and infiltration (inflammation) in vehicle (white bars), BCG (grey bars) or dBCG (black bars) vaccinated mice were quantified using Aperio Imagescope by calculating the area of

inflammatory foci (i.e. involvement) divided by the total area of the lung. Representative images at a final magnification of 20X. Pooled results from n=2 with 4-5 mice/group per time-point, mean ± SEM; One way ANOVA with Tukey's post-hoc test, *p<0.05, **p<0.01; ns: not significant.

18. Figures 7A, 7B, 7C, and 7D show that pulmonary vaccination with dBCG boosts CD69 and IL-17A, but not IFNy, responses in the lung oiM.tb infected mice. Figure 7A shows a timeline showing experimental design. C57BL/6J mice were intranasally inoculated with vehicle (white circle) or 5xl0 ⁵ viable BCG (grey square) or dBCG (black diamond) bacilli. Figure 7b shows T cell activation based on the expression of CD69 on CD4+ and CD8+ T cells at 50 DPV and at 10 and 21 DPI. CD69 was significantly increased in dBCG-vaccinated mice at 21 DPI. Figure 7C shows the total number of CD4+ and CD8+ T cells staining positive for IFNy after stimulation with CD3/CD28 in the presence of monensin. Figure 7D shows the total number of CD4+ and CD8+ T cells staining positive for or IL-17A after stimulation with CD3/CD28 in the presence of monensin. Mice vaccinated with dBCG had significantly higher proportions of CD8+IL-17A+ cells (10 DPI) and CD4+IL-17A+ and CD8+IL-17A+ (21 DPI) in the lung, while IFNy+ cells were not significantly elevated in either vaccination group. Pooled experiment from n=2 with 4-5 mice/group, mean ± SEM; One-way ANOVA with Tukey's post hoc test, *p<0.05, **p <0.01, ***p<0.001.

V. DETAILED DESCRIPTION

19. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. Definitions

20. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

21. Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value " 10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point " 10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

22. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

23. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

24. A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

25. "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

26. An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

27. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

B. Compositions

28. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular delipidated BCG vaccine is disclosed and discussed and a number of modifications that can be made to a number of molecules including the delipidated BCG vaccine are discussed, specifically contemplated is each and every combination and permutation of delipidated BCG vaccine and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

29. One potential explanation for BCG not being efficacious for pulmonary TB can lie in the route of immunization as BCG is administered intradermally, whereas M.tb is a natural airborne pathogen. This noted disparity may fail to confer optimum anti -mycobacterial immunity in the lung. To circumvent this issue research has shifted toward direct pulmonary vaccination with BCG. Evidence suggests that BCG is too pathogenic to be utilized as a direct pulmonary vaccine as it can induce significant pulmonary immunopathology. For this reason no human clinical trial has yet been implemented to evaluate the efficacy of pulmonary BCG vaccination against M.tb.

30. The primary reason behind the immunopathology caused by delivery of mycobacteria to the lung is attributed to potent inflammatory lipids such as trehalose dimycolate (TDM) (Geisel et al., 2005), di- and tri-acylglycerols (DAG/sTAGs) (Chauhan et al., 2013), phthiocerol dimycocerosates (PDMIs) (Astarie-Dequeker et al., 2009), phenolic glycolipids (PGLs)

(Sinsimer et al., 2008), among others, present on the BCG cell wall. These lipids induce rapid and robust innate immune responses that lead to tissue inflammation and damage (Reed et al., 2004). However, recombinant BCG strains deficient in specific lipids or a combination of these lipids are incapable of mounting sufficient immune responses to protect mice against M.tb (Tran et al., 2016). Thus, the total lack of several inflammatory lipids is detrimental to the generation of immunity, but their presence is responsible for much of the pathology observed. Thus, a balance must be struck to attain optimal immunity to M.tb. Accordingly, in one aspect, disclosed herein are attenuated Mycobacterium bovis Bacille Calmette et Guerin (BCG)

31. Herein is shown that treatment of BCG with delipidating agents (such as, for example, the organic solvent petroleum ether (PE)) can selectively extract many of the inflammatory lipids including trehalose dimycolate (TDM), phenolic glycolipid (PGL),

Mycoside B (MycB), and some tri-acylglycerols (TAGs) and phthiocerol dimycocerosates (PDMIs) without affecting the bacillus viability. Thus, in one aspect, disclosed herein are attenuated BCG wherein the amount of one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall of the BCG has been reduced. It is understood and herein contemplated that the amount of delipidation can vary amongst lipids each having a different impact on the attenuation and reduced inflammation of the delipidated BCG (dBCG). Disclosed herein in one aspect the attenuated BCG (i.e., the dBCG) can comprise a reduction in one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall, wherein the reduction of one or more of TDM, PGL, MycB, TAG, and/or PDMI can independently be at least a 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 percent reduction. For example, disclosed herein are attenuated BCG, wherein greater than 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 of the TDM, PGL, and/or MycB have been removed from the cell wall of the BCG by the petroleum ether treatment relative to an untreated control. In one aspect, the reduction of TDM, PGL, and/or MycB on the cell wall can be between 60 and 95%, more prefereably between 70 and 90%. Also disclosed are attenuated BCG (i.e., dBCG) wherein at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70% of the TAG and/or PDMI has been removed from the cell wall of the BCG by the petroleum ether treatment relative to an untreated control. In one aspect, the reduction of TAG and/or PDMI on the cell wall can be between 20 and 65%, more prefereably between 25 and 50%.

32. In one aspect, the delipidating agent can be any solvent known to remove lipids including but not limited any organic solvent including but not limited to aliphatic hydrocarbons [such as petroleum ether, sevoflurane (a nonflammable fluorinated ether)], chloroform:methanol, β-d-octyl glucoside, hexanes:isopropanol, and n-butanol among others . Thus, in one aspect, disclosed herein are attenuated BCGs of any preceding aspect, wherein the reduction of one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall of the BCG occurs due to exposure of a delipidating agent. In one aspect, disclosed herein are attenuated BCGs wherein the delipidating agent is selected from the group consisting of petroleum ether sevoflurane (a nonflammable fluorinated ether)], chloroform methanol, β-d-octyl glucoside,

hexanes:isopropanol, and n-butanol.

33. Using primary human cells it was demonstrated that delipidated BCG (dBCG) was significantly attenuated compared to conventional BCG (see Figure 2) having both reduced viability and a reduction in the amount of inflammation in infected tissue. Thus, in one aspect, disclosed herein are methods of attenuating BCG, comprising treating the BCG with a delipidating agent (such as, for example, petroleum ether) and reducing the amount of one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall of the BCG. 34. As noted above, the delipidation of BCG results in a reduction in inflammatory cytokines. Inflammatory cytokines such as TNF , IFNy, ILip, IL-2, IL-3, IL-6, IL-7, IL-9, IL- 12, IL-17, and IL-18 can play a significant role in the tissue damage associated with an infection contributing to the bulk of the cytopathology. As BCG is administered to subjects as a vaccine, reducing the amount of inflammation associated with the immunization represents a significant benefit to the recipient by reducing the pathological implications of receiving BCG. Thus, in one aspect disclosed herein are methods of reducing the amount of inflammatory cytokines (such as, for example, TNFa, Π β, IL-6, and/or IL-10) released at the site of a BCG infection in a subject comprising treating the BCG with a delipidating agent (such as, for example, petroleum ether) thereby reducing the amount of one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall of the BCG prior to administration of the BCG and administering to the subject the delipidated BCG.

35. The observation that delipidation resulted in an attenuated BCG with reduced inflammation led to the hypothesis that delipidation of BCG could be used as an effective pulmonary vaccine as it would bypass the pathology associated with this route of vaccination while retaining the efficacy associated with direct inoculation of the lung (i.e., it can be administered intranasally where non-delipidated BCG and M.tb cannot be so administered). Accordingly, in one aspect, disclosed herein are vaccines against Mycobacterium tuberculosis comprising an attenuated BCG. This is significant as all prior M.tb vaccines (including prior BCG vaccines) could not be administered intranasally. The delipidation methods disclosed herein and the resulting delipidated BCG disclosed herein overcome this issue and can be administered intranasally. This is a huge advance in the art. Specifically, in one aspect, disclosed herein are vaccines against Mycobacterium tuberculosis comprising an attenuated BCG wherein the amount of one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall of the BCG has been reduced (i.e., a delipidated BCG (dBCG)).

36. Because the efficacy of BCG against pulmonary TB infections is dubious at best, the efficacy of the dBCG against pulmonary infections was of key importance as well as the ability to deliver said dBCG intranasally. Herein is demonstrated that direct inoculation of the lung with dBCG significantly reduced the immunopathology of the lung of vaccinated mice compared to conventional BCG. Accordingly disclosed herein are methods of inhibiting a Mycobacterium tuberculosis infection (including, but not limited to infections wherein the Mycobacterium tuberculosis infection is a pulmonary infection) in a subject comprising administering to the subject a vaccine comprising a dBCG (for example, an attenuated BCG wherein the amount of one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall of the BCG has been reduced). In one aspect, the attenuated BCG (i.e., dBCG) vaccine can be administered intranasally.

37. Furthermore, vaccination with dBCG conferred superior protection relative to conventional BCG against challenge with M.tb. Protection against M.tb infection by dBCG vaccination was associated with increased numbers of effector and central memory T cell populations in the lung, specifically with increased CD69+ and IL-17A+, but not ΠΤΝΓγ+, T cell responses. Thus, the data indicate that the superior ability of dBCG to protect against M.tb was not due to enhanced IFNy responses, but may be associated with increased IL-17A. Together the results provide proof that dBCG can be easily adapted into a pulmonary vaccine with minimal safety concerns and enhanced efficacy against M.tb. That is, dBCG avoids the

immunopathology associated with intrapulmonary administration (for example, intranasal, aerosolized administration, topical administration to the nares, bronchial tubes or other direct intrapulmonary administration) and can be a pulmonary vaccine. Therefore, in one aspect, disclosed herein are methods of increasing the amount of effector memory (CD62L-CD44+) and central memory (CD62L+CD44+) T cells that arise in a subject following a BCG vaccination, comprising treating the BCG with a delipidating agent (such as, for example, petroleum ether) thereby reducing the amount of one or more of TDM, PGL, MycB, TAG, and/or PDMI on the cell wall of the BCG and administering to the subject the dBCG.

38. Although the disclosed dBCG can be used alone in a single application, it is understood and herein contemplated that the disclosed attenuated BCG (i.e., dBCG) can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times in a prime boost scenario. In one aspect, it is understood and herein contemplated that in a prime/boost inoculation strategy, the priming agent and boosting agent can be different. For example, the dBCGs used for the prime and each subsequent boost can comprise a difference in the number or combination of lipids reduced on the cell wall or the amount of reduction. Additionally, it is contemplated herein that a non- delipidated BCG can be used as a boost following a dBCG prime.

39. It is understood and herein contemplated that the disclosed attenuated BCG (i.e., dBCG) can be used prophylactically to prevent future exposure to M.tb. It is also contemplated herein that due to the dBCG ability to mount a protective T cell response which is often evaded by M.tb infections, dBCG or any vaccine or composition comprising said dBCG can be administered as a therapeutic immunotherapy to a subject already infected with M.tb. Accordingly disclosed herein are methods of inhibiting, reducing, or treating aM.tb infection comprising administering to a subject one or more of the dBCG vaccines disclosed herein.

1. Pharmaceutical carriers/Delivery of pharmaceutical products

40. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

41. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, intranasally, including topical intranasal administration, intranasal spray, or

administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

42. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. 43. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.

Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

44. The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

45. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA

1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

46. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly, intradermally, or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

47. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.

Pharmaceutical compositions may also include one or more active ingredients such as

antimicrobial agents, anti -inflammatory agents, anesthetics, and the like.

48. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

49. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

50. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. 51. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..

52. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

b) Therapeutic Uses

53. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.

54. In one aspect, it is understood and herein contemplated that the use of the delipidated BCG disclosed herein allows for a more effective inoculation, but also allows for a greater amount of bacteria to be administered to a subject without risk of disease or complications irrespective of administration route. In one aspect, the intranasal dosage can be 10 ³ , 10 ⁴ , 2xl0 ⁴ , 3xl0 ⁴ , 4xl0 ⁴ , 5xl0 ⁴ , 6xl0 ⁴ , 7xl0 ⁴ , 8xl0 ⁴ , 9xl0 ⁴ , 10 ⁵ , 2xl0 ⁵ , 3xl0 ⁵ , 4xl0 ⁵ , or 5xl0 ⁵ cfu. Also, disclosed are subcutaneous dosage ranges of 10 ³ , 10 ⁴ , 2xl0 ⁴ , 3xl0 ⁴ , 4xl0 ⁴ , 5xl0 ⁴ , 6xl0 ⁴ , 7xl0 ⁴ , 8xl0 ⁴ , 9xl0 ⁴ , 10 ⁵ , 2xl0 ⁵ , 3xl0 ⁵ , 4,xl0 ⁵ , or 5xl0 ⁵ , 6xl0 ⁵ , 7xl0 ⁵ , 8,xl0 ⁵ , or 9xl0 ⁵ , 10 ⁶ , 2xl0 ⁶ , 3xl0 ⁶ , 4xl0 ⁶ , or 5xl0 ⁶ cfu.

C. Examples

55. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

1. Example 1: Selective Delipidation of Mycobacterium bovis BCG Enhances its Vaccine Potential Against Mycobacterium tuberculosis Infection

a) RESULTS

(1) Petroleum ether treatment extracts non-polar lipids from BCG without affecting viability

56. Lipid extraction from PE treated BCG was assessed by thin layer chromatography (TLC). Three independent PE extractions are shown (PE BCG Extract). BCG total lipids (TL) were included on TLCs as controls. To assess the amount and makeup of the remaining lipids on BCG after PE extraction, bacteria were further extracted with chloroform: methanol (2: 1, v/v) (C:M BCG Extract). As described in the literature, TDM was highly extractable using PE resulting in a consistent removal of 70%-90% TDM from BCG (Fig. 1 A,D) (Indrigo et al., 2003). Due to the hydrophobic nature of PE, significant extraction of phenolic glycolipid Tb-1 (PGL) and mycoside B (MycB) was observed, both important virulence factors associated with the mycobacterial cell wall (Fig. lA,D). Additionally, some phthiocerol dimycocerosates

(PDFMs) and triacylglycerol's (TAGs) were also partially extractable (Fig. lB,D). As expected, PE was unable to remove phospholipids such as phosphatidyl-wyo-inositol mannosides (PFMs) from the BCG cell wall, and PFMs were only extractable after C:M extraction (Fig. lE,D). It was further confirmed that large chain carbohydrates such as mannose-capped lipoarabinomannan (ManLAM) were not directly or indirectly extractable with PE and remained on the PE treated BCG cell wall. 57. To confirm that lipid extraction by PE was not lethal or inhibitory for optimal growth of BCG, serial dilutions of PBS-treated, PE-treated, or PE+C:M-treated BCG were plated onto OADC supplemented 7H11 agar. No significant differences in growth between PBS and PE treated BCG was observed whereas PE+C:M-treated BCG did not grow (Fig. IE). Therefore, PE extraction of BCG reduced several cytotoxic non-polar molecules from the BCG cell wall (delipidated BCG or dBCG) with high reproducibility, and without affecting BCG viability.

(2) Delipidation of BCG attenuates uptake and growth, resulting in reduced inflammation in vitro

58. The consequence of removing non-polar lipids on the viability and immunogenicity of BCG was determined in vitro in human macrophages. Despite equal inoculum of BCG and dBCG (Fig.2A), dBCG was significantly less capable of surviving within human macrophages when compared to conventional BCG (Fig.2B). BCG and dBCG had an average uptake of 9.66 ± 5.51 and 3.00 ± 1.32 (M ± SD), respectively, highlighting that non-polar lipids are important for entry and/or association with human macrophages. dBCG infected macrophages had

significantly decreased release of TNFa, IL-Ιβ, IL-6, and IL-10, but not IL-12p40 (Fig.2C,D). Moreover, lower levels of secreted lactose dehydrogenase (LDH, cytotoxicity indicator) were detected in dBCG infected macrophages, indicating that loss of non-polar lipids renders macrophages less likely to die due to infection (Fig.2E). Images of infected macrophages captured at 120 h post infection corroborated these finding (Fig.2F).

(3) Delipidation of BCG attenuates pulmonary inflammation in vivo following intranasal vaccination and alters cell populations of the lung

59. The in vitro data indicate that removal of non-polar lipids reduced inflammatory cytokine secretion and therefore, the potential of dBCG as a safer pulmonary vaccine was evaluated. C57BL/6 mice were inoculated intranasally with 5.0xl0 ⁵ BCG or dBCG bacilli.

Despite equal inoculums (Fig.3 A), the ability of dBCG to persist in the lung was significantly reduced by day 2 post vaccination (DPV), a trend that continued for up to 150 days, with a continuous decrease in bacteria burden (Fig.3B). By 150 day post-vaccination (DPV), CFUs in the lung of dBCG-vaccinated mice were below accurate detection levels, whereas BCG- vaccinated mice had 2-3 logio CFUs in the lung (Fig.3B). The levels of TNFa and IL-6 in the lungs were significantly decreased as early as 7 DPV and the levels of IL-Ιβ, IL-10, and IFNy displayed the same reduced trend. By 50 DPV, all measured cytokines except for IL-12p40 were significantly decreased. Altogether, the data indicate that chemical removal of non-polar lipids from the BCG cell wall significantly impacts its ability to persist in vivo and diminishes inflammatory responses within the lung. To corroborate this, the immunopathology of the lung in BCG- or dBCG-vaccinated mice was analyzed. Lung inflammation was determined by quantifying the size of the inflammatory foci over the total area of the lobe. The lung of BCG vaccinated mice had larger areas of inflammation and perivascular cuffing compared to dBCG vaccinated mice (Fig.3C,D). This same trend was observed at 21 and 50 DPV with larger foci visible in BCG-vaccinated mice compared to dBCG-vaccinated animals. By 150 DPV, inflammation in the lung of BCG-vaccinated mice had decreased, but remained significantly higher compared to dBCG-vaccinated mice. Together, the data supports that dBCG reduces immunopathology when it is administered directly into the lung, and thus is a safe pulmonary vaccine.

60.

61. Immune cell kinetics was assessed in the lung of vaccinated mice across 50 days. Early post vaccination (7 and 21 DPV) significantly reduced percentages of monocytes and neutrophils were found in the lung of mice vaccinated with dBCG relative to BCG, resonating with findings (Fig. 2C) that vaccination with dBCG was associated with lower levels of inflammation. No significant difference in percentages of AMs and DCs was observed in the lung between groups, however the percentage of AMs decreased significantly from 7 to 21 and 50 DPV. The percentage of DCs increased across time in both groups relative to naive mice and were found to be significantly elevated in the lung of dBCG vaccinated mice at 50 DPV. B cell numbers increased significantly across time in both vaccination groups, though this was only significant between groups at 7 DPV. The number of NK cells decreased across time, but was significantly increased in BCG-vaccinated mice relative to dBCG vaccinated mice at 50 DPV, while the percentage of γδ+ Τ cells was not affected by either formulation despite overall significant increases across time.

(4) Pulmonary vaccination with dBCG increases memory T cell populations in the lung

62. BCG is the only licensed vaccine that primarily mediates protection via activation of CD4+, and to a lesser extent CD8+ T cells (Smith et al., 2012). BCG exerts its function as a vaccine against TB by generating a pool of memory T cells that respond rapidly upon infection with Mrf> (Vogelzang et al., 2014). For these reasons, the proportions of CD4+ and CD8+ T cells expressing markers associated with undifferentiated T cells (naive -CD62L+CD44), effector memory (TEM- CD62L CD44+) and central memory (TCM -CD62L ₊ CD44 ₊ ) in the lung of BCG- and dBCG-vaccinated mice were observed. The proportion of CD4+ and CD8+ T cells increased across time following vaccination in BCG and dBCG vaccinated mice but mice vaccinated with dBCG had significantly increased numbers of CD4+ and CD8+ T cells in the lung at 50 DPV (Fig.4A). No change to the proportion of naive cell in either subset across time (Fig.4B) was observed. Proportions of TEM CD4+ and CD8+ cells in the lung increased across time in BCG and dBCG vaccinated mice, with dBCG having significantly more at CD4+ TEM 50 DPV (Fig.4C). TEM cells are believed to possess short lived immunological memory, while tissue-resident TCM cells, though far fewer than TEM in tissue, are longer-lived (Mueller et al., 2013). dBCG induced greater proportions of CD4+ and CD8+ TCM cells in the lung compared to BCG vaccination (Fig.4D).

(5) M.tb challenge after pulmonary vaccination with dBCG attenuates pulmonary immunopathology and affords superior protection relative to conventional BCG

63. In order to replace the current TB vaccine the new candidate must be more efficacious at reducing M.tb bacterial burden and preventing TB pathology. Mice were randomized into vehicle (saline), BCG, or dBCG groups and vaccinated with 5xl0 ⁵ bacteria via intranasal inoculation of the lung. 50 days post vaccination mice were infected with a low dose aerosol of M. tb. Bacterial burden was assessed in the lung, spleen, liver, and mediastinal lymph node (MLN) at 21, 60, and 150 days post infection (DPI). A significant decrease in the bacterial burden of lung, spleen and liver was observed at all time points studied in mice vaccinated with dBCG, compared to vehicle control and conventional BCG (Fig.5). Also a significant delay was observed in colonization of the spleen (Fig.5B) and liver (Fig.5C) in dBCG-vaccinated mice. Furthermore, though no significant differences m M. tb burden in the MLN at 21 and 60 DPI were seen between BCG and dBCG, this was significantly decreased in mice vaccinated with dBCG at 150 DPI (Fig.5D). Together, these data indicate that removal of non-polar lipids from BCG (dBCG) augments its efficacy as a vaccine against M.tb challenge.

64. Importantly, it was determined if dBCG vaccination was able to reduce tissue damage (symptom of M.tb infection progression to disease) by quantifying areas of cellular aggregation relative to the total size of the lung (Fig.6). dBCG vaccination attenuated pulmonary

inflammation at every time point studied. At 21 DPI small areas of cellular aggregation were visible in all three groups, but foci in the lung of vehicle-vaccinated and BCG mice were significantly larger compared to dBCG (Fig.6A,B). Inflammatory foci doubled in size in the lung of vehicle-treated and BCG vaccinated mice at 60 DPI, whereas foci in the lung of dBCG- vaccinated mice remained small. Significantly decreased pulmonary inflammation was observed in the lung of dBCG vaccinated mice compared to BCG-vaccinated mice at this time point. At 150 DPI, the mean area of the inflammatory foci was 20.84 ± 4.66% in vehicle-treated mice and 19.14 ± 13.54% in BCG-vaccinated mice, whereas in dBCG-vaccinated was 8.30 ± 3.04%. Together the data indicate that dBCG is superior to BCG at reducing bacterial burden in the lung, and also more effective at preventing pulmonary immunopathology.

(6) Pulmonary vaccination with dBCG accelerates effector T cell 231 responses in the lung upon challenge with M.tb

65. Finally, effector T cells responses were evaluated in the lung of mice that had been vaccinated (DPV 50) or vaccinated and then challenged with Mrf> (DPI 10 and 21) (Fig.7A). The expression of CD69 on CD4 and CD8 T cells was studied, and the ability of T cells to produce IFNy or IL-17A. In BCG and dBCG groups, the number of resident CD69 expressing CD4 and CD8 T cells in the lung prior to M.tb challenge was increased relative to vehicle but did not differ between BCG and dBCG vaccinated mice. Infection with Mrf> increased the number of CD69+CD4+ and CD69+CD8+ T cells in the lung of vehicle control mice but the number of CD69 ₊ CD4 ₊ and CD69+CD8+ T cells in the lung was enhanced further with dBCG at 21 DPI (Fig.7B). To further assess T cell activation in the lung two important effector mechanisms required for M.tb control were evaluated, the production of IFNy and IL-17A by T cells (Aguilo et al., 2016; Flynn et al., 1993; Khader et al., 2007). Although the number of CD4+IFNY+ and CD8+IFNY+ T cells in the lung was elevated 50 DPV in BCG and dBCG vaccinated mice, statistically significant increases in the number of CD4+IFNY+ and CD8+IFNY+ T cells in the lung were not observed between BCG- or dBCG-vaccinated mice at any time point postMrf> infection (Fig.7C). However significant increases were observed in the number of CD4+IL-17A+ and CD8+IL-17A+ T cells in the lung (Fig.7D), with significant increases in the number of CD8+IL-17A+ T cells at 10 DPI and significant increases in CD4+IL-17A+ and CD8+IL-17A+ T cells at 21 DPI in the lung of dBCG-vaccinated mice. Overall, the results indicate that IL-17A actively participates in the control of M.tb and is a better correlate of vaccine efficacy,

b) DISCUSSION

66. BCG remains the only vaccine available for the prevention of TB, yet it fails to confer long term immunity against PTB. Despite its success against meningeal- and military- TB, its efficacy against PTB must be improved if M.tb is to be eradicated. One underlying factor that can contribute to this phenomenon is the route of immunization with BCG vs. the route οΐΜ.ώ infection. M.tb has evolved to be a pulmonary pathogen by taking advantage of the immunoprivileged status of the lung, where inflammatory responses are tightly regulated to prevent excessive damaging inflammation (Cooper, 2009). Vaccination with BCG, on the other hand, is administered into the dermal layer of the skin (Moliva et al., 2015). The immune cell composition of these tissues has been shown to vary significantly, and within them, the way immune responses to pathogens are generated (Hussell and Bell, 2014). Although other vaccines administered via percutaneous injection such as the measles-mumps-rubella (MMR) or the diphtheria-tetanus-pertussis (DTP) vaccines have been extremely successful, their primary target for immunological memory is through the generation of long lived plasma B cells that produce high-affinity antibodies capable of rapidly neutralizing their target pathogen (Moliva et al., 2017). In contrast, the BCG vaccine relies primarily on CD4 and CD8 T cell memory for protection against M.tb. Multiple studies have suggested that BCG is more efficacious against M.tb when delivered directly into the lung (Derrick et al., 2014; Perdomo et al., 2016; Aguilo et al., 2016). However, the presence of inflammatory lipids on the mycobacterial cell wall has inhibited the use of BCG as a pulmonary vaccine. As a result, removal of inflammatory lipids from the cell wall of BCG will enable direct pulmonary vaccination with BCG.

67. The results show that selective delipidation of BCG with petroleum ether (PE) effectively serves as a more potent vaccine against M.tb when administered directly into the lung. Pulmonary vaccination with dBCG was more effective than conventional BCG at reducing M.tb bacterial burden in the lung, spleen, and liver at the initial stage of infection (DPI 21, 60) and in all organs including the MLN at later stages (DPI 150). In addition to the observation that pulmonary vaccination with dBCG provided better protection than conventional BCG, it is also highly significant that pulmonary vaccination with dBCG led to a significant decrease in BCG specific lung immunopathology as well as M.tb induced immunopathology. Pulmonary immunopathology is the primary reason that BCG has not been repurposed as a pulmonary vaccine (Nuermberger et al., 2004; Tree et al., 2004) and immunopathology of M.tb infected animal models is a major correlate of morbidity and mortality (Moliva et al., 2015). The dBCG pulmonary vaccine is therefore superior at reducing one of the most important processes that can lead to TB disease in animal models and presumably also in humans.

68. The improved protection and reduce pulmonary inflammation (to vaccine and M.tb challenge) that was observed with dBCG vaccination is likely attributed to the chemical removal of virulent lipids including TDM, MycB, PGL, TAGs, and PDEVIs from the cell wall that inhibit the development of effective immunity. In this context, TDM has been associated with the ability to inhibit fusion between phospholipid vesicles such as those required for fusion of phagosomes with lysosomes (Spargo et al., 1991). Phagosome-lysosome (P-L) fusion is required for the killing of intracellular pathogens and for the subsequent presentation of foreign peptides along with MHC-class II to adaptive immune cells (Pieters, 2008). TDM can also inhibit cellular energy metabolism by stimulating NADase activity lowering the levels of NAD and thereby, reducing the activity of NAD-dependent enzymes which can also affect the generation of adaptive immune responses (Fox et al., 2005). Furthermore, macrophages infected with TDM- expressed higher levels of MHC-II, CD Id, CD40, CD80, and CD86 and as a result were more capable of stimulating CD4+ T cell responses (Kan-Sutton et al., 2009). TDM can also induce apoptosis of lymphocytes in the thymus leading to atrophy, indicating TDM can also inhibit T cell development (Ozeki et al., 1997).

69. Some M. tb clinical isolates synthesize the trisaccharide form of PGL (PGL) and a clinical isolate of M. tb belonging to the East- Asian lineage (i.e. HN878) was found to be hypervirulent in animal models due to the presence of PGL on its cell wall (Reed et al., 2004). The trisaccharide domain of PGL was able to inhibit Toll-like receptor 2 (TLR2)-induced NF-kB activation, and thus production of IL-Ι β, TNFa, IL-6, and CCL2, suggesting PGL enhances mycobacterial subversion of the immune system (Arbues et al., 2014). Similar to PGL, the accumulation of TAGs on the mycobacterial cell surface can increase virulence (Reed et al., 2007). In addition, purified MycB, a monosaccharide PGL produced by all sub-strains ofM bovis (including BCG) but not by M.tb (Jarnagin et al., 1983) was incapable of stimulating IL- 1 β and IL-6 secretion from macrophages, but could induce TNFa (Geisel et al., 2005). Thus, the accumulation of PGLs and TAGs can confer an adaptive advantage for growth in stressful environments. Similar to other lipids on the outer surface of M. tb, PDFMs also appear to play a role in the virulence of mycobacteria. M.tb strains lacking PDFM were shown to be less capable of causing disease in mice (Cox et al., 1999). Genetic Mrf> mutants lacking PDFM were less capable of binding to the plasma membrane of host macrophages and studies concluded that PDFMs directly contribute to the initial step of macrophage infection and participated in preventing phagosomal acidification. Other studies have shown that lack of PDFM on M. tb reduces the survival of the bacteria within macrophages, suggesting PDFMs protect M.tb from early innate host responses. Therefore, chemical delipidation of BCG has a significant impact on multiple virulence factors that can be involved in both innate and adaptive immune function.

70. BCG mediates immunity through the development of antigen-specific memory T cells that respond rapidly following infection with M. tb (Shen et al., 2002). Evidence suggests the success of BCG against meningeal- and miliary- TB is due to the rapid proliferation of memory T cells that quickly contain the infection, bypassing the delay in T cell priming in the lymph nodes that occurs in naive hosts (Kipnis et al., 2005). In this context, memory T cells exist within two populations: Central memory (TCM) and effector memory (TEM). TcMare abundant within lymphoid tissue, respond faster to stimulus, have the ability to self-renew, and are long- lived. On the other hand, TEM cells reside in larger numbers within non-lymphoid tissues, respond more slowly to stimulus, and are short-lived (Mueller et al., 2013). Effector memory T (TEM) cells accumulate in the lung of mice vaccinated with conventional BCG via the subcutaneous route, but central memory T (TCM) cells do not (Henao-Tamayo et al., 2010). This phenomenon was also observed in humans (Purwar et al., 2011). Thus, if numbers of TCM cells are increased in the lung it can lead to faster immune responses to pathogens. The data indicate that pulmonary vaccination with dBCG amplifies the number of effector memory CD4+ T cells, but more importantly, increases the number of central memory CD4+ and CD8+ T cells within the lung.

71. In addition to increasing the number of TCM and TEM cells within the lung, pulmonary vaccination with dBCG accelerated T cell activation in the lung following M.tb infection. A significant increase in the number of CD4 and CD8 T cells positive for CD69 was observed in both BCG- and dBCG-vaccinated groups at 50 DPV or 10 DPI relative to control groups.

However, dBCG vaccinated mice had higher numbers of CD4+CD69+ and CD8+CD69+ T cells in the lung 21 DPI, indicating that vaccination with dBCG does indeed accelerate T cell activation upon M.tb infection. The number of T cells capable of producing IFNy and IL-17A in the lung was also assessed, as evidence suggests they are critical for M.tb containment (Gopal et al.,

2014; Pitt et al., 2012). Unexpectedly, no significant differences were observed between BCG- and dBCG vaccinated mice in the number of T cells capable of producing IFNy prior to, or following, infection with M.tb in the lung. IFNy responses develop slowly in mice upon infection with M.tb, reaching equal proportions in vaccinated mice at 21 days post M.tb challenge. The fact that no significant difference was observed in IFNy responses in the lung of BCG- and dBCG-vaccinated mice indicates that IFNy can have limited ability to contribute to the additional protective immunity conferred by dBCG (Sakai et al., 2016). Since IFNy was not mediating the additional protection against M.tb associated with dBCG vaccination, IL-17A responses were assessed as they have been implicated in conferring immunity to M.tb at mucosal sites (Gopal et al., 2014; Khader et al., 2007). Significant increases were observed in the number of CD8+IL- 17A+ T cells in the lung of dBCG-vaccinated mice at 10 DPI, and significant increases in the number of CD4+IL-17A+ and CD8+IL-17A+ cells in the lung at 21 DPI; indicating that IL-17A responses may well be responsible for the increased protection observed in dBCG-vaccinated mice. Thus, the data indicates IFNy has a limited ability to protect against M.tb, while IL-17A responses can be further manipulated to improve protection.

72. As high incidence of TB occurs in developing countries, particularly within rural communities that may lack access to routine health care, TB vaccine design should aim to develop single dose formulations. Though some TB vaccine boosters have shown promising results, implementation of large-scale vaccination programs may be difficult (Andersen and Kaufmann, 2014). Additionally, vaccines that require administration of components over a span of a few days/weeks pose significant logistical challenges. For these reasons, the design was centered on developing a single-dose vaccine that controls M.tb and importantly reduces pathology upon infection, a trade mark of progression to disease. TB vaccines that are inoculated into the lung have been shown to be more effective than percutaneous injections but toxic lipids on the BCG cell wall have prevented administration of BCG directly into the lung. Both of these challenges were overcome via a simple lipid extraction using the readily available petroleum ether. The procedure was fast, reproducible, inexpensive, and can be easily implemented into current vaccine production. It was shown herein that pulmonary vaccination with dBC G was more effective than BCG at reducing M.tb bacterial burden, and more importantly, dBCG was quickly cleared from the lung after inoculation while lung inflammation was significantly reduced. Thus, BCG was selectively attenuated while simultaneously increasing its protective efficacy against M.tb. Additionally dBCG effectively abolishes the primary concern for moving BCG from an intradermal vaccine to pulmonary one.

73. The data indicate that BCG cell wall lipids directly inhibit the generation of protective immune responses to M.tb, and that modification of the mycobacterial cell wall can be further exploited to improve protective immunity to mycobacteria. Whereas current vaccines typically fall under the categories of attenuated, inactivated, or subunit/conjugate, dBCG was classified under a new category called "selective attenuation". dBCG was classified this way because the bacterium remains alive, and therefore is able to replenish the extracted lipids on its cell wall. However, the removal of cell wall lipids via PE extraction significantly reduces its ability to infect cells and induce immunopathology, thus attenuating it. This allowed for direct vaccination into the lung, the most immunogenic route of vaccination for protection against M.tb. Given that cell wall molecules are utilized by the bacteria to engage immune cells, the initial contact between the host and the bacterium will mediate the success of the vaccine. Not only was vaccination with dBCG more effective against M.tb infection, it also opened the possibility of pulmonary vaccination in humans due to the absence of excessive inflammation to the lung. Furthermore, the observations revealed that targeting IFNy responses does not necessarily increase immunity to mycobacteria. Instead, IL-17A responses appear to contribute significantly to mycobacterial immunity in the lung in the context of vaccination. c) METHODS

(1) Mice.

74. Specific- pathogen-free, female mice aged 6-8 weeks of the C57BL/6 background were purchased from Jackson Laboratories (Bar Harbor, ME). Upon arrival, mice were supplied with sterilized water and chow ad libitum and acclimatized for at least one week prior to experimental manipulation. Mice were maintained in micro-isolator cages located in either a standard vivarium for all noninfectious studies or in a biosafety level three (ABSL-3) core facilities for all studies involving Mrf>. Mice were divided into three groups: Mock-vaccinated (vehicle), PBS treated BCG-vaccinated (BCG), or PE-treated BCG-vaccinated (dBCG). All experimental procedures were approved by The Ohio State University Institutional Laboratory Animal Care and Use Committee.

(2) Mycobacteria Growth and delipidation.

75. GFV-M.tb Erdman (provided by Dr. Horwitz, UCLA, CA) andM bovis BCG Pasteur strains [American Type Culture Collection (ATCC), #35734] were grown.

76. To delipidate M bovis BCG, freshly plated M. bovis BCG were harvested between 9- 14 days of growth into siliconated tubes (Fisher Scientific, Hampton, NH), suspended in 1ml of petroleum ether (PE) or phosphate buffered saline (PBS) and vortexed for two minutes, rested for five minutes, and then pelleted at 6000 g for five minutes. The procedure was repeated three times. The supernatants from the PE treated bacteria were collected, dried under N2, and kept at - 20°C until further analysis. Treated bacteria used for in vitro or in vivo studies were dried briefly in the biosafety cabinet to evaporate excess solvent, washed twice in PBS, and suspended in PBS prior to use. The viability of BCG and dBCG was assessed by performing serial dilutions, plating the bacteria onto Middlebrook 7H11 agar supplemented with OADC and counting colonies three to four weeks later.

(3) Analysis of extracted lipids.

77. To assess the remaining lipids on the cell wall of BCG post PE extraction, PE-treated bacteria were further extracted with chloroform -methanol (C:M, 2:, v/v) at 37°C for 12 h. PE and C:M extracted lipids (spotted 100 μg per lane) were analyzed by thin layer chromatography (TLC) using aluminum -backed TLC plates with the following solvent systems: For TDM, mycoside B, and PGL (chloroform/methanol 95 :5, v/v); for phthiocerol dimycocerosates (PDIMs) and triglycerides (TAGs; petroleum ether/acetone 96:4, v/v); and phosphatidyl myoinositol mannosides (PIMs; chloroform/acetic acid/methanol/water 40:25 :3 :6, v/v/v/v).

Quantification was performed using the NIH software ImageJ. Total BCG extracts were obtained by sequential extractions using C:M (2: 1, v/v); C:M (1 :2, v/v) and C:M:water (10: 10:3, v/v/v) and later combined.

(4) Vaccination and M.tb aerosol infection.

78. C57BL/6 mice were anesthetized 737 with an aerosolized solution containing 2-5% isoflurane. A single cell suspension of BCG or dBCG containing approximately 5xl0 ⁵ viable bacilli in 50 μΐ was intranasally administrated evenly between the two nostrils allowing for inhalation into the lungs. Following administration, mice were held in an upright position for 15 seconds to ensure the entire inoculum was inhaled. Mice were then returned to their cage and monitored until recovery. At the indicated time post vaccination, mice were euthanized, and the lung was removed and processed for histological analysis, CFU enumeration, or lung cell isolation as described below. Bacterial burden in the lung of vaccinated mice was assessed by culturing serial dilutions of organ homogenates onto Middlebrook 7H1 1 agar, supplemented with OADC. Colonies were enumerated after 3-4 weeks incubation at 37°C. Data are expressed as the logio value of the mean number of CFU recovered per organ («=4-5 mice). A separate group of mice were immunized with BCG or dBCG, rested for 50 days, and then infected via aerosol with a low dose of M.tb using the Glas-Col (Terre Haute, IN) inhalation exposure system. Briefly, the nebulizer compartment was filled with a suspension of M.tb calculated to deliver 40-100 viable bacteria into the lung. Mice were sacrificed at various time points post infection, the lung, spleen, liver, and mediastinal lymph node (MLN), were aseptically removed into sterile saline and the bacterial burden was assessed by culturing serial dilutions of organ homogenates onto OADC supplemented Middlebrook 7H1 1. Colonies were enumerated after 3- 4 weeks incubation at 37°C. Data are expressed as the logio value of the mean number of CFU recovered per organ («=4-5 mice). ΈοχΜ.ώ challenge studies, organ homogenates were also plated onto OADC supplemented 7H1 1 media containing 2 μg/ml of 2-thiophenecarboxylic acid hydrazide (TCH; Sigma-Aldrich, St. Louis, MO) to exclude BCG growth (Flaherty et al., 2006).

(5) Histopathology.

79. The middle right lung was isolated from each individual mouse and inflated with and stored in 10% neutral buffered formalin. Lung tissue was processed, sectioned at 4-5 μπι, and stained with hematoxylin and eosin (H&E) for light microscopy with lobe orientation designed to allow for maximum surface area of each lobe to be visualized. Sections were examined by a board-certified veterinary pathologist (G.B.) without prior knowledge of the experimental groups and graded according to severity, granuloma size and number. H&E stained slides were digitized for morphometric analysis using Aperio ScanScope XT slide scanner (Leica, Buffalo Grove, IL) at 40X magnification. Immune cell infiltration and granulomatous tissue was calculated by manually outlining all foci and determining the total area of inflammation as a percentage of the total area of the lung.

(6) SDS-PAGE and whole-cell ELISA for ManLAM.

80. Several loopfulls of BCG underwent PE extraction and subsequently C:M (2: 1, v/v) extraction as described above. Extracts were dried under N2 and suspend in dimethyl sulfoxide

(DMSO) at a final concentration of 10 μg/μl. 100 μg of each sample were separated by SDS- PAGE (12%) and stained with periodic acid silver nitrate. Surface-exposed ManLAM on treated M. bovis BCG bacilli was also analyzed by ELISA using anti-ManLAM monoclonal antibody CS-35. Briefly, BCG was subjected to PE treatment or PBS as described, immobilized on a medium binding ELISA plate and dried overnight. The plates were blocked with 1% BS A/PBS with 0.05% Tween-20. The plates were then probed with CS-35 overnight. The following day, the binding was assayed by standard ELISA technique as described (Arcos et al., 2011).

(7) In vitro infection of human macrophages.

81. Human monocyte-derived macrophage (MDM) monolayers for cytokine and CFUs enumeration were prepared from healthy tuberculin negative human volunteers. Briefly, whole blood was collected and separated using Ficoll-Paque differential centrifugation. Peripheral blood mononuclear cells (PBMCs) were isolated and differentiated over a five day period in Teflon wells using RPMI 1640 (Thermo Fisher Scientific, Waltham, MA) containing 20% autologous serum at 37°C, 5% CO2. Macrophages were then collected and plated onto tissue culture plates and allowed to further differentiate for an additional seven days in RPMI 1640 containing 20% autologous serum. Macrophages were infected with a single cell suspension of conventional (untreated) BCG or PE-treated BCG dissolved in PBS at a multiplicity of infection (MOI) 1 : 1 or 10: 1. Supernatants were collect at each time point and frozen at -80°C until further analysis. To enumerate CFUs, infected macrophage monolayers were lysed and serial dilutions were plated onto OADC supplemented 7H11 agar plates as described (Arcos et al., 2011). CFUs were counted 3-4 weeks later. Images of macrophage monolayers were obtained on an Olympus CKX41 SF2 microscope using an Olympus DP71 digital camera at a final magnification of lOOx.

(8) Lung cell isolation. 82. Lung cell were isolated. The lungs were cleared of blood via perfusion through the pulmonary artery with 10 ml PBS containing 50 U/ml heparin and placed into 2 ml cold complete-DMEM (c-DMEM) [DMEM (Mediatech, Herndon, VA), supplemented with 10% heat-inactivated FBS (Atlas Biologicals, Ft. Collins, CO), 1% HEPES buffer (1 M, Sigma- Aldrich), 10 ml lOOx nonessential amino acid solution (Sigma- Aldrich), 5 ml

penicillin/streptomycin solution (50,000 U penicillin, 50 mg streptomycin, Sigma-Aldrich), and 0.1% 2-mercaptoethanol (50 mM, Sigma-Aldrich)]. The lungs were then supplemented with 2 ml complete DMEM containing collagenase XI and type IV bovine pancreatic DNase, before being partially dissociated using the mouse lung dissociator program one on the gentleMACS tissue dissociator (Miltenyi Biotec). Following 30 min of incubation at 37°C, 5% CC , the tissue was further dissociated by using program two on the gentle MACS dissociator. Digested lungs were dispersed gently through a 70 μιη nylon mesh to obtain a single-cell suspension. Red blood cells were lysed using Gey's lysis buffer, and washed with c-DMEM. Cell suspensions were counted using trypan blue to exclude dead cells and resuspended at a working concentration in c- DMEM or fixed for flow cytometry as described below.

(9) Analysis of immune cells by flow cytometry.

83. Cells were prepared for flow cytometry. Briefly, lung cell suspensions were adjusted to lxlO ⁷ cells/ml with FACS buffer supplemented with 0.1% sodium azide and incubated at 4°C for one hour. For intracellular staining, 2.5xl0 ⁶ unfixed cells were first stimulated with 10 μg/ml CD3s, 1 μg/ml of CD28 and 3 μΜ of monensin for 4h at 37°C, 5% C0 ₂ (Cyktor et al., 2013a). Cells were then labeled with cell surface markers, permeabilized using BD Cytofix/Cytoperm™ Plus and then labeled with intracellular markers as directed by manufacturer for 25 min at 4°C in the dark. Cells (Ι ^χ ΙΟβ) were labeled with 25 μg/ml of specific fluorescent-labeled antibody for 30 min at 4°C in the dark followed by two washes with FACS buffer. Samples were read on a Becton Dickinson LSRII flow cytometer, and data were analyzed using FlowJo version 10 software. Lymphocytes were gated according to their forward- and side-scatter profiles, and CD4 or CD8 T cells were identified by the presence of specific, fluorescent-labeled antibody in combination with CD3s. Innate immune cells were blocked with CD16/CD32 (Fc block-BD Biosciences, East Rutherford, NJ) prior to staining. Antibodies used for phenotyping were: FITC-conjugated Gr-1 (RB6-8C5- BioLegend), FITC -conjugated CD3s (145-2C11-BD

Biosciences), FITC-conjugated CD44 (FM7-BD Biosciences), FITC-conjugated CD8a (53-6.7- BioLegend), PE-conjugated CD4 (H129.19-BD Biosciences), PE-conjugated CD4 (RM4-5-BD Biosciences), PE-conjugated IL-17A (TCl l-18H10.1-BioLegend) PerCP-Cy 5.5 -conjugated CDl lb (M1/70-BD Biosciences), PerCP-Cy 5.5 -conjugated TCR y/δ (GL3-BioLegend), PerCP- Cy 5.5 -conjugated CD69 (H1.2F3-BioLegend), APC-conjugated CDl lc (HL3-BD Biosciences), APC-conjugated CD8a (53-6.7-BioLegend), APC-conjugated IFNy (XMG1.2-BioLegend), PE- Cy7-conjuated CD19 (6D5-BioLegend), PE-Cy7-conjuated NK1.1 (PK136-BioLegend), PE- Cy7-conjuated CD3s (17A2-BD Biosciences), APC-Cy7-conjugated CD62L (MEL-14- BioLegend), and APC-Cy7-conjugated CD4 (GK1.5-BD Biosciences). Appropriate isotype controls recommended by the manufacturer were included in each experiment and used to set gates for analysis.

(10) Cytokine/LDH quantification by ELISA.

84. Concentrations of IFNy, TNFa, IL-10, IL-12p40, IL-12p70, IL-6, and IL-Ιβ in mouse organ homogenates or human macrophage supernatants were assessed by ELISA (BD

Biosciences for mouse and R&D Systems, Minneapolis, MN for human) following the manufacturer's protocol. Briefly, 96-well plates were coated with antibodies designed to detect the specific cytokine and incubated for 12-16 h. Organ homogenates or macrophage supernatants were then overlaid and incubated for 2 h at room temperature. Colorimetric analysis at OD450 along with a standard curve was used to determine the concentration of each cytokine.

Concentration of LDH was assessed using CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Fitchburg, WI) following manufacturer's protocol. Colorimetric assays were read on a Spectramax M2 Microplate reader (Molecular Devices LLC, Sunnyvale, CA).

(11) Quantification and Statistical Analysis Statistical analysis.

85. Statistical significance was determined using Prism 4 software (GraphPad Software, San Diego, CA). The unpaired, two-tailed Student's t-test was used for two group comparisons. Multiple comparisons were analyzed using one-way ANOVA with Tukey's post hoc test.

Statistical significance was reported as *, /><0.05; **, /><0.01; or ***, /><0.001. For 'n' values for each experiment performed see the respective Figure Legends.