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. 2003 Jun;71(6):3146-54.
doi: 10.1128/iai.71.6.3146-3154.2003.

The Mycobacterium Tuberculosis Recombinant 27-kilodalton Lipoprotein Induces a Strong Th1-type Immune Response Deleterious to Protection

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The Mycobacterium Tuberculosis Recombinant 27-kilodalton Lipoprotein Induces a Strong Th1-type Immune Response Deleterious to Protection

Avi-Hai Hovav et al. Infect Immun. .
Free PMC article

Abstract

Th1 immune response is essential in the protection against mycobacterial intracellular pathogens. Lipoproteins trigger both humoral and cellular immune responses and may be candidate protective antigens. We studied in BALB/c mice the immunogenicity and the protection offered by the recombinant 27-kDa Mycobacterium tuberculosis lipoprotein and the corresponding DNA vaccine. Immunization with the 27-kDa antigen resulted in high titers of immunoglobulin G1 (IgG1) and IgG2a with a typical Th1 profile and a strong delayed hypersensitivity response. A strong proliferation response was observed in splenocytes, and significant nitric oxide production and gamma interferon secretion but not interleukin 10 secretion were measured. Based on these criteria, the 27-kDa antigen induced a typical Th1-type immune response thought to be necessary for protection. Surprisingly, in 27-kDa-vaccinated mice (protein or DNA vaccines) challenged by M. tuberculosis H37Rv or BCG strains, there was a significant increase in the numbers of CFU in the spleen compared to that for control groups. Furthermore, the protection provided by BCG or other mycobacterial antigens was completely abolished once the 27-kDa antigen was added to the vaccine preparations. This study indicates that the 27-kDa antigen has an adverse effect on the protection afforded by recognized vaccines. We are currently studying how the 27-kDa antigen modulates the mouse immune response.

Figures

FIG. 1.
FIG. 1.
Purification and metabolic radiolabeling of the recombinant 27-kDa antigen in E. coli and expression of the native form in BCG and M. tuberculosis. E. coli SG13009 cells harboring the plasmid pRHB27 were grown to an OD600 of 0.7 with or without [3H]palmitic acid. Protein production was induced by IPTG. The induced protein was purified using a nickel-nitrilotriacetic acid column, and samples were analyzed by SDS-PAGE and stained with Coomassie blue. The radioactively labeled samples were exposed to an X-ray film for 1 month. Numbers at the left indicate molecular masses in kilodaltons. Lane A, SDS-PAGE of the purified recombinant 27-kDa lipoprotein; lane B, an autoradiogram of the purified radiolabeled 27-kDa lipoprotein after SDS-PAGE analysis; lanes C and D, Western blot of BCG and M. tuberculosis H37Rv lysates, respectively, exposed to rabbit-anti-27-kDa serum; lane E, RT-PCR of the LprG gene in HeLa cells transfected with pIHB27 (1), pRHB27 as a control vector (2), and λ HindIII DNA marker (m).
FIG. 2.
FIG. 2.
Antibody response to the 27-kDa antigen in vaccinated mice. BALB/c mice were immunized subcutaneously with the 27-kDa antigen, Ribi-27-kDa, DDA-27-kDa, Ribi, DDA, and saline three times 2 weeks apart. Sera were collected and analyzed by ELISA for the presence of anti-27-kDa IgG1 and IgG2a antibodies. Each point represents the average and standard deviation of three independent experiments; in each experiment five individual mice were tested.
FIG. 3.
FIG. 3.
Proliferation response and IFN-γ and NO secretion in 27-kDa-immunized mice after in vitro stimulation of the mouse splenocytes with the 27-kDa antigen. (A) Splenocyte proliferation for immunized mice exposed to 27-kDa antigen (20 μg/ml). The proliferation results presented are from one representative experiment (out of three experiments) with five mice in each group. IFN-γ secretion (B) and nitric oxide production (C) from splenocytes of immunized mice after 96 h of in vitro stimulation with the 27-kDa antigen (20 μg/ml) are shown. The IFN-γ and NO secretion results shown are the mean and standard deviation for seven experiments with three to five mice in each group. Data were analyzed by Student's t test. ∗, P < 0.05; ∗∗, P < 0.01; #, P < 0.005 (compared to the naive group).
FIG. 4.
FIG. 4.
DTH evaluation in 27-kDa- or BCG-vaccinated mice. Mice were injected in the footpads with the 27-kDa antigen (10 μg) 4 weeks after the last vaccination or 1 and 3 months after BCG vaccination. Footpad swelling was measured, and the results shown are the means and standard deviations for three to five experiments with three to seven mice in each group. Data were analyzed by Student's t test. ∗, P < 0.001 (compared to naive control mice after 24 h).
FIG. 5.
FIG. 5.
Intravenous M. tuberculosis H37Rv challenge. Mice were immunized twice with different formulations of the 27-kDa antigen; 4 weeks after the last injection, mice were challenged (intravenously) with 5 × 105 CFU of M. tuberculosis strain H37Rv. Four weeks (A) or seven weeks (B) after infection, numbers of CFU in mouse spleens were determined. (C) Mice were immunized subcutaneously with 2 × 105 CFU of BCG Pasteur with or without the 27-kDa antigen. After 2 weeks, parts of the groups (BCG/27 and BCG + 27/27) were injected with the 27-kDa antigen alone, and 6 weeks later mice were challenged (intravenously) with 5 × 105 CFU of M. tuberculosis H37Rv. (D) Timelines of the vaccination protocols used in Fig. 5C. Four weeks after the challenge, CFU at the spleens were counted. Data were analyzed by Student's t test. ∗, P < 0.05; ∗∗, P < 0.01; #, P < 0.005 (compared to the naive group).
FIG. 6.
FIG. 6.
Intravenous BCG Pasteur challenge. (A) Mice were immunized three times with the different 27-kDa antigen preparations. Four weeks after the last injection, mice were challenged intravenously with 5 × 106 CFU of BCG Pasteur. Four weeks after the infection, CFU in mouse spleens were measured. (B) Mice were injected twice with 85B, Esat6, and L7/L12 antigens in the absence (3Ags) or the presence of the 27-kDa antigen (4Ags). Another group was vaccinated subcutaneously with 2 × 105 CFU of BCG Pasteur. Four weeks after the last injection, mice were challenged intravenously with 5 × 106 CFU of BCG Pasteur, and after 4 weeks, CFU values in the spleen were determined. Data were analyzed by Student's t test. ∗, P < 0.05; ∗∗, P < 0.0001 (compared to the naive group). #, P < 0.01; ##, P < 0.005 (compared to the relevant 4Ags group).

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