Immune Netw.  2015 Apr;15(2):83-90. 10.4110/in.2015.15.2.83.

Investigation of Immune Biomarkers Using Subcutaneous Model of M. tuberculosis Infection in BALB/c Mice: A Preliminary Report

• Husain AA
• Daginawala HF
• Warke SR
• Kalorey DR
• Kurkure NV
• Purohit HJ
• Taori GM
• Kashyap RS

• KMID: 2168033
• DOI: http://doi.org/10.4110/in.2015.15.2.83

Abstract

Evaluation and screening of vaccines against tuberculosis depends on development of proper cost effective disease models along with identification of different immune markers that can be used as surrogate endpoints of protection in preclinical and clinical studies. The objective of the present study was therefore evaluation of subcutaneous model of M.tuberculosis infection along with investigation of different immune biomarkers of tuberculosis infection in BALB/c mice. Groups of mice were infected subcutaneously with two different doses : high (2x10(6) CFU) and low doses (2x10(2) CFU) of M.tuberculosis and immune markers including humoral and cellular markers were evaluated 30 days post M.tuberculosis infections. Based on results, we found that high dose of subcutaneous infection produced chronic disease with significant (p<0.001) production of immune markers of infection like IFNgamma, heat shock antigens (65, 71) and antibody titres against panel of M.tuberculosis antigens (ESAT-6, CFP-10, Ag85B, 45kDa, GroES, Hsp-16) all of which correlated with high bacterial burden in lungs and spleen. To conclude high dose of subcutaneous infection produces chronic TB infection in mice and can be used as convenient alternative to aerosol models in resource limited settings. Moreover assessment of immune markers namely mycobacterial antigens and antibodies can provide us valuable insights on modulation of immune response post infection. However further investigations along with optimization of study protocols are needed to justify the outcome of present study and establish such markers as surrogate endpoints of vaccine protection in preclinical and clinical studies in future.

Keyword

Mice; Tuberculosis; Subcutaneous route; Immune markers

MeSH Terms

Animals
Antibodies
Biomarkers*
Chronic Disease
Hot Temperature
Lung
Mass Screening
Mice*
Shock
Spleen
Tuberculosis*
Vaccines
Antibodies
Vaccines

Figure

• 

  Figure 1 (A) Mean Total IgG and (B) anti-PPD levels in serum of mice (n=7, each group) in high dose, low dose and control groups. Antibody levels were estimated 30 days after development of TB infection. Paired t-test was used to compare and obtain statistical significance. Error bars indicate standard error of mean. **Represents significant (p<0.05) and ***represents highly significant (p<0.001) values.

• 

  Figure 2 Scatter plot of antibody levels against panel of MTB H37Rv antigens (a) Ag85B (b) 45kDa (c) Hsp 16 (d) GroEs (e) ESAT-6 and (f) CFP-10 in serum of mice (n=7, each group) after TB infection. Paired t-test was used to compare and obtain statistical significance. Error bars indicate standard error of mean. **Represent significant (p<0.05) and ***represents highly significant (p<0.001) values.

• 

  Figure 3 Mean mycobacterial Hsp levels (a) mHsp 16 (b) mHsp-65 and (c) mHsp-71 in serum of mice (n=7, each groups) in different dose groups. Error bars indicate standard error of mean. **Represents significant (p<0.05) and ***represents highly significant (p<0.001) values.

• 

  Figure 4 Shows mean growth time of MTB in lung and spleen homogenates of mice (n=7) collected 30 post MTB infection in different dose group. Mice from respective groups were sacrificed and mycobacterial load was determined by inoculating serially diluted lung homogenates in Middle brook 7H9 liquid medium and incubating at 37℃ in BacT/Alert system (Biomerieux) for 30 days. Bars represent mean time required for growth in BacT was taken as a correlate of load in respective organs of mice group. Data are shown as ±SD. **Represent significant (p<0.05) values.

• 

  Figure 5 (A) Representative Histopathology of lungs section of mice (high dose, low dose and control) stained with hematoxylin and Eosin. (B) along with percentage lymphocytic infiltarion in different dose groups and control. Mice were sacrificed 30 days after MTB infection. Arrows indicate percentage of lesions covered in lungs. (C) Shows mean INF-γ levels in lung homogenates of mice at different dose groups. Errors bars indicate standard error of mean. **Represent significant (p<0.05) and ***represents highly significant (p<0.001) values.

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