Post-exposure vaccination against Mycobacterium tuberculosis

Abstract

Enhancing immunity to tuberculosis in animal models after exposure to the infection has proved difficult. In this study we used a newly described flow cytometric technique to monitor changes in cell populations accumulating in the lungs of guinea pigs challenged by low-dose aerosol infection with Mycobacterium tuberculosis and vaccinated 10 days later. On day 40 after infection the fusion protein F36 and a pool of Ag85A and ESAT6 vaccines had significant effects on the bacterial load, showed increased expression of the activation marker CD45+ on CD4+ T cells, and reduced numbers of heterophils. Lung pathology and pathology scores were marginally improved in animals given these vaccines, but lymph node pathology was not influenced. Despite early effects no changes in long-term survival were seen. These results suggest that a single post-exposure vaccination can initially slow the disease process. However, this effect is transient, but this could be of use in an multidrug resistant/extremely drug resistant outbreak situation because it could potentially slow the infection long enough to complete drug susceptibility testing and initiate effective chemotherapy.

Figure 1

Bacterial growth in the lungs and Kaplan-Meier analysis of the survival of guinea pigs infected with M. tuberculosis and receiving post-exposure vaccines. Bacterial counts in the lungs (panel A), on day 40 from guinea pigs infected with a low dose of M. tuberculosis and receiving post exposure vaccine 10 days after infection were compared. Results are expressed as the average (n=5) of the bacterial load in each group expressed as Log10CFU, standard error mean (SEM) and protection. Median survival days (panel B) of Hartley guinea pigs (n=10) vaccinated with, Ag85/ESAT6 (solid square), F36 (solid triangle), BCG control (solid inverted triangle), adjuvant control (solid diamond), and saline control (solid circle) are compared.

Figure 2

Light photomicrographs of lungs (panel A, A–E) from representative guinea pigs that were guinea pigs infected with M. tuberculosis and receiving post-exposure vaccines saline control (A, A), adjuvant control (A, B), BCG control (A, C), Ag85A+ESAT6 (A, D) and F36 (A, E) on day 40 are shown. [bar denotes H&E staining, 20X]. The lesion scores (panel B) of lungs were calculated as described in Materials and methods section for all the four groups on day 40 of the infection. The scores are presented as total mean lesion values. On day 40, smaller lesions (arrows) and reduced lesion scores were seen in the animals vaccinated with the F36 fusion protein compared to the saline control, adjuvant control, BCG control, Ag85A+ESAT6. Results are expressed as the mean lesion scores (±SEM, n=4). (Student t-test, *p≤0.05) compared to adjuvant control guinea pigs.

Figure 3

Light photomicrographs of spleens (panel A, A–E) from representative guinea pigs that were guinea pigs infected with M. tuberculosis and receiving post-exposure vaccines saline control (A, A), adjuvant control (A, B), BCG control (A, C), Ag85A+ESAT6 (A, D) and F36 (A, E) on day 40 are shown. [bar denotes H&E staining, 20X]. The lesion scores (panel B) of spleens were calculated as described in Materials and methods section for all the four groups on day 40 of the infection. The scores are presented as total mean lesion values. On day 40, smaller lesions (arrows) and reduced lesion scores were seen in the animals post-exposure vaccinated with the BCG control, F36 fusion protein and Ag85/ESAT6 compared to the saline control and adjuvant control. Results are expressed as the mean lesion scores (±SEM, n=4). (Student t-test, *p≤0.05) compared to adjuvant control guinea pigs.

Figure 4

Numbers of T cell expressing CD45+ in the lungs of guinea pigs infected with M. tuberculosis and receiving post-exposure vaccines. Lungs obtained from guinea pigs infected with M. tuberculosis and receiving post-exposure vaccines saline control (solid bar), adjuvant control (open bar), BCG control (dotted bar), Ag85A+ESAT6 (hatched bar) and F36 (line bar) on day 40 are shown. Guinea pig lungs (n=5) were assayed by flow cytometry, and numbers of cells per 1.0 gram of tissue were calculated from these assays. Panels A show the total numbers of MIL4^neg CD4+ cells and MIL4^neg CD8+ cells respectively. Panels B show the totalcells/1.0 gram of tissue of CD4⁺ CD45⁺ in the lungs of post-exposure vaccinated guinea pigs. Panel C and D shows a representative flow cytometric dot plot from isotype control and guinea pigs receiving post-exposure vaccines saline control, adjuvant control, BCG control, Ag85A+ESAT6 and F36. Results are expressed as the mean cells/1.0 gram of tissue of each analyzed cell population (±SEM, n=4). (Student t-test, *p≤0.05) compared to adjuvant control guinea pigs.

Figure 5

Numbers of B cell and MIL4⁺ heterophil (granulocyte) of guinea pigs infected with M. tuberculosis and receiving post-exposure vaccines. Lung obtained from guinea pigs infected with M. tuberculosis and receiving post-exposure vaccines saline control (solid bar), adjuvant control (open bar), BCG control (dotted bar), Ag85A+ESAT6 (hatched bar) and F36 (line bar) on day 40 are shown. Guinea pig lungs (n=4) were assayed by flow cytometry, and numbers of cells per 1.0 gram of tissue were calculated from these assays. Panels A show the total numbers of MIL4^neg B cells+ cells and panel B shows the MIL4+ cells respectively. Results are expressed as the numbers of MIL4^neg B cells and MIL4⁺ granulocytes (±SEM, n=4). (Student t-test, *p≤0.05) compared to adjuvant control guinea pigs.