Elizabeth Creissen

The use of novel vaccine delivery systems allows for the manipulation of the adaptive immune systems through the use of molecular adjuvants that target specific innate pathways. Such strategies have been used extensively for vaccines against cancer and multiple pathogens such as Mycobacterium tuberculosis. In the current study we used heat killed non-pathogenic recombinant Saccharomyces cerevisiae expressing M. tuberculosis antigen Rv1886c (fbpB, mpt59, Ag85B) as a delivery system in conjunction with its ability to stimulate innate immunity to determine its ability to induce immunity. We established that the recombinant yeast induced activated antigen specific T cells are capable of reducing the mycobacterial burden. Inoculation of the recombinant yeast after vaccination with BCG resulted in a systemic alteration of the phenotype of the immune response although this was not reflected in an increase in the reduction of the mycobacterial burden. Taken together the data suggest that heat killed yeast can induce multiple cytokines required for induction of protective immunity and can function as a vehicle for delivery of M. tuberculosis antigens in a vaccine formulation. In addition, while it can enhance the effector memory response induced by BCG, it had little effect on central memory responses.

Mycobacterium bovis Bacille Calmette Guérin (BCG) is a potent immune stimulator that activates innate and adaptive immunity. In the C57BL/6 mouse model of tuberculosis, BCG stimulated immunity causes a significant reduction of M. tuberculosis burden after pulmonary infection. The majority of work has focused on BCG induced T cell immunity as CD4+ T cells play a significant role in protection against tuberculosis. We hypothesized that BCG also induced a T cell independent mechanism that was sufficient to significantly restrict mycobacterial growth. BCG robustly activates innate immunity and our preliminary experiments showed that BCG induced a significant reduction in colony forming units (CFU) in the lungs of mice vaccinated 7 days before pulmonary infection. Our studies indicated that although BCG was administered through subcutaneous inoculation, increased numbers of Ly6C+ monocytes were observed in the lungs within 7 days. Selective depletion also determined that neutrophils play...

Mycobacterium tuberculosis is responsible for millions of deaths each year and with the rise of drug resistance, the need for an effective vaccine is paramount. Currently, the Mycobacterium bovis strain, BCG is the only vaccine available, which provides inconsistent efficacy against pulmonary tuberculosis. In the mouse model, BCG induces adequate protection against M. tuberculosis infection, even in the absence of T cells. In our preliminary studies, subcutaneous BCG vaccination sufficiently protected mice when given 7 days prior to pulmonary infection with M. tuberculosis, reinforcing the T cell-independent nature of immunity and leading us to hypothesize that innate immunity was responsible. Various studies have defined a significant role for innate immunity during BCG vaccination in what has been termed “trained innate immunity”. However, our studies suggest that whereas “trained innate immunity” may play a role in BCG induced protection against non-mycobacterial diseases, this m...

<p>A. Comparison of pro-inflammatory cytokines levels throughout the infection with pairs of clinical and laboratory clonal <i>Mtb</i> strains (t-test, *p<0.05). Limit of detection (LD) for IFN-γ: 0.5pg/mL, TNF-α: 0.9 pg/mL, and IL-6: 1.4 pg/mL.) Pair comparison between mice infected B). IL-10 (LD: 16.8 pg/mL) and IL-2 (LD: 0.1 pg/mL) levels in mice infected with laboratory and clinical <i>Mtb</i> pairs. Bars represent the mean values of cytokine concentration for five mice and the error bars represent the standard deviation.</p

<p>A and B). Western blot using anti-KatG (from mouse clone IT-57) from culture supernatants and cytosol fractions of <i>Mtb</i> clonal strains cultured in GAS media respectively (1 and 2 represent the two culture replicates of each strain used). Recombinant KatG was used as a positive control. C). Standard curve for peroxidase activity using 3,3′,5,5′-Tetramethylbenzidine (TMB) as substrate and recombinant KatG. D). KatG peroxidase activity (derived from the standard curve, C) from laboratory and clinical <i>Mtb</i> pairs using recombinant KatG as reference. Non-paired <i>t</i>-test without assuming consistent standard deviation, *<i>p</i><0.05.</p

<p>A) and C). Lung CFU count of mice infected with Laboratory and Clinical clonal strain pairs respectively. B) and D). Spleen CFU counts of Laboratory and Clinical clonal strain pairs respectively. Non-paired <i>t</i>-test without assuming consistent standard deviation (SD).*p<0.05, **p<0.001. Each time point represents the mean values for five mice infected with each of the four strains with the respective standard deviation shown in the error bars.</p

<p>Western blot using anti-AhpC<sub><i>Mtb</i></sub> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166807#pone.0166807.ref017" target="_blank">17</a>] from <b>c</b>ulture supernatants and cytosolic fraction of the four strains used in the study grown in GAS media (1 and 2 represent the two culture replicates of each strain used). CFP of H37Rv from BEI was used as a positive control. *indicates the lane for the ladder.</p

The microbial communities in gut and lung are important players that may modulate the immunity against tuberculosis or other infections as well as impact the vaccine efficacy. We discovered that vaccination through the subcutaneous route affect the composition of gut and lung bacteria, and this might influence susceptibility and defense mechanisms against tuberculosis.

ABSTRACTThe global epidemic caused by the bacterial pathogenMycobacterium tuberculosiscontinues unabated. Moreover, the only available vaccine against tuberculosis,Mycobacterium bovisbacillus Calmette-Guérin (BCG), demonstrates variable efficacy. To respond to this global threat, new animal models that mimic the pathological disease process in humans are required for vaccine testing. One new model, susceptible C3Heb/FeJ mice, is similar to human tuberculosis in that these animals are capable of forming necrotic tubercle granulomas, in contrast to resistant C3H/HeOuJ mice. In this study, we evaluated the impact of prior BCG vaccination of C3Heb/FeJ and C3H/HeOuJ mice on exposure to a low-dose aerosol ofMycobacterium tuberculosisW-Beijing strain SA161. Both BCG-vaccinated murine strains demonstrated reduced bacterial loads 25 days after infection compared to controls, indicating vaccine efficacy. However, during chronic infection, vaccine efficacy waned in C3H/HeOuJ but not in C3Heb/F...

A single intradermal vaccination with an antibiotic-less version of BCGΔBCG1419c given to guinea pigs conferred a significant improvement in outcome following a low dose aerosol exposure to M. tuberculosis compared to that provided by a single dose of BCG Pasteur. BCGΔBCG1419c was more attenuated than BCG in murine macrophages, athymic, BALB/c, and C57BL/6 mice. In guinea pigs, BCGΔBCG1419c was at least as attenuated as BCG and induced similar dermal reactivity to that of BCG. Vaccination of guinea pigs with BCGΔBCG1419c resulted in increased anti-PPD IgG compared with those receiving BCG. Guinea pigs vaccinated with BCGΔBCG1419c showed a significant reduction of M. tuberculosis replication in lungs and spleens compared with BCG, as well as a significant reduction of pulmonary and extrapulmonary tuberculosis (TB) pathology measured using pathology scores recorded at necropsy. Evaluation of cytokines produced in lungs of infected guinea pigs showed that BCGΔBCG1419c significantly red...

The guinea pig model of tuberculosis is used extensively to assess the efficacy of novel tuberculosis vaccines. There are established parameters to determine vaccine efficacy in this model, but the science community currently lacks established biomarkers for early detection and monitoring of experimental disease in guinea pigs. To define a set of biomarkers that could be used as benchmarks for disease progression and early endpoint criteria, we assessed serum biochemical and hematology parameters in 2 groups of guinea pigs—one vaccinated with the attenuated Mycobacterium bovis vaccine strain (BCG) and one sham-vaccinated with saline—and then experimentally infected with a virulent strain of Mycobacterium tuberculosis.After infection, WBC showed the strongest differences between saline-inoculated and vaccinated animals, with more subtle changes in other serum biochemical parameters, including ALT and ALP. Therefore, this study provides a starting point for evaluating the utility of b...

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4(+) and CD8(+) T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG-C, a liposome-based formulation containing the M. tuberculosis antigen ESAT-6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T-cell response. Humanized mice provide a crucial pre-cli...

Over the last 10 years, Mycobacterium abscessus group strains have emerged as important human pathogens, which are associated with significantly higher fatality rates than any other rapidly growing mycobacteria. These opportunistic pathogens are widespread in the environment and can cause a wide range of clinical diseases, including skin, soft tissue, central nervous system, and disseminated infections; by far, the most difficult to treat is the pulmonary form. Infections with M. abscessus are often multidrug-resistant (MDR) and require prolonged treatment with various regimens and, many times, result in high mortality despite maximal therapy. We report here the evaluation of diverse mouse infection models for their ability to produce a progressive high level of infection with M. abscessus. The nude (nu/nu), SCID (severe combined immunodeficiency), gamma interferon knockout (GKO), and granulocyte-macrophage colony-stimulating factor (GMCSF) knockout mice fulfilled the criteria for an optimal model for compound screening. Thus, we set out to assess the antimycobacterial activity of clarithromycin, clofazimine, bedaquiline, and clofazimine-bedaquiline combinations against M. abscessus-infected GKO and SCID murine infection models. Treatment of GKO and SCID mice with a combination of clofazimine and bedaquiline was the most effective in decreasing the M. abscessus organ burden.