The effects of Mycobacterium vaccae on allergen-induced airway responses in atopic asthma

Subjects

A total of 24 male atopic asthmatics (mean age 34.8 yrs, range 20–53 yrs) with mild-to-moderate disease were recruited (table 1⇓). All the subjects demonstrated an early (EAR) and late airway response (LAR). The subjects either had mild asthma (n=15), requiring only inhaled β₂-agonists, or moderately severe asthma (n=9), with daily symptoms requiring the use of low-to-moderate doses of inhaled corticosteroids (<600 µg of beclomethasone dipropionate or equivalent per day). Individuals with a history of smoking during the previous 12 months, alcohol or drug abuse, or serious adverse reactions to medications were excluded. None of the subjects reported a respiratory infection or required prescription medication, other than that for asthma, within 14 days prior to the study and none of the subjects had used oral corticosteroids within the previous 6 months.

The study was approved by the Southampton University (Southampton, UK) and the Hospital Ethics Committee and subjects gave their written informed consent.

Study design

This was a randomised, placebo-controlled, parallel-group Phase I study. Subjects attended the department on nine separate occasions (fig. 1⇓).

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Fig. 1.—

Study design. SRL172: Mycobacterium vaccae; PBMC: peripheral blood mononuclear cells; IgE: immunoglobulin E.

At the first screening visit, medical history was taken and a physical examination, routine haematological and biochemical tests, urinalysis and skin-prick tests were performed. In addition, baseline responsiveness to histamine was measured. After the first visit there was a run-in period of 28 days where the disease activity was recorded on diary cards in order to ascertain asthma stability. The first allergen challenge was performed at visit 2 using an incremental-dose challenge protocol. The following day (visit 3) subjects underwent a histamine challenge to assess any change in responsiveness, secondary to allergen challenge. Two weeks later (visit 4) blood was drawn for the first in a series of three PBMC cultures and serum IgE measurements. Subjects then received an intradermal injection of 0.1 mL (1 mg) of SRL172 or placebo into the lateral aspect of the deltoid muscle region on the left arm. After 3 days they returned to the department for recording of any local reaction (visit 5). Eighteen days after injection (visit 6), haematology and biochemistry blood tests, urinalysis and serum IgE measurement were repeated. At visit 7 (3 weeks after the injection), a second blood sample was taken for PBMC culture and allergen challenge was repeated. This was followed by a histamine challenge the next day (visit 8). Subjects were followed-up until 42 days postinjection (visit 9) when a full medical examination was conducted, a third blood sample was taken for serum IgE, PBMC culture, haematology, biochemistry and urinalysis was performed.

In order to avoid observer bias, a pharmacist at the Southampton General Hospital prepared the study medication and a physician blinded to the study administered the injections. Furthermore, the site of injection was concealed from other investigators with a bandage.

Allergen and histamine inhalation challenge

Allergen challenge was performed using a modification of an established protocol 19. If subjects were taking regular inhaled beclomethasone they stopped treatment 5 days before allergen challenge to enable an LAR to develop. Allergens that produced the greatest wheal response were used. After taking five breaths of physiological saline to ensure the absence of nonspecific airways irritability, subjects inhaled increasing concentrations of allergen solution (Aquagen: ALK, Horsholm, Denmark). Beginning with 1 Standardised Quality Units (SQ)·mL⁻¹ and increasing 10-fold at 15 min intervals up to 10,000 SQ units·mL⁻¹ or until a fall in forced expiratory volume in one second (FEV₁) of ≥25% from postsaline baseline had been achieved (EAR). FEV₁ measurements were recorded at 20, 30, 45 and 60 min and, thereafter, hourly for 8 h. An LAR was defined as a fall in FEV₁ >15% from postsaline baseline between 3 and 8 hr postchallenge. Subjects were then routinely given 2.5 mg of nebulised salbutamol and allowed home. They returned the following morning for histamine challenge using a previously reported protocol 19. A second allergen challenge was performed 3 weeks after the injection, using an identical increasing concentration regimen and the same total dose as used on the first occasion.

In vitro interleukin-5 responses to allergen

PBMC were isolated from 20 mL of heparinised venous blood by Ficoll-Hypaque gradient centrifugation (Lymphoprep Nycomed Pharma AS, Oslo, Norway). Cells were washed twice and resuspended at 2×10⁶ cells·mL⁻¹ in Rosswell Park Memorial Institute (RPMI) 1640 medium supplemented with 1 mM sodium pyruvate, 2 mM l-glutamine, 100 U·mL⁻¹ penicillin, 100 µg·mL⁻¹ l streptomycin, 50 µM 2-mercaptoethanoll (all from GibcoBRL, Life Technologies, Paisley, UK), and 5% heat-inactivated human AB serum (SIGMA Ltd, Poole, UK), and cultured for 48 hr at 37°C in 5% carbon dioxide (CO₂) in the presence or absence of house dust mite allergen extract (Dermatophagoides pteronyssinus; ALK) at a concentration of 5,000 SQ units·mL⁻¹. The dose of allergen was chosen from pilot experiments as causing the greatest increase in IL-5 synthesis. Culture supernatants were assayed by commercial enzyme-linked immunosorbent assay (ELISA) kits for IL-5 levels (CytoScreenTM; BioSource International, Inc. Camarillo, CA, USA) with sensitivity at 4 pg·mL⁻¹.

Serum immunoglobulin E

The total serum IgE was measured by the Regional Immunology Laboratory, Southampton General Hospital, by ELISA, using a monoclonal anti-IgE antibody developed in-house and a commercially available anti-IgE antibody (Dako, High Wycombe, UK).

Statistical analysis

The principal outcome variables were as follows: maximum percentage fall in FEV₁ during the first 2 h following allergen challenge (EAR) and between 3 and 8 h after allergen challenge (LAR), and changes in histamine responsiveness 24 h postallergen challenge. The primary outcome on which power calculation was performed was the maximum fall in FEV₁ during the LAR. Studies of intersubject variability and reproducibility of late-phase responses have shown that in order to demonstrate a 30% reduction in the LAR, with 95% power and probability of <5% of the difference occurring by chance, 12 subjects are needed 20. The changes in FEV₁ were also expressed as areas under the curve (AUC) for the EAR and LAR. The AUC was calculated using trapezoid integration as the area between the curve and the x-axis and divided by the duration (first 2 h for the EAR and 3–8 h for the LAR) over which the AUC was calculated. Comparisons of changes in maximum percentage falls in FEV₁, AUC for the EAR and LAR, and provocative dose causing a 20% fall in FEV₁ (PC₂₀) as a result of treatment with SRL172 or placebo were performed using formal analysis of variance models (ANOVA). Within-group changes in the same were made by paired t-test. The changes during the course of the study in IL-5 production in vitro and serum IgE levels relative to the first measurement were expressed as AUC by trapezoid integration of the percentage change at the three time-points. This test was used as it takes into account the dynamics of IgE and IL-5 over the whole course of the study and does not rely on individual time-points alone. The AUC in the two treatment arms were then compared by Mann-Whitney U-test, assuming a non-normal distribution. The changes in IgE levels and IL-5 synthesis in the two treatment groups were also compared using the Fisher's exact test.

Baseline airways responses

At baseline, all the subjects developed both an EAR and LAR. The mean (range) maximum percentage falls during the EAR were 33% (20.6–50.6) and 33% (20.6–42.7) in the SRL172 and placebo groups, respectively. During the LAR, the mean (range) maximum percentage FEV₁ fall was 35.8% (16.5–66.7) in subjects receiving SRL172 and 29.5% (16.9–39.4%) in subjects receiving placebo (fig. 2⇓).

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Fig. 2.—

Effects of a) Mycobacterium vaccae (SRL172) and b) placebo on allergen-induced airway responses. •: pretreatment; ○: post-treatment. The maximum fall in forced expiratory volume in one second (FEV₁) and the area under the curve of the FEV₁ changes during the late asthmatic response were significantly reduced after treatment with SRL172 (p=0.005 and p=0.02, respectively) but not after placebo.

Baseline geometric mean (range) PC₂₀ histamine, although lower in the subjects treated with SRL172 (1.17 mg·mL⁻¹ (0.65–2.69 mg·mL⁻¹)) when compared with those receiving placebo (1.82 mg·mL⁻¹ (0.35–13.49 mg·mL⁻¹)), was not significantly different (p=0.23).

Effects of treatment on airway responses to allergen challenge

Treatment with placebo or SRL172 had no significant effect on the EAR either in terms of the maximum percentage falls in FEV₁ or AUC. However, treatment with SRL172 caused a significant (p=0.005) reduction in the maximum fall in FEV₁ during the LAR from a mean (range) of 35.8% (16.5–66.7) before treatment to 26.7% (3.6–60.6) after injection of SRL172, a mean relative reduction of 25.4% (fig. 3⇓).

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Fig. 3.—

Effects of Mycobacterium vaccae (SRL172) on a) the area under the curve (AUC) and b) the maximum fall in forced expiratory volume in one second (FEV₁) during allergen-induced late phase asthmatic responses. ┘: SRL172; □: placebo. The AUC was calculated by trapezoid integration of the area between the curve and the x-axis, taking 3–8 h as the period of analysis. ^#: p=0.005; ^¶: p=0.002; ⁺: p=0.06; ^§: p>0.1. The p-values of 0.06 and 0.1 denote levels of significance when comparing the two groups with respect to the changes in AUC and maximum fall in FEV₁, respectively.

Similarly, the AUC of the percentage fall in FEV₁ was reduced significantly (p=0.02) from 19.4% (5.8–41.4) to 12.8% (0.4–39.1), a mean relative reduction of 34%. In contrast, in the subjects receiving placebo there was no significant change in either the maximum percentage fall in FEV₁ or AUC of the LAR. When comparing the magnitude of changes in the groups of subjects receiving SRL172 and those receiving placebo by ANOVA, the effect on the AUC during the LAR failed to reach the conventional 5% significance level (p=0.06), and the effect on the maximum percentage fall in FEV₁ was not significant.

Effect of treatment on airway responsiveness

The two groups of subjects were not different in respect of baseline PC₂₀ histamine. Allergen challenge performed before any treatment caused a significant (p=0.007) change in PC₂₀ from a geometric mean (range) of 1.17 mg·mL⁻¹ (0.64–-2.72 mg·mL⁻¹) to 0.55 mg·mL⁻¹ (0.1–1.68 mg·mL⁻¹) in the subjects that subsequently received SRL172 and a significant change (p=0.02) from 1.81 mg·mL⁻¹ (0.35–13.63 mg·mL⁻¹) to 0.78 mg·mL⁻¹ (0.16–8.84 mg·mL⁻¹) in the subjects that received placebo. The changes in PC₂₀ between the two treatment groups were not significantly different (p=0.98). Comparisons of the geometric means for postallergen challenge PC₂₀ recorded before and after treatment with placebo or SRL172 using ANOVA showed no difference between the two treatment arms, with the mean value changing from 0.55 mg·mL⁻¹ to 0.76 mg·mL⁻¹ in the actively treated subjects and from 0.77 to 1.41 mg·mL⁻¹ in the subjects receiving placebo.

Effect of treatment on IL-5 synthesis in vitro and serum IgE levels

Cultures of PBMC were performed on all three occasions in 19 subjects (10 treated with SRL172 and nine subjects receiving placebo). Subjects who were randomised to receive active treatment had a tendency to exhibit higher IL-5 responses to allergen in vitro, but these were not significantly different from subjects receiving placebo (fig. 4⇓).

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Fig. 4.—

Effects of Mycobacterium vaccae (SRL172) on allergen-induced a) interleukin (IL)-5 generation by peripheral blood mononuclear cells and b) total serum immunoglobulin (Ig)E levels during the study. V: visit. ┘: SRL172; □: placebo. ^#: p=0.07. The p-values indicate significance levels when comparing the changes in IL-5 and IgE concentrations in the SRL172 group with those in the placebo group that had occurred between day 0 and day 21 using the Fisher's exact test.

In seven out of 10 subjects treated with SRL172 there was a gradual decline in IL-5 synthesis over the course of the study. In contrast, there was a decrease in IL-5 in only two subjects receiving placebo. In the latter group, there was a tendency towards a transient increase at visit 7, i.e. just before the second allergen challenge, which was followed by a return to baseline levels at the end of the study. Comparisons by Mann-Whitney U-test of AUC of IL-5 changes that had occurred between visits 4 and the subsequent visits showed a tendency towards significant differences (p=0.07 for differences in AUC between visit 4 and 7 and 0.08 for the whole time course, i.e. between visits 4 and 9). Comparison of the changes in IL-5 and IgE concentrations in the SRL172 group with those in the placebo group that had occurred between visits 4 and 7 using the Fisher's exact test also showed a tendency for significant differences (p=0.07). Similar trends were seen for total serum IgE levels when comparing placebo and SRL172 treatment groups (p=0.07 when comparing visits 4 and 7 by Fisher's exact test; fig. 4⇑). Comparison of AUC showed no difference between the two treatment groups.