Environmental effects on protection against Mycobacterium tuberculosis after immunization with Ad85A

Abstract

Previously we have shown that intradermal (i.d.) immunization with a recombinant adenovirus expressing antigen 85A (Ad85A) induced a strong splenic CD8T cell response in BALB/c mice but a weak lung immune response and did not protect mice against challenge with Mycobacterium tuberculosis (Mtb). After moving to a new animal house, the same i.d. immunization induced a strong lung immune response and the mice were protected against Mtb challenge. Increased numbers of antigen 85A-specific CD8 cells were present in lung tissue but were not recoverable by bronchoalveolar lavage (BAL). Mycobacterial growth was inhibited 21 days after Mtb challenge. In contrast, the effects of intranasal (i.n.) immunization did not change between the animal houses; 85A-specific T cells were recovered by BAL and were able to inhibit Mtb growth early after challenge. The effect of alterations to the environment was investigated by administering BCG or Mycobacterium abscessus in the drinking water, which induced protection against Mtb challenge, while Mycobacterium smegmatis did not. However, when Ad85A was given i.d. at the same time as BCG or M. abscessus, but not M. smegmatis, the protection induced by Ad85A was abolished. Treatment of mice with a CD25 antibody during the challenge period, abolished the suppressive effect of oral mycobacterial administration, suggesting that regulatory T cells (T regs) were involved. These results showed that exposure to environmental microorganisms can alter the protective immune response to a parenterally administered subunit vaccine, a result with important implications for the use of such vaccines in humans.

Fig. 1

T cells responses and protection against Mtb in different animal facilities. (A) Cytokine responses of T cells to antigen 85A. BALB/c mice were immunized with Ad85A i.d. or i.n. in an animal house with open top cages (OTC) or an animal house with individually ventilated cages (IVC). Lung and splenic lymphocytes were isolated 5 weeks post immunization and stimulated with pooled 85A peptides. The percentage of CD8+ cells expressing IFNγ was determined by flow cytometry. Error bars show SD. (B) Mtb growth after Ad85A i.d. or i.n. immunization in IVCs. In two experiments, BALB/c mice were immunized with Ad85A i.d. or Ad85A i.n. One month (B) or 8 months (C) later they were challenged with Mtb and lung CFU enumerated 5 weeks later. Data from the two experiments with 5–7 mice/group are shown. ***p < 0.001, **p < 0.01, *p < 0.05 from naïve or between the indicated groups, one-way ANOVA with Tukey's post test.

Fig. 2

Location of cells and kinetics of Mtb growth after Ad85A immunization. (A) BALB/c mice in IVCs were immunized with Ad85A i.d. or i.n. Four weeks post immunization BAL was collected from individual mice and the number of CD8+, CD8IFNγ+ and CD8CXCR6+ cells determined by flow cytometry. The figures show the mean number of each cell population recovered ±SD from 3 mice in one of three independent experiments ***p ≤ 0.005. (B) Kinetics of Mtb growth after Ad85A i.d. and i.n. immunization. BALB/C mice were immunized in IVCs once with Ad85A i.d. or i.n. Five weeks after immunization mice were challenged with Mtb and groups of 5 mice sacrificed 7, 14, 21 and 28 days later for enumeration of lung Mtb CFU. ***p < 0.001, *p < 0.05 compared to naïve animals, one-way ANOVA with Tukey's post test. Representative data from one of two experiments are shown.

Fig. 3

Effect of oral mycobacteria on Ad85A i.d. induced protection. Unimmunized (N) or Ad85A i.d. immunized BALB/c mice in IVCs were given drinking water with or without BCG (A), M. abscessus (M abs) or heat killed M. abscessus (HK M abs) (B) or M. smegmatis (M sm) (C). Five weeks after Ad85A i.d. immunization they were challenged with Mtb i.n. and after further 5 weeks sacrificed for enumeration of lung and spleen Mtb CFU. Data from one of two experiments with 5–7 mice/group are shown. ***p < 0.001, **p < 0.01, *p < 0.05 compared to naïve animals with clean water, one-way ANOVA with Tukey's post test.

Fig. 4

Mtb CFU after CD25 depletion. Unimmunized (N) or Ad85A i.d. immunized BALB/c mice in IVCs were given drinking water with or without M. abscessus (M abs). Five weeks after the immunization they were challenged with Mtb i.n. and after further 5 weeks sacrificed for enumeration of lung and spleen Mtb CFU. The day before and 7 and 14 after the Mtb challenge, mice were given CD25 antibody or control rat Ig i.p. Representative data from one of two experiments with 5–7 mice/group are shown. ***p < 0.001, **p < 0.01, *p < 0.05 compared to unimmunized animals with clean water, one-way ANOVA with Tukey's post test.