Abstract

Background

Major obstacle in control of tuberculosis being poor patient compliance with the protracted regimen in areas with limited resources which may lead to relapse of active disease, transmission of infection and development of drug resistant strains [1]. In such circumstances, immunotherapy to enhance the efficiency of the immune response in M. tuberculosis infected patients could be an additional valuable strategy to complement anti-bacterial chemotherapy. Even immunotherapy might shorten the duration of treatment for drug-susceptible tuberculosis, thereby reducing the cost and increasing treatment completion rates or might increase the cure rates in case of MDR tuberculosis. In last decade, various nonspecific or antigen specific immunological agents have been used either alone or as an adjunct to chemotherapeutic regimen with variable success [2,3] such as, DNA plasmids [4,5], detoxified M. tuberculosis extract in liposomes (RUTI) [6], Mycobacterium vaccae [7], cytokines [8], Immunoglobulins [9], mycobacterial antigens [10] etc, to name a few. Thus, considering the advancement in the field and keeping in view the potential clinical aims, further research on the concept of immunotherapy, or as an adjunct to chemotherapy of tuberculosis, seems to be valuable.

Ag85B, a 30 kDa fibronectin-binding protein with mycolyltransferase activity, is a major protein secreted by all mycobacterium species and belongs to the Ag85 family. Ag85B is highly immunogenic, as shown by the easy detection of specific humoral and cell-mediated immune responses both in latently and actively infected TB patients [11,12]. It has also been shown to induce a strong Th1-type immune response in mice as well as in humans. Several studies have shown a significant protective effect in the lungs of mice immunized with Ag85B [13-16], whereas a few contradictory reports on the efficacy of Ag85B protein vaccination have also been reported [17-19]. Recently we reported the immunotherapeutic effect of Ag85AB complex as a whole in mouse model [10].

Analysis of the immunological mechanism in various models suggest that the induction of Th1 immune response including antigen-specific CD8⁺/CD4^-/CD44^high memory type cytotoxic T cells producing IFNγ, is required for TB therapeutic vaccine efficacy in humans as well [20-22]. Since DNA vaccines are known to establish cellular immune responses, including cytotoxic T-lymphocyte (CTL) and Th1 responses, much interest is being given to them. Their prophylactic behaviour [15,17,23-25] was found to be effective at limiting the growth of M. tuberculosis in mice, but their therapeutic use has been largely controversial [2].

Present study was carried out to better investigate the immunotherapeutic effect of Ag85B protein and a DNA vaccine based on this protein in mouse model of tuberculosis. Its adjunctive immunotherapeutic effect with simultaneous conventional chemotherapy and moreover its effect on the persisters left after short-term, non-sterilizing chemotherapy was also investigated. This was done with the main objective of understanding the immunological mechanisms involved in the therapeutic anti-TB immune response.

Results

Therapeutic effect of the Ag85B protein and its DNA vaccine

Mice infected with the M. tuberculosis H₃₇Rv strain were treated with either rAg85B protein or Ag85B-DNA (Figure ​(Figure1),1), and the therapeutic effects were expressed as the bacterial load in the spleen and lung (Figure ​(Figure22 & Table ​Table1).1). Compared with the adjuvant immunized control group, rAg85B significantly reduced the bacterial numbers in the spleen and lung (p< 0.001), and was equally effective to that of 'short term' chemotherapy. The adjunctive effect of immunotherapy and chemotherapy was much more pronounced as compared to adjuvant immunized control group (p< 0.001) showing effective cumulative effect on bacterial eradication (Table ​(Table1).1). Although the effect was not additive as marginal decrease in CFU count was not statistically significant when compared to ATD or IT alone. However no significant reduction in CFUs with Ag85B-DNA immunotherapy was observed (data not shown). Further efforts were made to mimic the process of chemotherapy dosage reduction by delivery of drug dosage just once a week with simultaneous immunotherapy and a significant reduction in CFU was observed as compared to that of control (p< 0.01). These findings suggest that immunotherapy could be effectively used as an adjunct to chemotherapy and drug dosage can be considerably reduced to once a week with concomitant immunotherapy, as the CFU reduction was same as that of the group where ATD was given daily (Figure ​(Figure22).

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Figure 1

Time line representation of the experimental design. Schematic time line representation of the experimental design to analyze the immunotherapeutic effect of adjunctive immunotherapy (upper) and the effect of immunotherapy on the persister bacteria (lower). Animals treated with immunotherapy or chemotherapy alone, were treated along same timeline as shown in upper panel. (CFU: Colony Forming Units; IR: Immune Responses)

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Figure 2

Increased protection in animals as depicted by numbers of viable CFUs of M. tuberculosis H₃₇Rv in the lungs of M. tuberculosis H₃₇Rv infected animals receiving adjunctive immunotherapy. Two weeks post chemotherapy/immunotherapy, lungs and spleen from treated and untreated M. tuberculosis H₃₇Rv infected animals were isolated and cultured on Middlebrook 7H11 agar plates. Results are expressed as mean log₁₀ CFUs ± standard deviation of 5 animals per group tested individually. Statistical analysis of the results was carried out by Student's t test. ***p< 0.001, **p< 0.01 compared to untreated animals. Basal level represents CFU after two weeks of infection. (ATD: Antituberculous Drugs; IT: Immunotherapy; O/W: Once Weekly)

Table 1

Bacterial load in terms of CFU in lung and spleen of infected animals after immunotherapy and short-term chemotherapy

Groups	No. of CFU(× 105) in
	Lung	± 95% CI	P value	Spleen	± 95% CI	P value
Basal	2.66 ± 0.254	0.22 (5.20-5.64)		1.22 ± 0.616	0.54 (4.54-5.62)
Untreated	83.72 ± 0.527	0.46 (6.46-7.38)		12.5 ± 0.707	0.62 (5.47-6.71)
ATD dialy	0.196 ± 0.814	0.71 (3.58-5.00)	0.0003 *	0.080 ± 0.509	0.45 (3.45-4.35)	0.0005 *
IT	0.507 ± 0.639	0.56 (4.14-5.26)	0.0003 *	0.061 ± 0.47	0.41 (3.37-4.19)	0.0003 *
ATD dialy+ IT(o/w)	0.086 ± 0.523	0.46 (3.47-4.39)	0.0001 *	0.029 ± 0.448	0.39 (3.07-3.85)	0.0001 *
ATD(o/w) +IT (o/w)	0.435 ± 0.978	0.86 (3.77-5.49)	0.0017 *	0.114 ± 0.667	0.58 (3.47-4.63)	0.0016 *
Untreated (2 months)	147 ± 0.551	0.48 (6.68-7.64)		15.6 ± 0.028	0.02 (6.17-6.21)
ATD dialy 1 month + 1 month adjuvant immunized	1.29 ± 0.315	0.28 (4.83-5.39)		2.70 ± 0.99	0.87 (4.56-6.3)
ATD dialy (1 month) + IT next month (o/w)	0.31 ± 0.319	0.28 (4.21-4.77)	0.0001 $ 0.014 #	0.088 ± 0.44	0.39 (3.55-4.33)	0.0001 $ 0.015 #

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* With respect to Untreated

$ With respect to group Untreated (2 months)

# With respect to group ATD daily 1 month + 1 month adjuvant immunized

CFU: colony Forming Units; ATD: Anti Tuberculous Drugs; IT: Immunotherapy; o/w: Once Weekly;

Immunotherapeutic effect on persister bacteria

After the completion of conventional chemotherapy, the bacteria that survive are usually persisters and in order to check the efficacy of current immunotherapy on those persisters, mice were administered a four week dose of chemotherapy to eliminate the drug susceptible bacteria and then were subjected to immunotherapy. Significant reduction in bacterial CFUs was observed as compared to group where mice were adjuvant immunized for 1 month after chemotherapy; implying that immunotherapy with Ag85B protein was effective in inhibiting the persisters to regrow, that could resist the 'short term' chemotherapy (Figure ​(Figure33).

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Figure 3

Effect of immunotherapy on persisters in terms of Log₁₀ CFUs of Mtb at two weeks post treatment in the lungs and spleen of Mtb infected animals. Two weeks post challenge with Mtb, mice were treated with ATD for 4 weeks and then 4 doses of rAg85B weekly. Two weeks post treatment lungs and spleen from M. tuberculosis H₃₇Rv infected animals were isolated and cultured on Middlebrook 7H11 agar plates. Results are expressed as mean log₁₀ CFUs ± standard deviation of 5 animals per group tested individually. Statistical analysis of the results was carried out by Student's t test. ***p< 0.001, compared to animals that were left untreated during the treatment period of eight weeks. #p< 0.05, compared to animals receiving ATD for four weeks. (ATD: Antituberculous Drugs; IT: Immunotherapy; O/W: Once Weekly)

Cytokine profile after Immunotherapy with rAg85B

The levels of Th1 (IFNγ and IL-12) and Th2 (IL-4) cytokines were monitored before and after immunotherapy. The levels of antigen specific IFNγ and IL-12 were significantly increased after immunotherapy as compared to that of untreated group. Moreover, antigen specific IL-4 levels declined upon immunotherapy with Ag85B (Figure ​(Figure4).4). On the contrary there was no significant level of antigen induced cytokines in the mice receiving immunotherapy with plasmid expressing Ag85B (data not shown).

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Figure 4

Cytokine secretion by PBMCs isolated from untreated/treated tuberculous mice. Two weeks after the treatment, PBMCs were obtained from the untreated/treated tuberculous mice (5 animals per group) and in vitro stimulated with or without rAg85B in quintuplicates. Supernatants were collected from the cultures, and the levels of respective cytokines were measured by ELISA assay. The results are expressed as mean ± standard deviation of five OD values after deducting the values of unstimulated wells. Statistical analysis of the results was carried out by Student's t test. The basal levels of cytokines were estimated two weeks after infection with M. tuberculosis, just before the treatment. (ATD: Antituberculous Drugs; IT: Immunotherapy; O/W: Once Weekly)

Discussion

Immunotherapy that modulates or enhances the host immune response to M. tuberculosis has proven to be an effective method for treatment of tuberculosis in mice [26,27]. The long treatment course, along with the side effects, often results in treatment failure. These limitations together with the increasing incidence of drug resistant strains and co infection with HIV point to an urgent need for additional immunotherapeutic regimens [28,29].

Effective antimycobacterial immunity is presumed to be due to Th1 response, which is dominated by antigen-specific T lymphocytes that produce IFNγ and are cytotoxic towards infected cells [20-22]. Th2 response characterized by IL-4 production, which is predominant during infection with M. tuberculosis, has been reported to be non-protective in TB [20,30,31]. A shift in the balance towards Th1 response may be beneficial, laying down a criterion for the selection of the antigens to be protective in case of tuberculosis. Their prophylactic behaviour being the guiding line for their probable therapeutic use on basis of the principle 'diamond cuts diamond' on the contrary to that of 'adding fuel to the fire'.

Compared to antituberculosis drugs, the natural immune response has a minor impact on mycobacterial elimination during the early phase of therapy [32]. It is expected that immune modulation by immunotherapy will lead to significant increase in the cure rates. The present study involved the evaluation of immunotherapeutic effect of rAg85B and a DNA vaccine expressing this protein. Immunotherapy with rAg85B led to significant decrease in bacterial load in both lungs and spleen which was comparable to that of short term chemotherapy (Figure ​(Figure2).2). A two log reduction observed in the lungs of mice receiving immunotherapy with rAg85B indicated it as an effective measure, as suggested by Orme et al [33] according to which measures reducing pulmonary bacterial loads by 0.7 logs are considered to be effective. On the contrary, no such protection was observed in case of pVAX85. This absence of protection in case of DNA vaccination is inconsistent with the results from the published literature [26] but still, though hard to justify, has supporting evidence [2]. Further, Ag85B immunotherapy, combined with short term chemotherapy over 4 weeks showed a stronger therapeutic effect than chemotherapy or immunotherapy alone when compared to that of adjuvant immunized control group (Table ​(Table1).1). Protective effect was still significant even when the chemotherapeutic dose was reduced to once weekly instead of daily, thus suggesting the possibility that immunotherapy including Ag85B combined with chemotherapy might shorten the period of conventional chemotherapy (Figure ​(Figure2).2). However, a time and antigen titration approach may determine an optimal combined regimen sufficient to confer superior therapeutic activity.

In this study a short dose of non-sterilizing chemotherapy was administered so that only drug susceptible bacteria are eliminated. The persisters left out were treated with rAg85B immunotherapy and a significant reduction in persister bacterial count was observed (Figure ​(Figure3).3). These findings suggest utility of Ag85B based immunotherapy against regrowth of persisters which are the major cause of protracted drug treatment and even relapse of active disease. This role of immunotherapy seems to be particularly important because the most organisms being extracellular during the initial phase of therapy are vulnerable to drugs but immunotherapy stimulates the immune cells (T cells and NK cells) to kill intracellular M. tuberculosis bacilli which are usually more or less defiant to drugs. Thus a distinctive role for immunotherapy might be to kill slowly replicating or "dormant" organisms more effectively than current antituberculosis agents, perhaps by administering immunotherapy after the initial phase of treatment. In the present study we evaluated the efficacy only after 4 weeks; this short term immunotherapeutic effect now needs to be corelated in long term experiments.

The reduced systemic Th1 response in tuberculosis [30,31] provides a rationale for using rAg85B as an immunotherapeutic adjunct to treat tuberculosis. We observed a significant shift towards Th1 type of immune response after the immunotherapeutic dosage as there was significant increase in the release of IFNγ and IL-12 with the concomitant abrogation of Th2 response envisaged by the repression in IL-4 levels (Figure ​(Figure4).4). This switch from Th2 to Th1 has been observed to be associated with a substantial reduction of pathology in the animals as reported by various groups [5,27,34], thus suggesting that the therapeutic effect of rAg85B may, at least partly, be due to the rectification of immunopathological subversion and the restoration of the Th1/Th2 balance. On the contrary the immune response upon DNA vaccination was in discord of expectations as no antigen specific cytokine production was observed. Even though there exists a lot of uncertainty regarding the immunotherapeutic use of DNA vaccines [35], still we cannot justify the current observation as we did not investigate both for the pathology as well as expression of antigen by DNA vaccine in the affected organs.

Conclusion

It seems imperative to explore the mechanisms of therapeutic vaccines for future investigations and moreover administration of single antigen induced immunotherapy is expected to alter only one aspect of a complex immune response. An alternative approach will be to generate a multifaceted favourable response. Application of such immunotherapy alone or as an adjunct to conventional chemotherapeutic regimen may result in explicit cure for tuberculosis.

Materials and methods

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JAS was involved in performing the experiments and analysis of results while GKK and IV participated in the design of experiments, analysis of results and manuscript writing. All authors have read and approved the final manuscript.

References