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, 72 (5), 3031-7

Protection Elicited by a Double Leucine and Pantothenate Auxotroph of Mycobacterium Tuberculosis in Guinea Pigs

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Protection Elicited by a Double Leucine and Pantothenate Auxotroph of Mycobacterium Tuberculosis in Guinea Pigs

Samantha L Sampson et al. Infect Immun.

Abstract

We developed a live, fully attenuated Mycobacterium tuberculosis vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. The deltaleuD deltapanCD double auxotroph is fully attenuated in the SCID mouse model and highly immunogenic and protective in the extremely sensitive guinea pig tuberculosis model, reducing both bacterial burden and disease pathology.

Figures

FIG. 1.
FIG. 1.
Immune response to PPD in guinea pigs immunized with the ΔleuD ΔpanCD strain. Outbred female Hartley guinea pigs were immunized i.d. with 5 × 105 CFU of the ΔleuD ΔpanCD strain, followed 6 weeks later by a second immunization with 3 × 106 CFU of the same strain. At the time that this group received the booster vaccination, a second group received a single immunization with 3 × 106 CFU of the ΔleuD ΔpanCD strain, while a third group received 3 × 106 CFU of BCG-P. A control group was unimmunized. Each experimental group consisted of five animals. (A) Five weeks after the last immunization, PBMCs were isolated from whole blood by Ficoll-Histopaque (Sigma, St. Louis, Mo.) density-gradient centrifugation. Two individual guinea pigs were tested per treatment group. PBMCs (2 × 105/well) were incubated at 37°C in 5% CO2 for 5 days with medium alone, phytohemagglutinin A (Sigma) (data not shown), or 5 μg of PPD (Mycos Research) per well. Proliferation was assessed on day 5 by [3H]thymidine (0.5 μCi/well) incorporation for 6 h. Cells were harvested with a Skatron cell harvester, and incorporated thymidine was measured using a beta-plate liquid scintillation counter. Values are expressed as mean counts per minute of triplicate samples. Error bars represent standard deviations from the mean. (B) DTH responses were determined 7 weeks after the last immunization. Four TU (data not shown) or 40 TU of PPD (FDA/Center for Biologics Evaluation and Research, Bethesda, Md.) was injected i.d. in the right flank, and the diameter of induration was measured at 24 h. Mean induration diameters (in millimeters) (indicated by horizontal lines) were 11.6 ± 2.3 for BCG-P-immunized animals, 11.0 ± 2.3 for animals immunized once with the ΔleuD ΔpanCD strain, and 13 ± 2.4 for animals immunized twice with the ΔleuD ΔpanCD strain. *, P < 0.001 as determined by one-way analysis of variance.
FIG. 2.
FIG. 2.
Protection of guinea pigs against aerosol challenge with M. tuberculosis H37Rv. Outbred female Hartley guinea pigs (n = 5 per group) were immunized i.d. once with M. bovis BCG-P, once with the ΔleuD ΔpanCD strain, or twice (6 weeks apart) with the ΔleuD ΔpanCD strain. A negative control group was unimmunized. Seven weeks subsequent to the last immunization, animals were challenged via the aerosol route with 20 CFU of M. tuberculosis H37Rv. Spleen and right caudal lung bacterial burdens were determined 5 weeks postchallenge. Error bars represent standard deviations from the mean. *, P < 0.001; **, P < 0.01; determined by one-way analysis of variance.
FIG. 3.
FIG. 3.
Histopathology of the lungs of vaccinated and unvaccinated guinea pigs 5 weeks after an aerosol challenge with M. tuberculosis. (A and B) Left caudal lung section from an unvaccinated guinea pig showing severe multifocal and spreading granulomatous pneumonia with central caseation necrosis. (B) Higher-power photomicrograph showing the spreading granulomatous reaction composed of epithelioid cells accompaniedby large numbers of macrophages and lymphocytes surrounding the central caseation necrosis. (C and D) Lung of a guinea pig vaccinated with BCG-P. The lower-power photomicrograph (C) shows multiple sites of focal perivascular and interstitial discrete accumulations of moderate to large numbers of lymphocytes (arrowheads). Also shown are scattered granulomatous foci composed of epithelioid cells and numerous lymphocytes (arrows). Very mild caseation necrosis was evident in a few of the granulomas. The higher-power photomicrograph (D) shows perivascular lymphoid accumulations (arrows). (E and F) Lung of a guinea pig vaccinated once with the ΔleuD ΔpanCD strain. (E) The lungs had lower numbers of discrete focal granulomas widely scattered in the parenchyma (arrows). Multifocal small, localized lymphocytic accumulations (arrowheads) were apparent, similar to those in the BCG-vaccinated guinea pigs. (F) Higher-power photomicrograph showing a discrete granuloma composed of predominantly epithelioid cells accompanied by low to moderate numbers of lymphocytes and focal lymphocytic accumulations in the parenchyma and perivascular regions. A few granulomas had mild central caseation necrosis. (G and H) Lung from a guinea pig vaccinated twice with the ΔleuD ΔpanCD strain, exhibiting extensive coalescing granulomatous pneumonia, particularly along airways (panel G). The spreading areas of inflammation were composed of epithelioid cells and moderate numbers of lymphocytes, accompanied by macrophages. Many of the granulomas showed mild to moderate central caseation necrosis, considerably less pronounced than in the unvaccinated guinea pigs, but more prominent than in BCG- and singly immunized animals.
FIG. 4.
FIG. 4.
Morphometric analysis of lung pathology. The areas of lung lesions were measured on hematoxylin- and eosin-stained left caudal lobe sections by use of Scion Image software. These measurements were used to calculate the total area of the lung section occupied by diseased tissue. The percentage of diseased tissue was significantly reduced in animals immunized with BCG-P and the ΔleuD ΔpanCD strain relative to naive animals. *, P < 0.01 as determined by one-way analysis of variance.

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