+-------------+--------------------------------------------------------+
| **          | Numerous novel tuberculosis vaccines and immunization  |
| Abstract:** | strategies are being developed to combat the           |
|             | devastating TB epidemic. Many of these new vaccine     |
|             | preparations are being targeted for use in both        |
|             | immune-naïve and TB infected persons. Recent reports,  |
|             | however, suggest that some of these new vaccines may   |
|             | induce Koch-like reactions and actually exacerbate the |
|             | tuberculous disease when administered to infected      |
|             | animals. In this proposal, we plan to establish animal |
|             | models at CBER to study the vaccine-mediated induction |
|             | of Koch responses in latently and chronically infected |
|             | mice. We will assess whether the administration of new |
|             | vaccine candidates into infected mice evokes Koch      |
|             | responses by evaluating lung pathology and organ       |
|             | bacterial burden after vaccination. After validating   |
|             | the models, we plan to test BCG recombinant vaccines,  |
|             | live attenuated *M. tuberculosis* strains, DNA         |
|             | vaccines, and subunit vaccine-adjuvant formulations    |
|             | for their capacity to induce Koch reactions in         |
|             | infected mice. It is anticipated that these safety     |
|             | tests will be needed before proceeding into clinical   |
|             | testing of PPD+ individuals.                           |
+-------------+--------------------------------------------------------+
| **Specific  | 1.  To establish and validate models of latent and     |
| aims:**     |     chronic TB infection for evaluating                |
|             |     vaccine-mediated induction of Koch reactions.      |
|             |                                                        |
|             | 2.  To evaluate promising TB vaccine candidates in     |
|             |     these Koch response models.                        |
+-------------+--------------------------------------------------------+
| **Ba        | Even in the 21^st^ century, disease caused by          |
| ckground:** | *Mycobacterium tuberculosis* is the leading cause of   |
|             | death from a bacterial infection with more than 2      |
|             | million people worldwide dying from tuberculosis each  |
|             | year. Furthermore, the WHO estimates that more than    |
|             | one billion people are infected with *M.               |
|             | tuberculosis*; these latently infected individuals are |
|             | at risk for reactivational TB disease. Largely because |
|             | of this global public health tragedy, substantial      |
|             | research efforts have been directed toward the         |
|             | development of new improved tuberculosis vaccines.     |
|             | Recently, several attractive TB vaccines candidates    |
|             | have been generated and characterized. One of these    |
|             | new vaccines is currently being evaluated clinically   |
|             | in Europe and the clinical testing of 4-5 other        |
|             | preparations will likely begin in the United States    |
|             | within the next year.                                  |
|             |                                                        |
|             | Most of these new vaccines have been developed and     |
|             | characterized for use in immune-naive populations.     |
|             | However, given the enormous number of latently         |
|             | infected people, many investigators have expressed     |
|             | interest in testing their vaccines in individuals      |
|             | previously exposed to mycobacteria \[PPD(+)\] as well  |
|             | as mycobacterial-naïve \[PPD(-)\] subjects. Clinical   |
|             | evaluation of these vaccines in infected individuals   |
|             | is problematic because of safety concerns associated   |
|             | with the potential for induction of Koch reactions.    |
|             | More than one hundred years ago, Robert Koch reported  |
|             | that administration of crude tuberculin preparations   |
|             | could cure tuberculosis in guinea pigs (1).            |
|             | Unfortunately, subsequent use of this treatment in     |
|             | patients with mild disease caused florid local and     |
|             | systemic reactions. Additionally, when 230 patients    |
|             | with cavitary disease were treated with this           |
|             | tuberculin, several deaths were noted. Subsequent      |
|             | analyses of these reactions have suggested that severe |
|             | necrosis evoked in deep lesions within the spine and   |
|             | lung resulted in the more advanced disease after the   |
|             | tuberculin treatment.                                  |
|             |                                                        |
|             | Recently, intense reactions to *M. tuberculosis*       |
|             | extracts and associated clinical worsening have been   |
|             | described in pre-clinical testing of new vaccines in   |
|             | animal models. Moreira et al. have reported that       |
|             | injection of mycobacterial antigens to mice with prior |
|             | *M*. *tuberculosis* infection lead to immune           |
|             | activation that exacerbated lung pathology without     |
|             | reducing the bacillary load (2). For example, when *M. |
|             | tuberculosis* infected mice were treated with BCG      |
|             | engineered to secrete cytokines a significant increase |
|             | in the granuloma size in the lungs was detected. In    |
|             | another study, Taylor et al. found that modestly       |
|             | infected mice immunized with a mycobacterial hsp60 DNA |
|             | vaccine developed Koch reactions which were            |
|             | characterized by substantial cellular necrosis         |
|             | throughout the lung granulomas (3). Similar severe     |
|             | pathology was seen when mice were given the highly     |
|             | protective 85B TB DNA vaccine. The authors concluded   |
|             | that because of these potential safety problems "DNA   |
|             | vaccines should be used with caution in individuals    |
|             | who may have been exposed to tuberculosis". It is      |
|             | important to note that other vaccines (eg., adjuvanted |
|             | subunit preparations) have caused no adverse events in |
|             | pre-infected animals.                                  |
|             |                                                        |
|             | These recent reports have increased concerns about the |
|             | exacerbation of disease via Koch-like reactions after  |
|             | vaccination of PPD (+) individuals. In this            |
|             | submission, we propose to develop a model at CBER to   |
|             | assess whether specific new TB vaccines will evoke     |
|             | Koch reactions in previously infected animals. After   |
|             | standardizing the model, we propose to investigate     |
|             | whether an array of novel TB vaccines including BCG    |
|             | recombinants, live attenuated *M. tuberculosis*        |
|             | strains, DNA vaccines, and subunit vaccines formulated |
|             | with potent adjuvants will increase disease severity   |
|             | when administered therapeutically. Overall, the        |
|             | development and use of a standardized test for         |
|             | measuring Koch responses should help identify vaccines |
|             | which can be safely administered to latently infected  |
|             | persons; this enhanced safety profile should           |
|             | facilitate the clinical testing and introduction of    |
|             | these much-needed vaccines in areas where the          |
|             | incidence of TB is high.                               |
+-------------+--------------------------------------------------------+

+-------------+--------------------------------------------------------+
| **Study     | #                                                      |
| des         |                                                        |
| ign/Plan:** | # [LATENT INFECTION MODEL]{.underline}                 |
|             |                                                        |
|             | We propose to use a variation of the widely used       |
|             | Cornell model of dormant TB to evaluate whether        |
|             | specific vaccines induce Koch-like reactions in        |
|             | previously infected animals (4). In the Cornell model, |
|             | mice are infected and then are given chemotherapy to   |
|             | reduce bacterial burden below detectable levels.       |
|             | Subsequently steroid immunosuppression can be given to |
|             | reactivate surviving bacilli. We have previously used  |
|             | this model to demonstrate that immunization with a DNA |
|             | vaccine cocktail (which protected naïve animals) did   |
|             | not prevent reactivation in latently infected mice     |
|             | (5).                                                   |
|             |                                                        |
|             | Specifically, we plan to aerogenically infect C57BL/6  |
|             | and Balb/c mice with a low dose of *M. tuberculosis*   |
|             | Erdman. One month after the initial infection, the     |
|             | mice will be given chemotherapy by adding INH and RIF  |
|             | to the drinking water for 12 weeks. We showed earlier  |
|             | that this drug combination reduces the organ bacterial |
|             | burden to undetectable levels and that subsequent      |
|             | injections of dexamethasone reactivates the disease    |
|             | (5). After completion of the chemotherapy, the mice    |
|             | will be immunized with the candidate vaccines using    |
|             | standard immunization schedules. Four and eight weeks  |
|             | after the final vaccination, lung pathology and        |
|             | bacterial burden in lungs and spleens of immunized and |
|             | control mice will be compared. For the lung pathology, |
|             | H&E stained slides will be evaluated in a blinded      |
|             | fashion by trained investigators. Worsening lung       |
|             | pathology (larger granulomas, greater cellular         |
|             | infiltrate, more extensive necrosis, etc.) and         |
|             | increased bacterial burdens in vaccinated animals      |
|             | relative to controls would be evidence of              |
|             | deteriorating clinical disease after immunization.     |
|             |                                                        |
|             | Our initial efforts in establishing the model will be  |
|             | focused toward the identification of a preparation     |
|             | that consistently induces Koch-like reactions and      |
|             | could serve as a positive control. We plan to first    |
|             | investigate whether the hsp60 DNA vaccine, *M.         |
|             | tuberculosis* crude extracts (previously shown to      |
|             | induce Koch responses) or heat-killed *M.              |
|             | tuberculosis* bacilli will reliably induce worsening   |
|             | clinical disease. Once the positive control has been   |
|             | established, we will begin investigating the capacity  |
|             | of new vaccine candidates to evoke Koch responses.     |
|             | Initially, we will test the recombinant BCG vaccine    |
|             | over-expressing Antigen 85B and its parent strain      |
|             | BCG-Tice in the model system. The BCG-85B preparation  |
|             | is a novel TB vaccine that is scheduled to be          |
|             | evaluated in clinical trials in 2004 (6).              |
|             | Subsequently, we will examine vaccines that have been  |
|             | shown to be effective in pre-clinical studies in our   |
|             | laboratory. Among these vaccines are live attenuated   |
|             | *M. tuberculosis* vaccine strains and a DNA vaccine    |
|             | cocktail (7, 8). In later studies we will test novel   |
|             | subunit vaccine-adjuvant formulations that have        |
|             | induced substantial protection in pre-clinical         |
|             | experiments. We are particularly interested in         |
|             | evaluating the activity of the alpha-crystallin        |
|             | protein in this model; a recent report has suggested   |
|             | that reactivational disease is limited after injection |
|             | of this protein into mice infected with *M.            |
|             | tuberculosis* (9).                                     |
|             |                                                        |
|             | # [CHRONIC INFECTION MODEL]{.underline}                |
|             |                                                        |
|             | It is possible that persons with undiagnosed active    |
|             | disease will be present in the PPD(+) cohort.          |
|             | Therefore, the capacity of the new vaccines to         |
|             | exacerbate disease in animals with active chronic      |
|             | infections should be evaluated. In these experiments,  |
|             | mice will again be aerogenically infected with a low   |
|             | dose of *M. tuberculosis* Erdman. One month after the  |
|             | aerosol infection, the mice will be treated with       |
|             | isoniazid for 60 days. Data from a recent report       |
|             | suggest that chemotherapy with isoniazid only will     |
|             | reduce the organ bacterial burden to modest levels     |
|             | (3). After completion of the chemotherapy, the         |
|             | vaccines will be administered using standard           |
|             | immunization schedules. At two, four, and eight weeks  |
|             | after the immunization, lung pathology and bacterial   |
|             | burden will be compared in vaccinated and control      |
|             | animals. Worsening lung pathology and increased        |
|             | bacterial CFU values in the vaccinated mice would      |
|             | indicate that the vaccines had exacerbated disease in  |
|             | animals with active disease.                           |
+-------------+--------------------------------------------------------+

####### BIBLIOGRAPHY

1.  Iseman, M. D. 2000. A Physician's Guide to Tuberculosis. Lippincott,
    Williams, & Wilkins, Philadelphia, Pa.

2.  Moreira A. et al. 2002. Mycobacterial antigens exacerbate disease
    manifestations in *Mycobacterium* *tuberculosis*-infected mice.
    Infect. Immun. 70:2100-2107.

3.  Taylor J. et al. 2003. Pulmonary necrosis resulting from DNA
    vaccination against tuberculosis. Infect. Immun. 71;2192-2198.

4.  McCune R. et al. 1966. Microbial persistence. I. The capacity of
    tubercle bacilli to survive sterilization in mouse tissues. J. Exp.
    Med. 123:445-468.

5.  Repique C. et al. 2002. DNA immunization in a mouse model of latent
    tuberculosis: Effect of DNA vaccination on reactivation of disease
    and on reinfection with a secondary challenge. Infect. Immun.
    70:3318-3323.

6.  Horwitz M. et al. 2000. Recombinant bacillus Calmette-Guerin (BCG)
    vaccines expressing the *Mycobacterium tuberculosis* 30-kDa major
    secretory protein induces greater protective immunity against
    tuberculosis than conventional BCG vaccines in a highly susceptible
    animal model. Proc. Natl. Acad. Sci. 97:13853-13858.

7.  Delogu G. et al. 2002. DNA vaccine combinations expressing either
    tissue plasmingen activator signal sequence fusion proteins or
    ubiquitin-conjugated antigens induce sustained protective immunity
    in a mouse model of pulmonary tuberculosis. Infect. Immun.
    70:292-302.

8.  Sambandamurthty V. et al. 2002. A pantothenate auxotroph of
    *Mycobacterium tuberculosis* is highly attenuated and protects mice
    against tuberculosis. Nature Med. 8:1171-1174.

9.  Andersen P. 2003. Tuberculosis vaccines -- designing vaccines for
    pre and post exposure vaccination strategies. The First
    International Conference on TB vaccines for the World.