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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 11  |  Issue : 1  |  Page : 108-112

Immunotherapeutic potential of n-terminally formylated ESAT-6 protein in murine tuberculosis


1 Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India; Department of Medical Laboratory Sciences, College of Applied Medical Science, Majmaah University, Al Majmaah, Saudi Arabia
2 Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission18-Dec-2021
Date of Decision20-Jan-2022
Date of Acceptance24-Feb-2022
Date of Web Publication12-Mar-2022

Correspondence Address:
Sadhna Sharma
Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
India
Shabir Ahmad Mir
Department of Medical Laboratory Sciences, College of Applied Medical Science, Majmaah University, Al Majmaah

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_39_21

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  Abstract 


Background: The early secreted antigenic target-6 kDa (ESAT-6) being one of the important antigens expressed by Mycobacterium tuberculosis (MTB) has been widely investigated for its strong immunmodulatory effects. We have previously evaluated the immunotherapeutic efficacy of ESAT-6 in the murine model of experimental tuberculosis (TB). Now in the present study, we have evaluated the immunotherapeutic efficacy of N-terminally formylated form of ESAT-6 (f-ESAT-6) in murine TB. Materials and Methods: The production and purification of f-ESAT-6 have been discussed in our earlier report (Mir SA and Sharma S, 2014). In the present study, the MTB H37Rv-infected mice were treated with f-ESAT-6 alone or in combination with anti-TB drugs (ATDs). Four weeks postinitiation of the treatment, the experimental mice were sacrificed, and the colony-forming units (CFUs) were enumerated in their lungs and spleen as described in “materials and methods” section. Results: The N-terminally formylated ESAT-6 protein (f-ESAT-6) induced a moderate reduction in the bacterial load in the target organs of infected mice. Compared to the dimethyldioctadecyl ammonium bromide treated and untreated groups, the f-ESAT-6 treatment significantly reduced the CFU in the spleen and lungs of infected mice by 0.377 log10 units (P < 0.05) and 0.396 log10 units (P < 0.01), respectively. The administration of f-ESAT-6 in combination with ATDs revealed an additional immunotherapeutic effect and elicited higher therapeutic efficacy over drugs (ATDs) alone. Conclusion: The results of the present study clearly indicate that f-ESAT-6 protein alone as well as in combination with the conventional ATDs induce moderate therapeutic effect against experimental TB.

Keywords: Anti-tuberculosis drugs, colony-forming unit, histopathology, immunotherapy, N-formylated-early secreted antigenic target-6, tuberculosis


How to cite this article:
Mir SA, Sharma S. Immunotherapeutic potential of n-terminally formylated ESAT-6 protein in murine tuberculosis. Int J Mycobacteriol 2022;11:108-12

How to cite this URL:
Mir SA, Sharma S. Immunotherapeutic potential of n-terminally formylated ESAT-6 protein in murine tuberculosis. Int J Mycobacteriol [serial online] 2022 [cited 2022 May 25];11:108-12. Available from: https://www.ijmyco.org/text.asp?2022/11/1/108/339519




  Introduction Top


Tuberculosis (TB) remains to be a prominent cause of worldwide morbidity and mortality. According to the World Health Organization Global TB Report 2018, an estimated 10 million individuals became ill with TB and 1.6 million died from the disease in 2017.[1],[2] Currently, Bacillus Calmette-Guerin (BCG) is the only available vaccine against TB with limited protective efficacy ranging from 0% to 80% across different regions.[3] Moreover, the rise of multi-drug resistant and extremely drug-resistant strains of Mycobacterium TB (MTB) have further worsened the control of this disease. Therefore, the development of therapies other than traditional pharmacotherapy is highly needed for improved treatment of TB, especially the drug-resistant TB. One of these alternative therapies is immunotherapy. Host immunity holds great significance for efficiently inhibiting the growth of intracellular pathogens including MTB.[4] Indeed, immunotherapy can improve the treatment of TB by augmenting host immunity to get rid of the bacteria and to shorten the lengthy chemotherapy needed for TB patients.[5],[6],[7],[8],[9],[10],[11]

The early secreted antigenic target-6 (ESAT-6) being one of the significant antigens expressed by MTB has been widely investigated for its strong immunomodulatory effects as a subunit vaccine.[12],[13],[14],[15] Recombinant BCG that exports/secretes ESAT-6 enhances defence against TB in the animal model.[16],[17],[18],[19] Recently, it has been demonstrated that the Mycobacterium bovis live-attenuated vaccine with deletion of esat6 and cfp10 genes failed to confer protection against virulent M. bovis challenge in a mouse model of TB.[20] In addition, ESAT6 DNA vaccines have been reported to possess immunotherapeutic as well as immunoprotective effects in the mice model of TB.[21],[22],[23],[24]

In addition, we have previously evaluated the immunotherapeutic efficacy of ESAT-6 in the murine model of TB.[25] Immunotherapy of MTB-infected mice with recombinant ESAT-6 significantly reduced the bacteria loads in the target organs (lungs and spleen) and fetched additional therapeutic effect to the anti TB drugs (ATDs) when given in combination.[25] We also reported the moderate immunotherapeutic effect of synthetic N-terminal formylated peptide of ESAT-6 in murine TB.[26] Here, in this study, we evaluated the immunotherapeutic efficacy of N-terminally formylated recombinant ESAT-6 protein (f-ESAT-6) against TB infection in mice.


  Methods Top


Animals and bacterial culture

Balb/c mice (aged 6–8 weeks) and MTB H37Rv used in this study were maintained as reported earlier.[25],[26],[27]

N-terminal formylation of early secreted antigenic target-6 and its purification

Expression and N-terminal formylation of ESAT-6 as well as its purification was performed as described previously.[28]

Preparation of a mouse model of tuberculosis and treatment

Naive Balb/c mice were infected with MTB H37Rv and the infection was confirmed as described earlier.[25],[26],[27]

After confirmation of the infection, the MTB infected mice were distributed into five groups (n = 6/group) and treated with different formulations [Table 1]. The f-ESAT-6 protein was subcutaneously given at a dose of 30 μg emulsified in 250 μg of dimethyldioctadecyl ammonium bromide (DDA) (three doses/animal at 10 days interval). The anti-tubercular drugs were given orally for 4 weeks on the daily basis as explained elsewhere.[25],[26],[27]
Table 1: Treatment protocol and groping of mice

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Bacterial enumeration and histopathological examination of lungs

Four weeks posttreatment initiation, the lungs and spleens were removed aseptically from the sacrificed mice. The bacterial enumeration and histopathological examination of lungs was performed as reported earlier.[25],[26],[27]

Statistical analysis

The colony-forming units (CFU) data are expressed as mean ± standard error of the mean the comparison between the groups was accomplished using student's t-test. The difference was considered significant at P values less than 0.05 (P < 0.05).


  Results Top


Cloning and N-terminal formylation of early secreted antigenic target-6

We have previously reported the data related to cloning and N-terminal formylation of ESAT-6 as well as the purification of the f-ESAT-6.[28]

Therapeutic efficacy of f-early secreted antigenic target-6 protein in terms of colony-forming units reduction

The MTB H37Rv infected mice were treated with different formulations as described in earlier sections. In comparison with the untreated infected mice, no reduction in the bacterial load was observed in the DDA treated group (P > 0.05). However, compared to the DDA treated and untreated groups, the f-ESAT-6 treatment significantly reduced the CFU in spleen and lungs of infected mice by 0.377 log10 units (P < 0.05) and 0.396 log10 units (P < 0.01), respectively. The treatment of infected mice with anti-tubercular drugs reduced CFU in respective organs by 1.681 and 2.316 log10 units (P < 0.001) [Figure 1] and [Table 2]. The administration of f-ESAT-6 in combination with ATDs revealed an additional immunotherapeutic effect and elicited higher therapeutic efficacy over drugs (ATDs) alone. The f-ESAT-6 in combination with ATDs significantly reduced the CFU in lungs by 2.757 log10 units (P < 0.001) and in spleen by 2.114 log10 units (P < 0.001) when compared to the DDA treated animals [Figure 1] and [Table 2]. Further, in comparison to the ATD treated mice, the ATD + f-ESAT-6 combination significantly reduced the CFU in the spleen by 0.432 log10 units (P < 0.05) and in lungs by 0.440 log10 units (P < 0.05) [Figure 1] and [Table 2].
Figure 1: Therapeutic efficacy of recombinant f-early secreted antigenic target-6 alone or in combination with anti-tuberculosis drugs: (a) In lungs; (b) in spleen. Results expressed as mean of log10 colony-forming units ± standard error of the mean of six mice per group. *P < 0.05, **P < 0.01, ***P < 0.001compared to Dimethyldioctadecyl ammonium bromide treated group and #P < 0.05 compared to anti-tuberculosis drugs treated group. CFU: Colony-forming unit, ATDs: Anti-tuberculosis drugs, DDA: Dimethyl dioctadecyl ammonium bromide, f-ESAT-6: N-terminally formylated ESAT-6, SEM: Standard error of the mean

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Table 2: Bacterial colony-forming unit in the lung and spleen of Mycobacterium tuberculosis infected Balb/c mice after immunotherapy and/or short-term chemotherapy

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Effect of f-early secreted antigenic target-6 immunization on lung pathology of Mycobacterium tuberculosis infected mice

On histopathological analysis, tuberculous lesions, causing consolidation in the lungs of the untreated infected mice, were observed. In comparison, we observed total clearance of the tuberculous lesions in the lung sections of ATD treated as well as ATD plus f-ESAT-6 treated infected mice [Figure 2].
Figure 2: Histopathological alterations in lungs of experimental mice. Representative photomicrographs [H and E, ×20] of lung tissues of mice in each group: (i) Dimethyldioctadecyl ammonium bromide treated; (ii) f-early secreted antigenic target-6 treated; (iii) anti-tuberculosis drugs treated; (iv) anti-tuberculosis drugs + f-early secreted antigenic target-6 treated. ATDs: Anti-tuberculosis drugs, H and E: Hematoxylin and eosin, DDA: Dimethyldioctadecyl ammonium bromide, f-ESAT-6: N-terminally formylated ESAT-6

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  Discussion Top


TB is a worldwide threat to human health. There is an urgent necessity to develop novel treatment therapies for TB. Therapeutic vaccines have the potential to defend against TB by provoking the protective immunity of the host. These vaccines can overcome the drug resistance issues and will also help to shorten the duration of TB treatment.[29],[30],[31]

It is well established in the literature that ESAT6, an immuno-dominant antigen of M. tuberculosis, can induce potent T-cell stimulation and provide protection against TB.[32],[33],[34],[35],[36] ESAT-6-induces apoptosis in macrophages[37] and the fusion protein consisting of Ag85B and ESAT6 has been reported to induce long-lasting immune memory against MTB in mice.[17],[38] In our previous study, we evaluated the recombinant ESAT-6 protein for its therapeutic efficacy in murine TB.[25] The recombinant ESAT-6 protein induced moderate adjunctive immunotherapeutic effect in the mouse model of experimental TB.[25] The current study demonstrates the immunotherapeutic efficacy of N-terminally formylated form of ESAT-6 protein (f-ESAT-6). We have previously reported the cloning and expression of ESAT-6 gene (Rv3875) in  Escherichia More Details coli and the protocol for N-terminal formylation of the ESAT-6 as well as the production of N-formylated ESAT-6 has also been reported therein.[28]

In the present study, we now demonstrate the therapeutic efficacy of the f-ESAT-6 protein in MTB infected mice. Immunotherapy with f-ESAT-6 moderately decreased the bacterial load in spleen (P < 0.05) and lungs (P < 0.01) of infected mice [Figure 1] and [Table 2]. Further, f-ESAT-6 immunotherapy resulted in an additional therapeutic outcome when combined with short-term chemotherapy (P < 0.05) [Figure 1]. The results of this study show that f-ESAT-6 protein can act as an adjunct to the available anti-TB chemotherapy and help in shortening the treatment period of TB patients. The immunotherapeutic efficacy of f-ESAT-6 was further confirmed by the histopathological analysis of lungs of the experimental mice [Figure 2]. However, the immunotherapeutic efficacy of f-ESAT-6, although insignificantly, but was lower than its nonformylated form (ESAT-6), the data for which has already been published.[22]


  Conclusion Top


Our results show that N-terminally formylated ESAT-6 alone or in combination with the conventional ATDs induce moderate immunotherapeutic effect against experimental TB. Its therapeutic efficacy could be improved further by using adjuvants that are more efficient.

Compliance with ethical standards

The animal handling protocol was approved in the 39th meeting of the Institutional Animal Ethics Committee (IAEC) of PGIMER with IAEC number 140.

Financial support and sponsorship

This study was financially supported by Indian Council of Medical Research, New Delhi, India.

Conflicts of interest

There are no conflicts of interest.



 
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