Drug treatment combined with BCG vaccination reduces disease reactivation in guinea pigs infected with Mycobacterium tuberculosis

Abstract

Bacillus-Calmette-Guerin (BCG), the only human tuberculosis vaccine, primes a partially protective immune response against Mycobacterium tuberculosis infection in humans and animals. In guinea pigs, BCG vaccination slows the progression of disease and reduces the severity of necrotic granulomas, which harbor a population of drug-tolerant bacilli. The objective of this study was to determine if reducing disease severity by BCG vaccination of guinea pigs prior to M. tuberculosis challenge enhanced the efficacy of combination drug therapy. At 20 days of infection, treatment of vaccinated and non-vaccinated animals with rifampin, isoniazid, and pyrizinamide (RHZ) was initiated for 4 or 8 weeks. On days 50, 80 and 190 of infection (10 weeks after drug were withdrawn), treatment efficacy was evaluated by quantifying clinical condition, bacterial loads, lesion severity, and dynamic changes in peripheral blood and lung leukocyte numbers by flow cytometry. In a separate, long-term survival study, treatment efficacy was evaluated by determining disease reactivation frequency post-mortem. BCG vaccination alone delayed pulmonary and extra-pulmonary disease progression, but failed to prevent dissemination of bacilli and the formation of necrotic granulomas. Drug therapy either alone or in combination with BCG, was more effective at lessening clinical disease and lesion severity compared to control animals or those receiving BCG alone. Fewer residual lesions in BCG vaccinated and drug treated animals, equated to a reduced frequency of reactivation disease and improvement in survival even out to 500 days of infection. The combining of BCG vaccination and drug therapy was more effective at resolving granulomas such that fewer animals had evidence of residual infection and thus less reactivation disease.

Figure 1

Combination drug therapy significantly reduces the bacterial burden in the lungs, spleen and lymph node in BCG vaccinated and non-vaccinated guinea pigs infected with the Erdman KO1 strain of M. tuberculosis. Bacterial counts in the lungs (A), spleen (B) and lymph node (C), on days 20, 50, and 80 from guinea pigs infected with M. tuberculosis Erdman-KO1 by low dose aerosol. Treatment groups include guinea pigs (n=5) that were sham vaccinated with saline and received 40% sucrose and nutrient vehicle orally as a drug carrier (solid squares), those receiving BCG and drug carrier (open squares), those receiving saline and chemotherapy with RHZ in drug carrier (open diamonds) and those receiving the combination of BCG and RHZ chemotherapy in drug carrier (solid diamonds). Results are expressed as the mean (n=5) log10 CFU (± SEM) for each treatment group. * = p <0.050.

Figure 2

Combination drug therapy and BCG vaccination prolongs survival of guinea pigs infected with the Erdman KO1 strain of M. tuberculosis. The survival of individual guinea pigs (n=10 per group) were either sham vaccinated with saline and received 40% sucrose and nutrient vehicle orally as a drug carrier (solid squares), BCG and drug carrier (open squares), or saline and chemotherapy with RHZ in drug carrier (open diamonds) or the combination of BCG and RHZ chemotherapy in drug carrier (solid diamonds). Kaplan Meier analysis, n=10. * = p <0.05.

Figure 3

Combination of BCG vaccination and RHZ chemotherapy prevents progressive clinical disease in M. tuberculosis infected guinea pigs as measured by changes in body weight, percent peripheral blood oxygen saturation, and body temperature. Treatment groups included M. tuberculosis infected guinea pigs (n=5) that were either sham vaccinated with saline and received 40% sucrose and nutrient vehicle orally as a drug carrier (solid squares), BCG and drug carrier (open squares), or saline and chemotherapy with RHZ in drug carrier (open diamonds) or the combination of BCG and RHZ chemotherapy in drug carrier (solid diamonds). Guinea pigs that received RHZ chemotherapy either alone (open diamonds) or in combination with BCG vaccination (solid diamonds) gained weight (A), retained normal percent oxygen saturation (B) and normal body temperature out to 190 days of infection. Untreated control animals (solid squares) or animals that received BCG alone (open squares) showed evidence of progressive disease with a progressive decrease in body weight (A), percent oxygen saturation (B) and body temperature. Results are expressed as the mean (n=5) of body weight in grams, percent oxygen saturation and body temperature [degree C] (± SEM). * = p<0.05.

Figure 4

Treatment of BCG vaccinated guinea pigs with RHZ drug therapy significantly reduces the lesion burden in M. tuberculosis infected guinea pigs. On day 20, 50, 80 and 190 of infection, the lung, spleen and lymph node lesion severity was quantified in M. tuberculosis infected guinea pigs that were either sham vaccinated with saline and received 40% sucrose and nutrient vehicle orally as a drug carrier (control), BCG and drug carrier (BCG), saline and chemotherapy with RHZ in drug carrier (RHZ) or the combination of BCG and RHZ chemotherapy in drug carrier (BCG/RHZ). Data is expressed as the mean value for the percent area of lung, lymph node and spleen affected by granulomatous inflammation and the area of lesion affected by necrosis. The data is the mean value for 5 animals per treatment group and at each time point. * = p<0.05.

Figure 5

Treatment of BCG vaccinated guinea pigs with RHZ drug therapy significantly reduces the lung lesion severity in M. tuberculosis infected guinea pigs on days 50 and 80 of infection. Photomicrographs are representative sections of lung from individual animals that best represents the mean values shown in panel 4A. Panels C,G represent sections were taken from M. tuberculosis infected guinea pigs that were either sham vaccinated with saline and received 40% sucrose and nutrient vehicle orally as a drug carrier (saline/No RHZ). Panels C and G represent infected animals that received BCG and drug carrier (BCG/No RHZ). Panels B and F were from infected animals that received, saline and chemotherapy with RHZ in drug carrier (saline/RHZ). Panels D and H are from infected animals that received a combination of BCG and RHZ chemotherapy in drug carrier (BCG/RHZ). Images illustrate that BCG vaccination failed to prevent foci of granulomatous inflammation from developing central necrosis on days 50 (C, inset) and 80 (G, inset) compared to those that received BCG and RHZ chemotherapy D and H out to 50 and 80 days respectively. RHZ chemotherapy alone failed to resolve primary lesions within the perivascular and peribronchial lymphatics that became calcified and persisted to day 50 (B) and 80 (F) of infection. Combination BCG and RHZ almost completely resolved lung lesions by day 80 (H) leaving only residual foci of interstitial fibrosis (H, inset). Hematoxylin and eosin staining. Magnification = 40X, inset magnification 200X.

Figure 6

Combination BCG and RHZ chemotherapy prevented the relapse of inflammation in the perivascular and peribronchial lymphatics of M. tuberculosis infected guinea pigs. At 500 days after infection with the Erdman KO strain of M. tuberculosis, 4 of 5 animals mock-vaccinated with saline prior to treatment with RHZ had residual lesions within perivascular and peribronchial lymphoid aggregates (A, white arrows) that were either calcified (C) or showed evidence of reactivation of granulomatous inflammation (G) in panel B. In contrast, 4 of 5 guinea pigs vaccinated with BCG prior to infection followed by 8 weeks of treatment with RHZ showed no evidence of residual lesions (panel C) but had normal lung associated lymphoid aggregates (white arrow) associated with peribronchial and perivascular lymphatics (black arrows). Hematoxylin and eosin. Magnification = 200X.

Figure 7

Flow cytometric analysis of lung cells collected from M. tuberculosis infected guinea pigs that were CD4+ CD45+ (panel A), CD4+ CT4+ (panel B), CD8+ CT4+ (panel C), MR-1+ and MHC class II+ macrophages (panel D), B cells (panel E) and MIL4+ neutrophils on days 20, 50, 80 and 190 of infection. Guinea pigs infected with Erdman KO1 strain of M. tuberculosis were sham vaccinated with saline and received 40% sucrose and nutrient vehicle orally as a drug carrier (solid squares), received BCG and drug carrier (open squares), received saline and chemotherapy with RHZ in drug carrier (open diamonds) or received combination of BCG and RHZ chemotherapy in drug carrier (solid diamonds). Each data point represents mean (± SEM) total cell numbers per gram of tissue from 5 guinea pigs for each treatment and each time point. * = p<0.05.

Figure 8

Flow cytometric analysis of peripheral blood cells collected from M. tuberculosis infected guinea pigs that were CD4+ CD45+ (panel A), CD4+ CT4+ (panel B), CD8+ CT4+ (panel C), MR-1+ and MHC class II+ macrophages (panel D), B cells (panel E) and MIL4+ neutrophils on days 20, 50, 80 and 190 of infection. Guinea pigs infected with Erdman KO1 strain of M. tuberculosis were sham vaccinated with saline and received 40% sucrose and nutrient vehicle orally as a drug carrier (solid squares), received BCG and drug carrier (open squares), received saline and chemotherapy with RHZ in drug carrier (open diamonds) or received combination of BCG and RHZ chemotherapy in drug carrier (solid diamonds). Each data point represents mean (± SEM) total cell numbers per gram of tissue from 5 guinea pigs for each treatment and each time point. * = p<0.05.