A New Rabbit-Skin Model to Evaluate Protective Efficacy of Tuberculosis Vaccines.

Background: BCG protection is suboptimal and there is significant interest to develop new tuberculosis (TB) vaccines. However, there are significant limitations of the current vaccine evaluation systems in the mouse model. Here, we developed a BCG-challenge rabbit skin model as a new way to evaluate the protective efficacy of selected TB subunit vaccine candidates. Methods: Rabbits were immunized with subunit vaccines, including EAMM (ESAT6-Ag85B-MPT64<190-198>-Mtb8.4), MH (Mtb10.4-HspX), and LT70 (ESAT6-Ag85B-MPT64<190-198>-Mtb8.4-Rv2626c) three times subcutaneously every 3-weeks and challenged with the attenuated Mycobacterium bovis BCG intradermally 6-weeks after last immunization. The immune response induced by the vaccine candidates was measured, the histopathology induced by the BCG challenge was studied, and the number of bacilli in the liquefied caseum was determined. Results: The subunit vaccines generated high antigen-specific IgG antibodies and fastened the liquefaction and healing process, and significantly reduced the viable BCG load. The subunit vaccine LT70 and EAMM-MH reduced BCG bacterial load in comparison to proteins EAMM, MH, Rv2626c, and also BCG itself. The Koch phenomena induced by the LT70 and combination of EAMM-MH were the same as that produced by BCG itself and were more rapid than those induced by the other proteins and the saline controls. Conclusions: The subunit vaccines LT70 and the combination of EAMM-MH showed promising protective efficacy as expected in the rabbit skin model, which can serve as a visual and convenient new model for evaluating TB vaccines.

Perspectives for Developing New Tuberculosis Vaccines Derived from the Pathogenesis of Tuberculosis: I. Basic Principles, II. Preclinical Testing, and III. Clinical Testing.

Part I. Basic Principles. TB vaccines cannot prevent establishment of the infection. They can only prevent an early pulmonary tubercle from developing into clinical disease. A more effective new vaccine should optimize both cell-mediated immunity (CMI) and delayed-type hypersensitivity (DTH) better than any existing vaccine. The rabbit is the only laboratory animal in which all aspects of the human disease can be reproduced: namely, the prevention of most primary tubercles, the arrestment of most primary tubercles, the formation of the tubercle's solid caseous center, the liquefaction of this center, the formation of cavities and the bronchial spread of the disease. In liquefied caseum, virulent tubercle bacilli can multiply extracellularly, especially in the liquefied caseum next to the inner wall of a cavity where oxygen is plentiful. The bacilli in liquefied caseum cannot be reached by the increased number of activated macrophages produced by TB vaccines. Therefore, new TB vaccines will have little or no effect on the extracellular bacillary growth within liquefied caseum. TB vaccines can only increase the host's ability to stop the development of new TB lesions that arise from the bronchial spread of tubercle bacilli from the cavity to other parts of the lung. Therefore, effective TB vaccines do not prevent the reactivation of latent TB. Such vaccines only control (or reduce) the number of metastatic lesions that result after the primary TB lesion was reactivated by the liquefaction process. (Note: the large number of tubercle bacilli growing extracellularly in liquefied caseum gives rise to mutations that enable antimicrobial resistance-which is a major reason why TB still exists today). Part II. Preclinical Testing. The counting of grossly visible tubercles in the lungs of rabbits after the inhalation of virulent human-type tubercle bacilli is the most pertinent preclinical method to assess the efficacy of new TB vaccines (because an effective vaccine will stop the growth of developing tubercles before while they are still microscopic in size). Unfortunately, rabbits are rarely used in preclinical vaccine trials, despite their relative ease of handling and human-like response to this infection. Mice do not generate an effective DTH response, and guinea pigs do not generate an effective CMI response. Only the rabbits and most humans can establish the proper amount of DTH and CMI that is necessary to contain this infection. Therefore, rabbits should be included in all pre-clinical testing of new TB vaccines. New drugs (and/or immunological procedures) to reduce liquefaction and cavity formation are urgently needed. A simple intradermal way to select such drugs or procedures is described herein. Part III. Clinical Testing. Vaccine trials would be much more precise if the variations in human populations (listed herein) were taken into consideration. BCG and successful new TB vaccines should always increase host resistance to TB in naive subjects. This is a basic immunological principle. The efficacies of new and old TB vaccines are often not recognized, because these variations were not identified in the populations evaluated.

Perspectives on clinical and preclinical testing of new tuberculosis vaccines.

This review hopes to improve the selection of new tuberculosis (TB) vaccines by providing several perspectives on the immunization of humans, mice, guinea pigs, rabbits, and monkeys which have not usually been considered. (i) In human TB vaccine trials, the low rate of healing of Mycobacterium bovis BCG lesions (used as the control group) would distinguish individuals who might be helped by vaccination from the 95% who do not need it and would make these trials more conclusive. (ii) The rabbit immune response to Mycobacterium tuberculosis is much more effective in arresting tuberculosis than those of other laboratory animals, so pulmonary tubercle counting in rabbits should be included in all preclinical TB vaccine testing. (iii) Both delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI) are necessary to control the growth of M. tuberculosis. The testing of new TB vaccines in mice or in guinea pigs may not detect important antigens needed for human immunization. Mice respond poorly to tuberculin-like antigens that cause DTH. Guinea pigs respond poorly to antigens that cause CMI. Rabbits and humans respond well to both DTH and CMI antigens. Since monkeys are very susceptible to M. tuberculosis, they may not be as useful as rabbits for preclinical vaccine evaluation. (iv) Critical antigens (possibly ESAT-6 or CFP-10) might increase the immunity of the host to a greater extent than that produced by a natural M. tuberculosis infection and therefore would be useful in both prophylaxis and immunotherapy. Such critical antigens would increase the host's ability to neutralize key components of M. tuberculosis that enable it to survive in both laboratory animals and humans.

Liquefaction and cavity formation in pulmonary TB: a simple method in rabbit skin to test inhibitors.

To control tuberculosis in the world today an additional approach would be most welcomed. Preventing (or reducing) pulmonary cavity formation is one such approach that has been almost completely neglected. Pulmonary cavity formation and the extracellular growth of tubercle bacilli in cavities cause bronchial spread of the disease in adult patients and spread of the bacillus to the environment where they infect other people. Therefore, cavity formation perpetuates tuberculosis in mankind. If no cavities form, the patient is much less infectious. Also, cavity formation often allows the tubercle bacillus to multiply (extracellularly) to tremendous numbers. Therefore, in humans almost all multidrug-resistant tubercle bacilli develop in cavities. This communication reviews the literature on liquefaction and cavity formation, and lists some of the responsible hydrolytic enzymes. It also describes a simple method to identify inhibitory pharmaceuticals, i.e., to observe their effect on the liquefaction and ulceration of skin lesions produced in rabbits by ascending concentrations of live or dead tubercle bacilli.

The aerosol rabbit model of TB latency, reactivation and immune reconstitution inflammatory syndrome.

The large reservoir of human latent tuberculosis (TB) contributes to the global success of the pathogen, Mycobacterium tuberculosis (Mtb). We sought to test whether aerosol infection of rabbits with Mtb H37Rv could model paucibacillary human latent TB. The lung burden of infection peaked at 5 weeks after aerosol infection followed by host containment of infection that was achieved in all rabbits. One-third of rabbits had at least one caseous granuloma with culturable bacilli at 36 weeks after infection suggesting persistent paucibacillary infection. Corticosteroid-induced immunosuppression initiated after disease containment resulted in reactivation of disease. Seventy-two percent of rabbits had culturable bacilli in the right upper lung lobe homogenates compared to none of the untreated controls. Discontinuation of dexamethasone led to predictable lymphoid recovery, with a proportion of rabbits developing multicentric large caseous granuloma. The development and severity of the immune reconstitution inflammatory syndrome (IRIS) was dependent on the antigen load at the time of immunosuppression and subsequent bacillary replication during corticosteroid-induced immunosuppression. Clinically, many aspects were similar to IRIS in severely immunosuppressed HIV-infected patients who have functional restoration of T cells in response to effective (highly active) antiretroviral therapy. This corticosteroid model is the only animal model of the IRIS. Further study of the rabbit model of TB latency, reactivation and IRIS may be important in understanding the immunopathogenesis of these poorly modeled states as well as for improved diagnostics for specific stages of disease.

Susceptibility to tuberculosis: composition of tuberculous granulomas in Thorbecke and outbred New Zealand White rabbits.

We sought to characterize the lung cellular immune responses to inhaled Mycobacterium tuberculosis (Mtb) of the susceptible inbred Thorbecke rabbit (the genomically sequenced strain, now unavailable) and compare it to outbred, Mtb-resistant, New Zealand White rabbits. Using Mtb CDC1551, we confirmed that the inbred rabbits allowed establishment of infection with this low virulence strain, compared to poor establishment in outbred rabbits. With a more virulent strain, Mtb Erdman, that establishes infection well in both rabbit strains, we analyzed granulomas from rabbit lungs 5 weeks after aerosol infection. The lung granulomas of inbred rabbits had significantly higher frequencies of cells expressing MHC Class II and CD11b, and lower frequencies of CD8+ T cells than the outbred controls. Macrophage-sized cells expressing MHC Class II in inbred rabbit granulomas showed significantly decreased intensity of expression, suggesting impaired maturation. Although the inbred dermal tuberculin reactions were decreased, the in vitro IFN-gamma mRNA responses of hilar node lymphocytes to tuberculin were higher than those of outbred rabbits. Further delineation of the outbred rabbit's resistant immune response to Mtb infection is warranted.

Effects of dexamethasone and transient malnutrition on rabbits infected with aerosolized Mycobacterium tuberculosis CDC1551.

Malnutrition is common in the developing world, where tuberculosis is often endemic. Rabbits infected with aerosolized Mycobacterium tuberculosis that subsequently became inadvertently and transiently malnourished had compromised cell-mediated immunity comparable to that of the rabbits immunosuppressed with dexamethasone. They had significant leukopenia and reduced delayed-type hypersensitivity responses. Malnutrition dampened cell-mediated immunity and would interfere with diagnostic tests that rely on intact CD4 T-cell responses.

Susceptibility to tuberculosis: clues from studies with inbred and outbred New Zealand White rabbits.

The rabbit model of tuberculosis (TB) is important because rabbits develop a disease that is similar to TB in humans, namely, granulomas with caseous necrosis, liquefaction, and cavities. We describe here a comparison of inbred and outbred New Zealand White rabbits infected by aerosol with either Mycobacterium tuberculosis Erdman or H37Rv strain. Five weeks after infection with either bacillary strain, the inbred rabbits had significantly larger pulmonary tubercles than did outbred rabbits (2.7 versus 1.4 mm in diameter; P < 0.01). After infection with H37Rv, the inbred rabbits had significantly more pulmonary tubercles than did the outbred rabbits (98 +/- 12 versus 33 +/- 13; P < 0.01), with more mycobacterial CFU per tubercle (809 +/- 210 versus 215 +/- 115; P = 0.027) (means +/- standard errors of the means). Compared with histologic examination of lung granulomas from outbred rabbits, histologic examination of those from inbred rabbits showed more caseous necrosis, more visible bacilli, and fewer mature epithelioid cells. The delayed-type hypersensitivity (DTH) responses to intradermal tuberculin were significantly lower, and peritoneal macrophages from uninfected inbred rabbits produced significantly less tumor necrosis factor alpha after lipopolysaccharide (LPS) stimulation in vitro than those from the outbred rabbits (2,413 +/- 1,154 versus 8,879 +/- 966 pg/ml). We conclude that these inbred rabbits were more susceptible to TB than their outbred counterparts and had an impaired ability to contain disease, resulting in more grossly visible tubercles that were larger than those observed in outbred rabbits. Preliminary evidence is presented for a cell-mediated immune defect with lower DTH responses and macrophages that have a decreased ability to respond to in vitro stimulation with LPS or M. tuberculosis infection.

Different strains of Mycobacterium tuberculosis cause various spectrums of disease in the rabbit model of tuberculosis.

The rabbit model of tuberculosis has been used historically to differentiate between Mycobacterium tuberculosis and Mycobacterium bovis based on their relative virulence in this animal host. M. tuberculosis infection in market rabbits is cleared over time, whereas infection with M. bovis results in chronic, progressive, cavitary disease leading to death. Because of the innate resistance of commercial rabbits to M. tuberculosis, 320 to 1,890 log-phase, actively growing inhaled bacilli were required to form one grossly visible pulmonary tubercle at 5 weeks. The range of inhaled doses required to make one tubercle allows us to determine the relative pathogenicities of different strains. Fewer inhaled organisms of the M. tuberculosis Erdman strain were required than of M. tuberculosis H37Rv to produce a visible lesion at 5 weeks. Furthermore, with the Erdman strain, only 7 of 15 rabbits had healed lesions at 16 to 18 weeks; among the other animals, two had chronic, progressive cavitary disease, a phenotype usually seen only with M. bovis infection. Genotypic investigation of the Erdman strain with an H37Rv-based microarray identified gene differences in the RD6 region. Southern blot and PCR structural genetic analysis showed significant differences between M. tuberculosis strains in this region. Correlation of the relative pathogenicity, including disease severity, in the rabbit model with the strain genotype may help identify stage-specific M. tuberculosis genes important in human disease.

Macrophage turnover, division and activation within developing, peak and "healed" tuberculous lesions produced in rabbits by BCG.

This review is a synthesis and analysis of our nine experimental pathology papers on macrophage kinetics in dermal tuberculous lesions produced in rabbits by BCG. It is presented at this time to summarize the macrophage kinetics in both active and essentially healed tuberculous lesions and to suggest that the bacilli frequently multiply and are destroyed in the viable granulation tissue of many small arrested tuberculous lesions. The turnover of mononuclear cells (MN)-which were mostly macrophages with some medium and large lymphocytes-was most rapid in BCG lesions at 2-3 weeks (when tuberculin sensitivity and acquired cellular resistance were at their peaks). At this time, more macrophages entered, more died or left, more remained at the site, and more became activated than before or afterwards. Before this time, the host had no delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI), so that no antigen-specific enhancement of the inflammatory response occurred. After this time, the bacilli and their antigenic products had decreased, so that the stimuli for cell infiltration and activation were reduced. In "healed" lesions, the MN turnover still occurred, but was decreased. The continuous entry of live non-activated macrophages into the viable parts of tuberculous lesions provides fresh intracellular sites where tubercle bacilli may multiply before they are again inhibited by the DTH and CMI of the host. In tuberculosis, bacillary dormancy of long duration may only be present in caseous necrotic tissue where no live host cells exist.