Treatment of latent tuberculosis with RUTI

The development of LTBI is related to its capacity for slow growth, induction of necrosis, resistance to stress, and constant reactivation and induction of new lesions in order to face the continuous cell turnover in the lungs. Immunotherapy with RUTI may prevent the transient lack of immunological response after a short-period chemotherapy, and thus the chance for latent bacilli to reactivate, and provide a feasible shorter treatment against LTBI.


2.-The origin of latent bacilli

At the beginning of M. tuberculosis infection, the development of intragranulomatous necrosis in most infected mammals suggests the occurrence of the Koch phenomenon [4]. This phenomenon probably arise from a local Schwartzman reaction [5], triggering a “first wave” of latent bacilli (i.e. bacilli surviving the initial inflammatory response), which are then trapped in the collagen fibers that “takes the place” of the destroyed infected macrophages. A “second wave” of latent bacilli is then triggered by the induction of the specific immunity, which activates the infected macrophages to destroy most of the initial bulk, in particular the 90% in the experimental murine model induced by aerosol. This reduction is mainly induced by specific T cells [6] that trigger a number of mechanisms that induces the death of most bacterial cells (i.e. low pH, reactive oxygen intermediates, reactive nitrogen intermediates, etc.). As a consequence of this strong immune response, the population in the chronic phase comes from bacilli able to adapt to stress and thus becomes “latent” bacilli.


3.-The role of foamy macrophages in local immunodeppression and constant bacillary dissemination

However, during the chronic phase, new cells appear at the outermost layer of the granulomas, occupying the alveolar spaces: these cells are the foamy macrophages (FM) [7]. As it is extensively reviewed elsewhere [8] some of these FM contain single bacillus that are carried outside of the granuloma, where they finally multiply and destroy the FM, thus allowing a major bacillary spread through the alveolar spaces. That’s why not the entire bacillary load in the chronic infection is in latent state. Furthermore, since FM produce nitric oxide (NO) and may suppress the activating specific T cells, both Th1 and Th2, they constitute an immunosuppressive outermost ring around the lesion.


4.-The dynamic nature of the LTBI and the importance of a short-period chemotherapy in its treatment

It is hardly understandable why the administration of isoniazid for 9 months is required to treat LTBI. Given that the latent bacilli are not susceptible to such drug, why is then administered for such a long period? They answer is firstly to destroy the population of actively multiplying bacilli that can be found initially, and secondly, to prevent reactivation of any latent bacilli that attempts to multiply afterwards, thus allowing the macrophages themselves to follow their normal dynamics and drain the bacilli.
By reducing inflammation and preventing multiplication of the bacilli, antibiotic treatment also prevents the continuous formation of FM and their accumulation or movement through the alveolar spaces, thus removing the outermost immunosuppressive of the granuloma. It also reduces the possibility of generating a toxic Koch-type reaction when a therapeutic vaccination is administered.
On the other hand, the administration of chemotherapy induces a immunosuppression by itself and allows the reactivation of local latent bacilli when the chemotherapy ends [9]. That’s also the explanation for the long-period chemotherapy.


5.-Filling the gap. The origin of RUTI.

RUTI was designed to fill the immunological gap left by short-term therapy against LTBI (Figure 1) as it is able to boost the immunological response, mostly focused against growing bacilli, as well as triggering a new one against structural and “stress induced” antigens, which are present in latent bacilli.

The manufacturing process of RUTI has been published elsewhere [10]. It is manufactured by Archivel Farma s.l. (Mataró. Catalonia. Spain) under GMP. Briefly, M. tuberculosis are cultured for three weeks in Middlebrook 7H11 agar at 37ºC, under a progressive conditions of low pH and pO2. Colonies are carefully removed and mechanically disrupted using silica-zirconia beads and a PBS buffer with 4% Triton-X114. After centrifuging at 3,000 g to remove the entire cells, the pellet is centrifuged at 27,000 g, the lipidic supernatant is removed and the final product is washed, pasteurized at 65ºC for 40 minutes and lyophilized, and then liposomed. The peptidical composition of RUTI is determined using SDS-PAGE, and the patterns were obtained by western blot analysis as previously described [10].


6.-Protection mechanisms and effectiveness of RUTI

Inoculation of RUTI in infected mice treated with short-period chemotherapy induces a strong immunological Th1/Th2/Th3 response against 13 M. tuberculosis antigens, which is able to induce a strong accumulation (10 times) of IFN-g CD4 and CD8+ T cells PPD specific in the infected lungs when compared with the control mice only treated with short-period chemotherapy (Figure 2) [8]. Interestingly, immunotherapy with BCG only increased significantly the recruitment of CD4+ T cells. On the other hand, passive serumtherapy with polyclonal antibodies obtained from mice treated with short-period chemotherapy and RUTI, was able to control the reactivation of infected SCID mice treated with short-period chemotherapy (Figure 3) thus demonstrating a protective role of the humoral response triggered by RUTI [12].

Effectiveness has been demonstrated in a wide range of mice strains. Some of the data has been already published [11]. Figure 4 shows a standard experiment, reflecting not only the control of the reactivation but the bactericidal effect induced by the immunotherapy of RUTI. This effect has also been demonstrated in guinea pigs, as reflected in Figure 5. In this model is especially difficult to obtain data about bactericidal effect, as guinea-pigs are able to better control the bacillary concentration, when compared with mice. Long progression experiments (i.e. 24 weeks evolution) show no difference in the bacillary counts comparing with controls. In this case the difference is in the survival and the degree of pulmonary pathology, which is better controlled in RUTI treated animals.

This is also the case of M. bovis infected goats (Figure 6) (data not published). A field study carried on with these animals also reflects this benefit in RUTI treated animals. Short-period chemotherapy reduced dramatically both pulmonary and extrapulmonary affectation. Moreover, treatment with Qx and RUTI was the one that also decreased the pathology scoring in the hilar lymph nodes, a paramount sign in the treatment against LTBI in humans (Figure 7).

Besides, immunotherapy with RUTI induced the increase of IFN-g in peripheral blood after ex-vivo stimulation with ESAT-6, bovine-PPD and RUTI (Figure 8). Inoculation of RUTI induced a transient increase of rectal temperature (1 ºC, 24 hours after inoculation) and a local tumefaction as consequence of a granulomatous reaction. No systemic toxicity or mortality was induced by the inoculation of RUTI. Culture of samples form hilar lymph nodes detected an average of 2 log10 CFUs in all experimental groups.


7.- Conclusions

Once preclinical assays (including the lack of toxicity of the treatment) have demonstrated the utility of RUTI in the control of latent bacilli, a clinical development has been initiated in order to demonstrated the utility of shorten the treatment of LTBI from 9 to 1 month with the inclusion of therapeutic vaccination with RUTI at the end. Phase I clinical trial is going to be launched in late 2006.


REFERENCES

1. World Health Organization. Global Tuberculosis Control. WHO Report 2001. Geneva, Switzerland, WHO/CDS/TB/2001.287.
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3. Grosset J. Bacteriologic basis of short-course chemotherapy for tuberculosis. Clin Chest Med 1980; 1: 231-241.
4. Thoen CO. Tuberculosis in wild and domestic mammals In: Tuberculosis Pathogenesis protection and control Bloom BR. (Ed.). ASM Press Washington DC 1994;11:157-164.
5. Cardona PJ, Llatjós R, Gordillo S,Viñado B, Díaz J, Ariza A, Ausina V, Towards a “human-like” model of tuberculosis: Local inoculation of LPS in lungs of Mycobacterium tuberculosis aerogenically infected mice induces intragranulomatous necrosis. Scand J Immunol 2001; 53: 65-71.
6. North RJ & Jung YJ. Immunity to tuberculosis Annu Rev Immunol 2004;22:599-623
7. Cardona PJ, Gordillo S, Díaz J, Tapia G, Amat I, Pallarés A, Vilaplana C, Ariza A, Ausina V Widespread bronchogenic dissemination makes DBA/2 mice more susceptible than C57BL/6 mice to experimental aerosol infection with Mycobacterium tuberculosis. Infect Immun. 2003; 71 (10): 5845-5854.
8. Cardona PJ. RUTI: a new chance to shorten the treatment of latent tubeculosis infection. Tuberculosis (Edimb.). 2006. In Press.
9. Cardona PJ, Julian E, Gordillo S, Díaz J, Vallès X, Luquín M, Ausina V. Production of antibodies against glycolipids from the cell wall of M. tuberculosis in a murine model of tuberculosis. Scand J Immunol. 2002; 55: 639-645.
10. Cardona PJ, Amat I. Origin and Development of RUTI, a new therapeutic vaccine against Mycobacterium tuberculosis infection. Arch Bronconeumol 2006; 42:25-32.
11. Cardona PJ, Amat I, Gordillo S, Arcos V, Guirado E, Diaz J, Vilaplana C, Tapia G, Ausina V. Immunotherapy with fragmented Mycobacterium tuberculosis cells increases the effectiveness of chemotherapy against a chronical infection in a murine model of tuberculosis. Vaccine 2005; 23, 1393-1398.
12. Guirado E, Amat I, Gil O, Díaz J, Arcos V, Caceres N, Ausina V, Cardona PJ. Passive serum-therapy with polyclonal antibodies against Mycobacterium tuberculosis protects against post-chemotherapy relapse of tuberculosis infection in SCID mice. Microbes & Infection available online 27 january 2006.

Figure 1. Temporal strategy for the use of RUTI, indicating the effects of short-course chemotherapy and the requirements for subsequent immunotherapy.


Figure 2. Evolution of TCD4+ and TCD8+ IFNg+ cells, stimulated ex vivo with PPD, from lungs of infected mice treated with chemotherapy from weeks 6 to 14, and treated with 2 subcutaneous (s.c.) inoculations of RUTI (245 g) at weeks 14 and 17; 1 s.c. inoculation of BCG (10e6 CFUs) at week 14 or 1 s.c. of empty liposomes (control) -red, greeen and black symbols, respectively. The results are given as mean values with standard deviations obtained from 4 mice for each time point. Differences with control were significant when marked with * for p < 0.05 [8].



Figure 3. Control of CFUs after serum-therapy in the lung of SCID mice. After infection, mice were treated with INH+RIF from week 3 to 8 (in orange), and treated with 4 intraperitoneal (i.p.) inoculations of serum (hyperimmune serum, HS) from mice treated with short-period chemotherapy and RUTI (in red) or from normal non-infected mice (in black). The results are given as mean values with standard deviations obtained from 4 mice for each time point (apart from week 19, where 10 mice were treated with immune serum and 11 mice were given normal serum). Differences with control were significant when marked with * for p < 0.05. [12].



Figure 4. Bactericidal activity of RUTI in the lung of C57BL/6 mice. After infection, mice were treated with INH+RIF from week 9 to 17 (in orange), and with 3 subcutaneous (s.c.) inoculations of RUTI (185 g per inoculation) at weeks 17, 19 and 21 (in red) or empty liposomes (in black). The results are given as mean values with standard deviations obtained from 4 to 6 mice for each time point. Differences with control were significant when marked with * for p < 0.05 [11].



Figure 5. Bactericidal activity of RUTI in the lung of guinea pigs. After infection, animals were treated with INH+RIF from week 4 to 8 (in orange), and with 3 subcutaneous (s.c.) inoculations of RUTI (185 g per inoculation) at weeks 8, 10 and 14 (in red) or empty liposomes (in black).The results are given as mean values with standard deviations obtained from 8 animals for each time point. Differences with control were significant when marked with * for p < 0.05 [8].



Figure 6. Therapeutic diagram of the field study in M.bovis infected goats. Some of the animals were treated with isoniazid (300 mg intramuscular twice a week, in orange) and some of them received 2 inoculations of RUTI (271 g each). Peripheral blood was obtained at different time points to check the production of IFN-g after being stimulated ex vivo with different M. tuberculosis antigens.



Figure 7. Field study in M.bovis infected goats. Pathology scoring values (adapted from Vordermeier et al IAI 2002) reflects the control of the pathology induced by RUTI therapy in the hilar lymph nodes compared with the only chemotherapy treatment (Picture A). Chemotherapy treatment clearly induce a control of the infection, as reflected in the pathology scoring in pulmonary and extrapulmonary sites (Picture B).



Figure 8. Field study in M.bovis infected goats. Production of IFN-g after ex vivo stimulation of peripheral blood with ESAT-6. Picture A reflects individual monitoring of each animal. Picture B shows the ratios between weeks to check the tendency of the values (<1 decrease; >1 increase). Orange box shows the chemotherapy period and blue arrows RUTI inoculation.