Given the number of new drugs in the TB drug development pipeline (46
), it is more crucial than ever to have validated preclinical testing methods that are predictive of clinical outcome and that provide the highest confidence that the most potent drug regimens are being advanced into clinical trials. This is especially important in light of a recent initiative by the Gates Foundation (Critical Path to New TB Regimens [CPTR]) that will stimulate development of new combination regimens (of new investigational drugs along with existing TB drugs) to avoid developing each drug sequentially over decades to come (47
). Although there is no doubt that the CPTR approach will lead to more effective drug regimens, it will become important to select the most promising combinations from preclinical studies for both bactericidal and sterilizing activity to advance and be tested clinically.
Our previously published studies focused on a multitude of variables in the methods used in preclinical drug testing in mice (9
), and these ultimately revealed that the strain used in animal infection models deserved greater investigation. To our knowledge, there are no in vivo
studies that have evaluated combination anti-TB drug regimens against various strains of M. tuberculosis
in a single head-to-head experiment. In the studies presented here, we evaluated the bactericidal activity of the drug combinations HRZ and RZ in the BALB/c mouse model, infected via aerosol using five different strains of M. tuberculosis
(JHU H37Rv, CSU H37Rv, Erdman, CDC1551, and HN878). The results showed that the in vivo
efficacy of the tested drug combinations was not always the same for the different M. tuberculosis
evaluated. In fact, the degrees of in vivo
bactericidal activity of RZ for two different H37Rv strains were different, with the efficacy in the lungs being significantly better in JHU H37Rv-infected mice than in CSU H37Rv-infected mice. Moreover, the rankings of the HRZ and RZ regimens were not the same for both H37Rv strains. Antagonism in the HRZ combination was seen only with the JHU H37Rv strain, not with any of the four other strains evaluated.
Recently, it has become clear that even among the H37Rv strains across different laboratories there is a significant genetic heterogeneity (22
). Loerger et al. urge investigators to use caution in referring to H37Rv as a standard reference strain, as experimental results derived with “H37Rv” may depend on the laboratory in which it is maintained and its associated genetic characteristics. The whole-genome sequencing results obtained by the same authors showed that differences among H37Rv strains have arisen over time, and while they are relatively few (on the order of 5 to 10 polymorphisms per strain), they still could be functionally relevant. Which genetic differences are responsible for the strain-based differences in efficacy presented here remains unclear and would have to be investigated further. Ongoing genetic evolution has been observed in vitro
via the accumulation of genetic differences during serial passaging of cultures (39
); therefore, keeping the passage number low has been shown to be a definite requirement. The methods of propagation, storage, and passaging of strains are highly variable across different laboratories. Some investigators passage M. tuberculosis
strains through animals to maintain virulence (8
), while others grow the strain in a pellicle for the same purpose (32
). In both cases, there is no published evidence that in fact shows that either protocol increases or maintains the virulence of the strain. In contrast, Converse et al. published results indicating that animal passaging of M. tuberculosis
strains prior to quantitative virulence testing in mouse and guinea pig models did not enhance or restore potency to strains that may have lost virulence due to in vitro
). The authors mention that it is critical to verify virulence of parental strains before any manipulation is undertaken. For the studies described here, there was a clear difference in the origins of the H37Rv strain used by both groups, as well as the method of strain propagation: the JHU H37Rv strain was serially passaged in mice and individual bacteria were isolated and propagated, whereas the CSU H37Rv was grown as a pellicle, seed lots were frozen, and the passage number of the working stocks never exceeded six. Identifying the most important variable which can influence the virulence and drug responses of the strains used in animal models (source/origin, method of maintaining virulence, protocol for propagation, etc.) in TB drug evaluation mouse studies would require a careful stepwise investigation.
The chain of custody of different M. tuberculosis
strains can become more uncertain over time and is often not reported in published works. For instance, in regard to the history of the strains, it is known that the parent strain of H37Rv (TMC102) was isolated by E. R. Baldwin from a human lung in 1905 and dissociated from this parent (H37) in 1934 at the Trudeau Institute, Saranac Lake, NY (27
). H37RV was continually passaged in Proskauer-Beck medium until 1970, when it was frozen at −70°C, and it is the neo-type strain from which ATCC 27294 was derived. M. tuberculosis
Erdman was originally isolated from human sputum by W. Feldman at the Mayo Clinic in 1945 (personal communication, Trudeau Medical Library). The following year, in 1946, it was transferred to the Trudeau Institute, where it was maintained in Proskauer-Beck medium until 1970, when it was frozen at −70°C. We were unable to find documentation of the exact details of the years of additional propagation for both the H37Rv and Erdman strains.
The results here also shed some light on the question of whether the antagonism seen within the standard HRZ regimen by some investigators (15
) but not others (2
) is a TB strain-specific phenomenon. Previous studies have often had widely differing experimental variables and endpoints, and therefore a direct comparison was not feasible. In the direct head-to-head-comparison studies described in this work, the combination of HRZ was as effective as or significantly more effective than RZ dual therapy for four of five TB strains tested, excepting only the JHU H37Rv strain, for which the efficacy of RZ under most conditions was better than that of HRZ. In none of the other four TB strains did we ever observe antagonism with the HRZ combination. Even in our laboratory, the antagonism in HRZ for JHU H37Rv is not seen to the same extent every time, and this may be dependent on the INH dose or drug exposure, as was previously reported (1
). Another older study reported that the antagonism observed is dependent on certain timing of the infection and administration of drugs (36
). Although the basis of the observed antagonism remains poorly understood, the results presented here make the point that the antagonism within the standard regimen appears to be a strain-specific phenomenon and is seen only under specific conditions. Murine models certainly make it possible to rank efficacies of drug regimens, if appropriate and sufficient control groups for each regimen are wisely chosen. If, for instance, the impact of a new compound is evaluated when it is added to the standard HRZ regimen against a particular TB strain, its activity should be compared to that of the standard regimen by itself. On the other hand, if the goal is to evaluate the efficacy of a new regimen where the new drug replaces a drug of the standard drug regimen (e.g., H) then the appropriate control (e.g., RZ) should be included alongside HRZ.
The studies presented here have some obvious limitations. Only a limited number of drug regimens were studied due to the labor-intensive nature of these comparative studies, and therefore the conclusions regarding the choice of M. tuberculosis
strains can at this point be made only for the regimens tested. In addition, the responses of the various strains were evaluated only in terms of the bactericidal activity of certain drug regimens, not the sterilizing activity in long-term relapse trials. In relapse studies, the choice of M. tuberculosis
strain might affect the outcome even more, as the presence of memory immunity for all strains is not equal. For instance, only clinical isolates of M. tuberculosis
have been described to induce a regulatory T-cell population, which influences the degree of relapse of infection (7
). HN878, a member of the W-Beijing genotype, is known to be a potent inducer of regulatory T cells and is associated with increased virulence and pathology in lungs of mice (42
). Additional mouse studies will be necessary to answer these important questions regarding the difference in responses of laboratory-adapted and clinical TB strains in relapse and for the assessment of other drug regimens. One limitation in our second study was that the inoculums for the various strains were not entirely identical, as they were in the first study. This was especially the case for HN878, which surprisingly grew less well than the comparator strains.
In conclusion, in the in vivo
head-to-head experiments described here, the M. tuberculosis
strain was found to be one of the most important variables that can change the outcome of the in vivo
efficacy trial in mice. Therefore, we highly recommend confirmation of in vivo
efficacy results in a late stage of preclinical testing against a second M. tuberculosis
strain, thereby increasing the confidence to advance a potent drug regimen to clinical trials. Moreover, we suggest that these studies include current clinical M. tuberculosis
strains (from one or more different clades based on their geographical locations) (14
). Given the widespread dissemination of Beijing strains in the world and the coadaptation with humans over time, strong consideration should be given to including a member of the Beijing family of M. tuberculosis
strains. In addition, it is highly recommended for all in vivo
studies to use only M. tuberculosis
strains that are fully characterized (genetically), have good growth characteristics and a limited passage number, have been propagated appropriately, and have a completely documented chain of custody.
The recommendation to evaluate and confirm anti-TB drug regimens against relevant M. tuberculosis strains in validated mouse models is of especially great importance given the CPTR effort to advance a combination of three novel drugs into human clinical trials in the next few years. In summary, we believe that all efficacy data generated with animal models should be confirmed in parallel mouse models in a second laboratory and should include a range of representative isolates.