Animal models for experimental tuberculosis.

Animal models for tuberculosis vaccine assays have evolved through a series of sequential experiments aimed at optimizing the activity of a particular vaccine product. As a result, studies that use different animal models do not agree on the potency ranking of antituberculosis vaccines, and each major test-system variable contributes to the disagreement. Disagreements among laboratories about the efficacy of vaccines are in part due to differences in test systems. A survey of potency assays of tuberculosis vaccines suggests that, based on the choice of a specific combination of variables in the test model, an investigator can show that any given vaccine product has superior potency. In view of these problems with animal models for research on tuberculosis vaccines, we recommend that attention should be focused on those animal models that replicate the key aspects of the natural history of human tuberculosis. The development of vaccines by means of new technologies requires animal models to assay the protective potency of vaccines that, ideally, inhibit tubercle bacilli at the point of infection. The development of new tuberculosis vaccines may be handled most efficiently in a joint venture that includes both laboratories involved in vaccine production and laboratories concerned with animal models.

An analysis of some hypotheses related to the Chingelput bacille Calmette-Guérin trial.

Investigation of several hypotheses related to the outcome of the Chingelput Bacille Calmette-Guérin (BCG) Trial suggests at least 2 factors that might explain the major scientific puzzle of a protective effect expected of 80% and a protective effect observed of 0% (i.e., equivalent protection for BCG and placebo). One factor that explains some of the low efficacy observed for BCG in this trial is the virtual saturation level of exposure to environmental mycobacteria (EM). Studies in animal models demonstrated that the protection afforded by infection with EM was equivalent to the protection that resulted from BCG vaccination. The second factor, pathogenetic pathway, explains why there was still a high case rate for tuberculosis, even though the population was fully vaccinated by EM. This hypothesis states that tuberculosis in India, as well as in most developing countries, results primarily from exogenous reinfection, a pathway against which BCG (or EM) exerts no protective effect beyond that induced by the first episode of infection with Mycobacterium tuberculosis.

Mycobacterial infection in guinea pigs.

We described published reports of the chaos which exists in research concerning laboratory animal models for assay of tuberculosis (TB) vaccines and proposed a "rational animal model" as a solution to the problem. This animal model, an aerosol challenge model in guinea pigs, was recently applied to the problem of differences in growth characteristics of sputum isolates of low and high virulence. The same model was used to investigate the protective effect of high dose BCG given aerogenically. Based on studies in the guinea pig model of experimental airborne TB, and a review of the literature on pathogenesis of human TB, we described an "integrated model" for the pathogenesis of TB, a model which includes a role for both the endogenous reactivation and the exogenous reinfection pathways. Our hypothesis is that tubercle bacilli must be able to gain access to the "vulnerable region" in the lung apex in order to survive the effects of the CMI response. In endogenous reactivation TB (virulent tubercle bacilli), this access occurs via the bloodstream. Whereas in exogenous reinfection TB, access to the vulnerable region occurs via multiple exposures via the respiratory tract. Central to our perspective is the acceptance of the evidence that during first infection with virulent organisms, tubercle bacilli enter the bloodstream via the efferent lymphatics. We believe the hypotheses we have proposed have the potential to lead to a further increase in our knowledge of these mechanisms and are a prerequisite to studies aimed at the development of new vaccines.

Pathogenesis of tuberculosis: pathway to apical localization.

We have examined the published work of investigators which dealt with the pathogenesis of tuberculosis, especially the following: the infective dose, the yield of bacilli from the primary lesion and primary complex, the predominant location of the minimal lesion, the hypotheses of a vulnerable region in the lung and the specific pathways (endogenous or exogenous) by which tubercle bacilli cause disease. More knowledge of the pathogenic pathway to tuberculosis would provide clues to the development of new vaccines and drug regimens that can intervene at a specific stage in the pathogenesis. Based on the examination of the literature on pathogenesis of human tuberculosis and our findings in a guinea-pig model of experimental airborne tuberculosis, we have proposed an hypothesis which integrates the endogenous and exogenous pathways to tuberculosis. This hypothesis is based on the following observations: 1. The infectious dose is very low, usually 1-5 tubercle bacilli. 2. The first implant can occur anywhere in the lungs. 3. The cavitary lesion, characteristic of tuberculous disease, is often located in the apical regions in the lungs. 4. Whereas the primary implant can occur anywhere in the lungs, for the progression from infection to disease, the tubercle bacilli must gain access to the 'vulnerable' regions in the apex of the lungs. Our hypothesis states that in areas of the world where there is a low risk of infection with tubercle bacilli low incidence of vaccination or sensitization to environmental mycobacteria, or high incidence of high virulent isolates, the virulent tubercle bacilli reach the vulnerable region via a bacillemia during the first infection. In areas of the world where there is a high risk of infection with tubercle bacilli, high incidence of vaccination or sensitization to environmental mycobacteria or a high incidence of low virulent isolates, the tubercle bacilli reach the vulnerable region via the airway, which requires repeated episodes of infection as the probability of a first implant occurring in the vulnerable regions is low.

Growth characteristics of recent sputum isolates of Mycobacterium tuberculosis in guinea pigs infected by the respiratory route.

The consideration of virulence must distinguish between infectivity and the ability to cause progressive disease once the infection is established. Several investigators have reported the presence of naturally occurring isolates which differ in virulence for guinea pigs. Isolates from south India which differed with respect to gross disease and number of bacilli recovered from spleen after an intramuscular infection also differed in their efficiencies to initiate an infection, once inhaled and retained. Also, this difference was correlated with differences in the rate of multiplication at the site of implantation and rate of multiplication at sites of hematogenous seeding, as well as the extent of hematogenous seeding. The number of metastatic foci was identified as a quantitative measure of hematogenous seeding, which was not confounded by the rate of multiplication of bacilli. Even allowing for the fourfold-reduced efficiency of low-virulence tubercle bacilli to produce a lesion, this measure clearly revealed a significantly reduced ability of the low-virulence tubercle bacilli to disseminate via the bloodstream.

Virulence of Mycobacterium tuberculosis for guinea pigs: a quantitative modification of the assay developed by Mitchison.

One of the more accepted methods of assay of virulence of tubercle bacilli is one developed by Mitchison in which guinea pigs were infected by the intramuscular route with 1.0 mg of tubercle bacilli freshly harvested from Lowenstein Jensen medium and in which virulence was based on a subjective score of the extent of gross disease in the animal 6 weeks after infection. Due to the practical difficulties involved in such an assay when routinely performed, the following modifications were made: frozen stocks of the culture were prepared prior to the date of infection, the inoculum was quantitated by means of colony forming units, and virulence was based on the number of tubercle bacilli recovered from the spleen of infected animals 6 weeks after infection. A significant correlation was obtained between the findings from the Mitchison assay of virulence and the modification. The latter assay is recommended for its quantitative and objective features.

A guinea pig model of experimental airborne tuberculosis for evaluation of the response to chemotherapy: the effect on bacilli in the initial phase of treatment.

The purpose of this study was to develop and evaluate a guinea pig model of experimental airborne tuberculosis for its ability to assess chemotherapeutic regimens for their efficacy against virulent tubercle bacilli in vivo during the initial phase of treatment. The tissues examined included primary lung lesions and the metastatic foci in lung and spleen which result from the naturally occurring bacillaemia. The treatments examined, INH + RIF, INH + EMB, EMB + RIF, were initiated 4 weeks after infection and were continued for 8 weeks. Although minor differences were observed in the time of onset of a significant bactericidal effect or in the rate of decline in the microbial population, all three treatment combinations resulted in a significant reduction in the number of M. tuberculosis H37Rv recovered from primary lung lesions, primary lesion-free lung lobes and spleen. X-rays taken of excised inflated lung lobes showed a relationship between the degree of calcification of primary lung lesions and the number of surviving bacilli.

What animal models can teach us about the pathogenesis of tuberculosis in humans.

Technology that permits the reproducible infection of laboratory animals with virulent tubercle bacilli under conditions that simulate those under which humans are infected is now available. This technology has been used to investigate a series of fundamental questions about the pathogenesis of tuberculosis. An integrated view of the pathogenesis of tuberculosis has been constructed that combines studies from animal models and our understanding of the key events in the development of cavitary pulmonary tuberculosis in humans. This view, developed as a guide for further hypothesis testing, indicates that whether cavitary pulmonary tuberculosis develops by endogenous reactivation or by exogenous reinfection is determined solely by the route by which the tubercle bacillus reaches the apical-subapical region of the lung. It is in this region that the bacillus survives the cell-mediated immune response. This view of the pathogenesis of tuberculosis permits identification of the factors in a given geographic region that govern the probability of the development of cavitary pulmonary tuberculosis by one or the other pathway. Knowledge of these factors permits the identification of the appropriate strategies for tuberculosis control.

Evaluation of the protective potency of new tuberculosis vaccines.

Animal models used to evaluate the relative protective potency of a panel of tuberculosis vaccines have yielded dissimilar data. Moreover, it is not known which animal model predicts the protective potency of vaccines for humans. Accordingly, animal models should be developed on the basis of an understanding of the key events in the pathogenesis of tuberculosis in humans. Vaccines such as bacille Calmette-Guérin (BCG) appear to protect against tuberculosis by inhibiting the bacillemic phase of primary infection with virulent tubercle bacilli; therefore, such vaccines can be expected to provide protection against tuberculosis developing via the endogenous reactivation pathway but not against disease developing via the exogenous reinfection pathway. To protect against disease developing by the latter pathway, it would appear that new vaccines produced by recombinant DNA technology or through use of monoclonal antibody would have to inhibit the implantation of bacilli at the portal of entry in the lungs. Experience with BCG vaccine indicates that factors other than the inherent potency of the vaccine play a decisive role in the outcome of vaccine trials in humans. These same factors will probably influence field trials of any new generation of tuberculosis vaccines.

Virulence for guinea pigs of tubercle bacilli isolated from the sputum of participants in the BCG trial, Chingleput District, South India.

This study, conducted in Madras, India and in Madison, Wisconsin, USA, was concerned with the virulence of isolates of Mycobacterium tuberculosis obtained from the sputum of individuals living in the Chingleput district of south India. The following results were obtained. 1. The findings of Mitchison with respect to the predominance of low virulence for guinea pigs among isolates from persons living Madras, were confirmed on isolates from the sputum of residents of the Chingleput district. 2. A high correlation was found between the log10 number of tubercle bacilli recovered from the spleen of guinea pigs infected intramuscularly with 1.0 mg of tubercle bacilli and the root index of virulence. 3. A high correlation was found between the log10 number of tubercle bacilli recovered from the spleen of guinea pigs infected intramuscularly with 1.0 mg of tubercle bacilli and the number recovered from the spleen of guinea pigs infected by the respiratory route with 5-10 tubercle bacilli. 4. Relatively low correlations were found between RIV and the susceptibility of isolates to thiophene-2 carboxylic acid hydrazide or to hydrogen peroxide.

A co-operative evaluation of test systems used to assay tuberculosis vaccines.

The results of research on the immunogenicity of experimental mycobacterial vaccines are characterized by a remarkable lack of agreement about which substances are most immunogenic. The disagreement has usually been attributed to the differences in the methods of preparing the vaccines. An alternative hypothesis is that the conflicting results are a product of the different methods used to assess the potency of the vaccines.To determine if the method by which a vaccine is tested is a major factor contributing to the disagreement, an experiment was conducted in which a series of five different vaccines was distributed to each of nine participating laboratories. Each investigator evaluated the potency of the vaccines in one or more animal models of his own choosing. This in effect held the method of vaccine preparation constant while permitting all other variables to change.The ranking of the five vaccines was random, thus demonstrating that the method by which a vaccine is tested influences the apparent potency of a vaccine. These results cast doubt on the conclusions about the relative potency of tuberculosis vaccines evaluated by different methods.