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. 2006 Oct 30;75(1):408–416. doi: 10.1128/IAI.01290-06

The Combined CTA1-DD/ISCOMs Vector Is an Effective Intranasal Adjuvant for Boosting Prior Mycobacterium bovis BCG Immunity to Mycobacterium tuberculosis▿

Claire Swetman Andersen 1,†, Jes Dietrich 1,*, Else Marie Agger 1, Nils Y Lycke 2, Karin Lövgren 3, Peter Andersen 1
PMCID: PMC1828435  PMID: 17074845

Abstract

Infection with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains one of the leading causes of mortality worldwide. The current “gold standard” vaccine Mycobacterium bovis BCG has a limited efficacy that wanes over time. The development of a vaccine to boost BCG-induced immunity is therefore a highly active area of research. Mucosal administration of vaccines is believed to provide better protection against pathogens, such as M. tuberculosis, that invade the host via mucosal surfaces. In this study we demonstrate that an intranasal vaccine, comprising the antigenic fusion protein Ag85B-ESAT-6 and the mucosal combined adjuvant vector CTA1-DD/ISCOMs, strongly promotes a Th1-specific immune response, dominated by gamma interferon-secreting CD4-positive T cells. Mucosal administration of Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs strongly boosted prior BCG immunity, leading to a highly increased recruitment of antigen-specific cells to the site of infection. Most importantly, we observed a significantly (P < 0.001) reduced bacterial burden in the lung compared to nonboosted control animals. Thus, the results demonstrate the effectiveness of mucosal vaccination with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs as adjuvant for stimulating TB-specific protective immunity in the lung.

Tuberculosis (TB) remains one of the leading causes of mortality worldwide, accounting for an estimated 2 to 3 million deaths each year (38). Furthermore, infection with Mycobacterium tuberculosis, the causative agent of TB, is a leading cause of mortality for individuals infected with human immunodeficiency virus (http://www.who.int/mediacenter/factsheets/fs104/en/). When taken together with the spread of multidrug-resistant mycobacterial strains and the reemergence of the disease in the developed world, it becomes clear why TB remains at the forefront of public health awareness campaigns (32).

While the Mycobacterium bovis BCG vaccine for TB remains the most extensively used vaccine worldwide and a large proportion of the population of most counties has already been vaccinated with BCG, the vaccine nonetheless has questionable efficacy. Current indications suggest that BCG does not convey life-long immunity and is of limited value in protecting against adult TB (7, 14, 26). Thus, there has recently been a growing interest in (i) the development of a TB vaccine that can effectively boost preexisting immunity generated by prior BCG vaccination and, hence, the discovery of a suitable adjuvant, or (ii) an improved BCG vaccine (8, 15, 18, 27, 33).

M. tuberculosis is a pathogen which infects the individual via the mucosal tissue of the respiratory tract following inhalation of infectious droplets. While the majority of approaches to the development of a novel TB vaccine have focused on subcutaneous (s.c.) administration, there is evidence to suggest that mucosal vaccination is an efficient route of administration for vaccines against TB (16, 39); however, an efficient intranasal (i.n.) vaccine against TB requires an efficient mucosal adjuvant. We have constructed a combined adjuvant vector composed of immune-stimulating complexes (ISCOMs) and the cholera toxin-derived fusion protein CTA1-DD. The CTA1-DD/ISCOMs vector is a rationally designed mucosal adjuvant that was developed in order to incorporate the distinctive properties of the individual adjuvant components in a synergistic manner and thereby further enhance mucosal immune responses (25, 28). CTA1-DD is a construct based on a gene fusion protein between the enzymatically active CTA1 protein A (2). The ADP-ribosylating CTA1-DD adjuvant enhances T-cell-dependent and -independent responses by direct action on B cells involving antiapoptotic Bcl-2- and germinal center-promoting effects (1). The cholera toxin-derived CTA1-DD vaccine adjuvant administered i.n. does not cause inflammation or accumulate in the nervous tissues (13). This combined adjuvant vector, CTA1-DD/ISCOMs, has proven exceptionally potent as a mucosal immunoenhancer of a wide range of immune responses, including specific antibodies, CD4 T-cell priming, and effective cytotoxic T lymphocytes (25, 28).

The fusion protein Ag85B-ESAT-6 has been demonstrated in a range of animal models to be a successful candidate for use in s.c.-delivered subunit vaccines against TB (24, 29, 31). Therefore, we have evaluated the immunoenhancing properties of the mucosal combined adjuvant vector CTA1-DD/ISCOMs in a formulation for i.n. administration together with the Ag85B-ESAT-6 hybrid antigen.

We investigated the potential of Ag85B-ESAT-6 mixed with the combined CTA1-DD-ISCOMs adjuvant vector for stimulation of TB-specific protective immunity in naïve as well as previously BCG-immunized mice. The results demonstrate that this vaccine combination induces an efficient immune response characterized by high levels of gamma interferon (IFN-γ) and the release of immunoglobulin A (IgA). Most importantly, the CTA1-DD/ISCOMs adjuvant was effective in boosting TB-specific protection in mice with prior BCG-induced immunity.

MATERIALS AND METHODS

Animals.

Female C57BL/6 mice, 8 to 12 weeks old, were obtained from Harlan Scandinavia (Denmark). Infected mice were kept in cages within a biosafety level 3 laminar flow safety enclosure.

Antigens.

The fusion protein of Ag85B and ESAT-6 (designated Ag85B-ESAT-6 here) was produced as recombinant proteins as described previously (30).

Adjuvant.

The CTA1-DD/ISCOMs combined adjuvant vector was prepared as previously described (1, 3). Briefly, ISCOMs containing the CTA1-DD fusion proteins were prepared by adding 1-mg aliquots of a purified, freeze-dried fraction of Quil A saponin (Quadri A) (22) to 1 ml of a 1-mg/ml solution of CTA1-DD protein at room temperature in 0.2 M phosphate-buffered saline (PBS), pH 6. After being allowed to dissolve using a magnetic stirrer, 40 μl of a lipid mixture containing 1% cholesterol and 1% phoshatidylcholine (Northern Lipids, Vancouver, Canada) dissolved in 20% Mega 10 (Bachem, Bubendorf, Switzerland) was then added, and the mixture was stirred for 3 h at room temperature, followed by dialysis against 0.2 M PBS, pH 6, at room temperature for tubes containing 25% sucrose (wt/wt) in 0.2 M PBS, pH 6. After centrifuging the gradients for 5 h at 257,000 × g at 20°C, fractions were collected from the bottom of the tubes by puncturing with a needle. The fractions were analyzed for total protein content by the Bradford reaction (Bio-Rad). The protein-rich ISCOMs fractions were pooled and dialyzed against 0.2 M PBS for 2 days at 4°C. Finally, the ISCOMs preparations were concentrated using a centrifugal filter device to obtain a total protein concentration of 0.5 mg/ml, and the formation of intact ISCOMs was confirmed by electron microscopy (data not shown), followed by further dialysis for another 2 to 3 h and then overnight at 4°C. The dialyzed material was then centrifuged for 5 min at 10,000 × g, and the supernatant was transferred in 300-μl aliquots to 4-ml plastic ultracentrifuge tubes.

Immunization.

Mice were immunized i.n. up to three times, with a 2-week interval between each immunization. The vaccines (15 to 20 μl/nostril) consisted of 25 μg of the fusion protein Ag85B-ESAT-6 alone or mixed with 5 μg CTA1-DD-ISCOMs, unless otherwise indicated. In some experiments groups of mice received one dose of BCG Danish 1331 of 5 × 106 injected s.c. at the base of the tail.

Cellular assays.

Blood samples, lymph nodes, and spleens were removed aseptically from mice 7 to 21 days after the last immunization or 7 days postchallenge and prepared as previously described (4, 34). Lungs were removed aseptically and perfused with PBS containing heparin to remove erythrocytes. Lungs were crushed through a mesh, tissue was passed through a single-cell strainer (pore size, 100 μm), and the cells were collected and enumerated. Cell cultures were performed in triplicate in round-bottom microtiter wells containing 2 × 105 cells in a volume of 200 μl RPMI supplemented with 2-mercaptoethanol, glutamine, penicillin-streptomycin, HEPES, and 10% fetal calf serum. Antigens were used in concentrations ranging from 5 to 0.05 μg/ml. Results using 5 μg/ml are shown. In some instances, cells were stimulated with the immunodominant epitopes in Ag85B and ESAT-6. The immunodominant epitope in Ag85B was the previously described CD4 epitope Ag85B241-255 (12). The immunodominant epitope in ESAT-6 was the previously described CD4 epitope ESAT-61-20 (6). Wells containing medium only or 5 μg/ml of concanavalin A were included in all experiments as negative and positive controls, respectively. Culture supernatants were harvested from parallel cultures after 72 h of incubation in the presence of antigen, and the amount of IFN-γ or interleukin-5 (IL-5) was determined by enzyme-linked immunosorbent assay (ELISA), as previously described (5). Enzyme-linked immunospot (ELISPOT) analyses were conducted with cells from individual mice as described previously (11).

Determination of antibody titers.

Serum was collected and stored at −20°C until analyzed. Ag85B-ESAT-6-specific IgG, IgG1, IgG2a, or IgA concentrations were determined by ELISA. Briefly, polystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated with Ag85B-ESAT-6 at 10 μg/ml in PBS to measure antigen-specific antibodies. Serum was diluted 1/500, while bronchial lavage fluids were diluted 1/10 in 0.1% bovine serum albumin-PBS, before being serially diluted. Alkaline phosphatase-conjugated isotype-specific rabbit anti-mouse antibodies (1:500; Southern Biotechnology), followed by 2.1 μg/ml extravidin-peroxidase (Sigma); nitrophenyl phosphatase or o-phenylenediamine substrates were used, and the enzymatic reactions were read in a Titertek Multiscan spectrophotometer (Labsystems, Stockholm, Sweden). TB-specific antibody titers were defined as the interpolated absorbance reading giving a rise of absorbance of 0.4 above background, which consistently gave absorbance readings on the linear part of the curve. Titers were given in log10 means ± the standard deviation.

Fluorescence-activated cell sorter (FACS) analysis.

Splenocytes from BCG-immunized mice that had received an i.n. booster vaccination were isolated 7 days after the first boost and restimulated in 96-well U-bottom plates containing 5 μg/ml Ag85B-ESAT-6 and 106 cells/well. Control wells without antigen or with 5 μg/ml concanavalin A were also included. After restimulation overnight, brefeldin A (Sigma) was added at a final concentration of 2.25 μg/well, and the cultures were further incubated for 4 h. After cells were washed, nonspecific binding was blocked by a 15-min incubation with the 24G2 clone (CD16/CD32; BD Pharmingen) and subsequently stained with peridinin chlorophyll protein-CD4 and fluorescein isothiocyanate-CD8 (both from BD Pharmingen) on ice for 30 min. Intracellular cytokine staining was performed using the Cytofix/Cytoperm kit available from BD Pharmingen according to the manufacturer's protocol and using phycoerythrin-IFN-γ (BD Pharmingen). Cells were finally washed three times, resuspended in 3.7% formaldehyde in PBS, and analyzed with a FACScan (Becton Dickinson Immunocytometry Systems, Mountain View, CA) by collecting 50,000 events.

Experimental infections.

For evaluation of vaccine efficacy, mice were challenged 10 weeks after the first immunization by the aerosol route in a Glas-Col inhalation exposure system calibrated to deposit approximately 25 CFU of virulent M. tuberculosis Erdman in the lungs. The bacterial loads in the spleen and lungs were determined 6 weeks later by plating serial dilutions onto Middlebrook 7H11 agar supplemented with 2 μl 2-thiophene-carboxylic acid hydrazide per ml to selectively inhibit the growth of BCG. Colonies were counted after 2 to 3 weeks of incubation at 37°C.

Statistical analyses.

Assessment of experiments was conducted by one-way analysis of variance (ANOVA). When significant effects were indicated, differences between means were assessed by Tukey's test. A value of P < 0.05 was considered significant. The computer program SigmaStat was used for these calculations.

RESULTS

Intranasal administration of the combined CTA1-DD/ISCOMs vector induces an effective Th1 immune response.

The combined vector CTA1-DD/ISCOMs has previously been shown to act as a potent adjuvant for i.n. vaccine administration (10, 25, 28). In this study we first decided to determine the effectiveness of CTA1-DD/ISCOMs as an adjuvant for i.n. administration in conjunction with the fusion molecule Ag85B-ESAT-6.

C57BL/6 mice were immunized with Ag85B-ESAT-6, over a dose range of 0.2 to 25 μg/mouse, either alone or in conjunction with a 5-μg/dose of CTA1-DD/ISCOMs. Three immunizations were administered via the i.n. route at two weekly intervals. The subsequent immune response was monitored by in vitro restimulation of peripheral blood mononuclear cells (PBMC) purified 1 week after the final immunization.

Administration of antigen alone did not stimulate the release of IFN-γ (Fig. 1A). However, when administered in conjunction with CTA1-DD/ISCOMs, a dose-dependent increase in IFN-γ production was observed. At the highest antigen concentration a level of ∼6,000 pg/ml IFN-γ was recorded (Fig. 1A). Although a significant response against the immunodominant epitope ESAT-61-20 of ESAT-6 was observed, the majority of the response was directed against the immunodominant epitope Ag85B241-255 of Ag85B, in agreement with previous studies using the adjuvant dimethyldioctadecylammonium bromide-monophosphoryl lipid A (Fig. 1B) (30, 37). To confirm this observation, the frequency of IFN-γ-producing T cells in the spleens of immunized mice was measured 2 weeks post-final vaccination. Again, after administration of 25 μg Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs, a significant increase in the immune response generated was observed, compared to mice that received antigen alone, with a level of ∼300 antigen-specific IFN-γ producing T cells per million cells recorded by in vitro ELISPOT of Ag85B-ESAT-6-stimulated cells (P < 0.01) (Fig. 1C). The combination of Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs was therefore highly immunogenic and stimulated strong responses in the blood and spleen. The concentration of 5 μg/dose CTA1-DD/ISCOMs was found to be optimal for inducing IFN-γ responses, with higher doses resulting in a reduction in the levels of IFN-γ measured and an increase in IL-5 production (data not shown).

FIG. 1.

FIG. 1.

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Dose response following i.n. administration of Ag85B-ESAT-6 in CTA1-DD/ISCOMs. C57BL/6 mice were immunized i.n. three times with 0.2 to 25 μg Ag85B-ESAT-6 alone or mixed with 5 μg CTA1-DD/ISCOMs. (A) Mean IFN-γ release ± standard error of the mean (SEM) by PBMC isolated from the blood 7 days postvaccination was measured. Cells from three or four mice were pooled and restimulated with Ag85B-ESAT-6 in triplicate wells. (B) Mean IFN-γ release ± SEM by PBMC isolated from the blood of three mice, immunized with 25 μg Ag85B-ESAT-6 mixed with 5 μg CTA1-DD/ISCOMs, 7 days postvaccination. Cells from three mice were pooled and restimulated with Ag85B241-255 or ESAT-61-20 in triplicate wells. (C) At 21 days post-final vaccination, cells isolated from the spleen were restimulated with 5 μg/ml Ag85B-ESAT-6 and the number of specific IFN-γ-secreting cells per million was assessed by ELISPOT. Values are the means of duplicate values for four individual mice ± SEM. Results are representative of three independent experiments. **, P < 0.01, ANOVA and Tukey's test.

Using the optimal combination of 25 μg Ag85B-ESAT-6 mixed with 5 μg CTA1-DD/ISCOMs, we next investigated the distribution and quality of the immune response induced by the vaccine Ag85B-ESAT-6 mixed with CTA1-DD-ISCOMs. One week post-final vaccination, the combination of Ag85B-ESAT-6 and CTA1-DD-ISCOMs was found to induce a notable recall response in the cervical lymph nodes draining the nasal cavity (Fig. 2A). No IFN-γ release was observed in the nondraining inguinal lymph nodes (data not shown). The high secretion of IFN-γ in the draining lymph nodes was also reflected by levels of ∼7,000 pg/ml IFN-γ in the supernatants of Ag85B-ESAT-6-stimulated PBMC isolated from the spleen (Fig. 2A). Measurements of the total serum Ig levels indicated that a significant humoral response was also induced upon vaccination. Notably, in three independent experiments, a mean log10 titer of IgA in bronchial lavages of 1.8 ± 0.15 was detected (Fig. 2B). Furthermore, while high levels of IFN-γ secretion by antigen-specific T cells in the blood 3 weeks post-final vaccination were recorded, very low levels of IL-5 were detected, indicating that the vaccine primarily induced a Th1 response (Fig. 2C).

FIG. 2.

FIG. 2.

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Characterization of the immune response induced by i.n. administration of Ag85B-ESAT-6 in CTA1-DD/ISCOMs. Mice were vaccinated with i.n. three times with 25 μg Ag85B-ESAT-6 mixed with 5 μg CTA1-DD/ISCOMs (C/I). (A) One week post-final vaccination cervical lymph nodes and spleens were harvested and restimulated with Ag85B-ESAT-6 in triplicate wells, and IFN-γ release was assessed by ELISA. Cells were pooled for five mice per group. Values represent means ± standard errors of the means (SEM) of triplicate values. (B) Total Ig and IgA were measured in serum and bronchial lavage samples, respectively. (C) IFN-γ and IL-5 responses of PBMC isolated from the blood 3 weeks post-final vaccination. Mean ± SEM values are indicated. Results are representative of three independent experiments. ***, P < 0.001, ANOVA and Tukey's test.

Boosting prior BCG immunity with Ag85B-ESAT-6 and CTA1-DD/ISCOMs.

Having demonstrated that vaccinating mice i.n. with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs promoted an effective immune response, we next examined the ability of this vaccine to boost prior BCG-induced immunity. Mice were vaccinated s.c. with BCG and rested for 8 months. Thereafter, the mice received two booster vaccinations, at a 2-week interval, with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs. One week following the first booster vaccination, the immune response in the blood was assessed (Fig. 3A). Mice that had not received a prior BCG vaccination demonstrated only a minor recall response to Ag85B-ESAT-6 1 week after a single dose of Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs. Mice that had not received a booster vaccination also showed limited recognition of Ag85B-ESAT-6 (despite Ag85B also being produced by BCG), as did mice that were boosted with CTA1-DD/ISCOMs alone. However, a notable recall response to Ag85B-ESAT-6 (15,498 pg/ml) was observed in BCG-vaccinated mice that had received a booster vaccination with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs. This finding was paralleled by high levels of IFN-γ-producing cells specific for the immunodominant epitope Ag85B241-255 in the blood (37) (Fig. 3B). Thus, in the spleen the number of IFN-γ-producing T cells specific for Ag85B241-255 was higher in the group that received Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs (P < 0.05) than for the other groups (Fig. 3C). We subsequently employed intracellular cytokine FACS analysis to determine the phenotype of the IFN-γ-secreting T cells 1 week after the first booster vaccination. This analysis revealed that the immune response induced by boosting already BCG-immune mice with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs led to an ∼3-fold increase in the number of IFN-γ-secreting antigen-specific CD4-positive T cells. While an increase in the frequency of IFN-γ-secreting antigen-specific CD8-positive T cells was also observed, only the CD4-positive T cells released greatly enhanced levels of IFN-γ (Fig. 3D). No increase in IFN-γ-secreting antigen-specific T cells above the levels recorded for naïve mice was detectable for nonboosted mice or mice boosted with adjuvant alone using this technique (data not shown).

FIG. 3.

FIG. 3.

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Intranasal administration of Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs boosts BCG-generated immunity. Mice were vaccinated subcutaneously with BCG or left untreated, rested for 8 months, and subsequently boosted i.n. with CTA1-DD/ISCOMs alone or Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs (C/I). Groups of naïve or nonboosted mice were also included. (A and B) One week post-first boost, PBMC were isolated from four mice to measure IFN-γ recall responses following in vitro restimulation with Ag85B-ESAT-6 (A) and the number of IFN-γ-secreting Ag85B241-255-specific cells (B). Mean values ± standard errors of the means (SEM) are shown. (C) The number of Ag85B241-255-specific cells in the spleen was simultaneously measured. Mean triplicate values for three individual mice ± SEM are shown. *, P < 0.05, ANOVA and Tukey's test. (D) FACS analysis of IFN-γ expression by CD4 and CD8 spleen cells from mice boosted with Ag85B241-255 mixed with CTA1-DD/ISCOMs and restimulated overnight in vitro as indicated. Cells were pooled from five mice per group. The figure shows the percent IFN-γ-positive cells. Results are representative of three independent experiments.

In order to further assess the quality of the immune response, we compared the recognition of Ag85B-ESAT-6 and TB10.4 6 weeks after the second booster vaccination by analyzing PMBC isolated from the blood. TB10.4 is expressed by BCG but is not part of the Ag85B-ESAT-6 antigen component of the vaccine and was included as a control for the specificity of the booster vaccinations. While Ag85B-ESAT-6 was strongly recognized following boosting with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs, as characterized by a significantly greater secretion of IFN-γ (6,845 pg/ml ± 1,366) (Fig. 4), compared to the nonboosted group or those mice boosted with adjuvant alone (P < 0.01), the recognition of TB10.4 was limited and was the same in boosted and nonboosted groups. This indicates that the booster vaccine of Ag85B-ESAT-6 mixed with CTA1-DD-ISCOMs boosted only the BCG-induced Ag85B-specific T cells.

FIG. 4.

FIG. 4.

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Recall responses to Ag85B-ESAT-6 and TB10.4. Mice were vaccinated s.c. with BCG, rested for 8 months, and subsequently boosted i.n. with CTA1-DD/ISCOMs alone or Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs (C/I). Groups of naïve or nonboosted mice were also included. The in vitro recognition of Ag85B-ESAT-6 (black bars) and TB10.4 (white bars) by cells isolated from the spleen 1 week post-first booster vaccination is shown. Mean triplicate values for three individual mice ± the standard error of the mean are shown. The results for one representative experiment of three independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (ANOVA and Tukey's test).

Ten weeks post-first booster vaccination, the mice received an aerosol challenge with virulent M. tuberculosis Erdman. One week after challenge, the recruitment of Ag85B-ESAT-6 T cells in the lungs and spleens was assessed by ELISPOT. Boosting with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs resulted in an accelerated recruitment of Ag85B-ESAT-6-specific T cells (1,405 ± 64 per 106 cells) compared to naïve, nonvaccinated challenge mice (P < 0.01) (Fig. 5A). Negligible levels of IFN-γ-secreting Ag85B-ESAT-6-specific T cells were found in the lungs 1 week prechallenge (data not shown). Furthermore, a significantly lower number of IFN-γ-secreting Ag85B-ESAT-6-specific T cells was observed in the spleen compared to the lungs in the boosted group (P < 0.05) (Fig. 5A). Again, as a control for the nonspecific boosting of prior BCG-induced immunity with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs, we determined the number of TB10.4-specific T cells recruited to the lungs and spleen following challenge. Since TB10.4 is expressed by BCG and M. tuberculosis but is not part of the vaccine construct, these levels would not be expected to be affected by the booster vaccine. As expected, the levels of TB10.4-specific IFN-γ-secreting T cells in the lungs and spleen of BCG-vaccinated mice that had or had not received a booster vaccination were directly comparable, and no significant difference between recruitment to the lungs and spleen was observed (Fig. 5B). The recruitment of IL-4-secreting Ag85B-ESAT-6- and TB10.4-specific T cells to the lungs and spleen was negligible in both the boosted and nonboosted groups (data not shown). Therefore, boosting the BCG response with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs resulted in enhanced recruitment of Th1 CD4 cells to the site of infection, the lungs, following an aerosol challenge with virulent M. tuberculosis Erdman.

FIG. 5.

FIG. 5.

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Postchallenge recruitment of antigen-specific T cells to the lungs and spleen. Mice were vaccinated s.c. with BCG, rested for 8 months, and subsequently boosted i.n. with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs (C/I). Groups of naïve or nonboosted mice were also included. Six weeks post-final booster vaccination, mice received a live M. tuberculosis challenge via the aerosol route. One week after challenge the numbers of Ag85B-ESAT-6-specific (A) and TB10.4-specific (B) IFN-γ-secreting cells in the lungs and spleen were analyzed by in vitro ELISPOT. Values are the means of triplicate values for three individual mice ± the standard errors of the means. The results for one representative experiment of three independent experiments are shown. *, P < 0.05; **, P < 0.01 (ANOVA and Tukey's test).

In order to determine whether the enhanced recruitment of Ag85B-ESAT-6-specific Th1 CD4 cells translated into enhanced protection against TB challenge, mice were challenged with virulent M. tuberculosis. Six weeks postchallenge, the bacterial numbers in the lungs and spleen were assessed. In the lungs of BCG-vaccinated mice boosted with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs, a significant reduction of 0.56 log10 CFU in bacterial numbers (P < 0.001) was recorded compared to nonboosted BCG-vaccinated mice (4.41 log10 ± 0.07 versus 4.97 log10 ± 0.05) (Fig. 6 A). Boosting BCG with the adjuvant CTA1-DD/ISCOMs alone also resulted in a slight reduction in bacterial numbers in the lungs compared to nonboosted mice. However, this difference was not significant. A significant difference between the protective effect of boosting with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs and boosting with adjuvant alone was recorded (P < 0.001). Moreover, only mice that had been boosted with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs showed a significant decrease in bacterial numbers compared to BCG-vaccinated mice (P < 0.001). It should be noted that a minor protective effect of 0.15 ± 0.012 log10 CFU reduction in the lung was recorded for mice administered two doses of Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs without receiving a prior BCG vaccination (data not shown). A similar pattern was observed in the spleen. Intranasal boosting of BCG with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs therefore resulted in an enhanced recruitment of IFN-γ-secreting T cells to the site of infection, leading to a significantly increased protection against M. tuberculosis compared to that afforded by BCG alone.

FIG. 6.

FIG. 6.

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Boosting BCG with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs leads to increased protection against subsequent infection with M. tuberculosis. Mice were vaccinated s.c. with BCG, rested for 8 months, and subsequently boosted i.n. with CTA1-DD/ISCOMs alone or Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs (C/I). Groups of naïve or nonboosted mice were also included. Six weeks post-second boost, mice received an aerosol challenge with live M. tuberculosis. Six weeks postchallenge the bacterial loads in the lungs (A) and spleen (B) were measured. Results are the individual log10 CFU value for individual mice (symbols) and the mean values for the mice in each group (bars). Results are representative of three independent experiments. *, P < 0.05; ***, P < 0.001 (ANOVA and Tukey's test).

DISCUSSION

The present study demonstrates the potential of the combined CTA1-DD/ISCOMs adjuvant vector for the stimulation of strong protective immunity against TB. The administration of Ag85B-ESAT-6 alone did not promote specific immunity, as evidenced by the weak in vitro recall response to the antigen. In contrast, the i.n. administration of Ag85B-ESAT-6 mixed with an optimal dose of 5 μg CTA1-DD/ISCOMs resulted in the induction of a significant immune response comprising both cell-mediated and humoral immunity. The cell-mediated component was dominated by CD4+ T cells and the release of the Th1 cytokine IFN-γ. Thus, in agreement with recent studies using the PR8-influenza virus antigens, CTA1-DD/ISCOMs promoted a strong Th1 response against the Ag85B-ESAT-6 antigen (21). Importantly, i.n. immunization augmented both strong systemic as well as local mucosal immune responses.

It is widely accepted that the BCG vaccine is the “gold standard” vaccine against which new vaccines should be compared and tested. However, while the BCG vaccine was developed in the 1920s and is still in widespread use today, it has a limited protective efficacy of 0 to 85% and is largely ineffective at preventing TB in adults. Indeed, the BCG vaccine is at best believed to provide effective protection against TB for 10 to 15 years, after which its protective efficacy wanes (9, 14). The development of a novel vaccine to supplement BCG vaccination is therefore much warranted. There have been several attempts to boost prior BCG immunity with an i.n. vaccine. Haile et al. met with limited success in boosting prior BCG immunity delivered via the s.c. route, followed by i.n. administration of either heat-killed BCG or arabinomannan-tetanus toxoid conjugate mixed with the Eurocine adjuvant. Although a significant reduction in the bacterial load in the spleen compared to nonboosted BCG controls was documented, no significant reduction in the lungs was observed (20). It should be noted that in this study mice were challenged by intravenous administration of M. tuberculosis H37Rv. In contrast, in a study by Goonetilleke et al., boosting i.n.-administered BCG with either the i.n. administration of a second boost of BCG or the recombinant modified vaccinia virus Ankara expressing M. tuberculosis Ag85A resulted in a protective effect in the lungs and spleen that was significantly greater than that afforded by a single BCG immunization (17). Moreover, Santosuosso et al. recently showed that BCG immunity could be boosted with an intranasal-administered recombinant adenovirus-based TB vaccine expressing Ag85A, resulting in increased protection against TB (35).

In the present study we successfully demonstrated that the i.n. vaccine based on Ag85B-ESAT-6 and the CTA1-DD-ISCOMs adjuvant vector effectively boosted prior BCG immunity. Using Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs to immunize mice that had received a previous BCG vaccination resulted in a strong boosting response characterized by the induction of Ag85B-ESAT-6-specific IFN-γ-secreting T cells and the release of IFN-γ. Very low IFN-γ release was observed following boosting with adjuvant alone or in mice that had not received a booster vaccination or a prior BCG vaccination, or in those that had received an i.n. boost with BCG or antigen alone (data not shown). FACS analysis confirmed this finding and revealed that CD4-positive T cells were the primary cell type to have been boosted. This was important to determine, as CD4-positive T cells play a crucial role in controlling TB infection (19). The observed correlation between IFN-γ-secreting cells recruited to the lungs and the protection against TB is in agreement with recent studies using an intranasal-administered recombinant adenovirus-based TB vaccine expressing Ag85A (35, 36). Interestingly, these studies also revealed that airway luminal T cells were of particular importance for the protection against TB irrespective of whether the vaccine was used as a prophylactic vaccine (36) or a BCG booster vaccine (35).

No increase in the release of IFN-γ in response to in vitro restimulation with TB10.4 was observed as a result of boosting with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs, demonstrating that the booster vaccination only boosted BCG-induced Ag85B-specific T cells, as would be anticipated given that TB10.4 is not part of the antigenic fusion protein used in this booster vaccine. Furthermore, poor responses were recorded against ESAT-6 in contrast to Ag85B, suggesting that the majority of the response was targeted against the immunodominant antigen Ag85B (23).

Following a challenge with live M. tuberculosis Erdman via the aerosol route, we observed a significant enhancement in the levels of recruitment of Ag85B-ESAT-6-specific IFN-γ-secreting CD4+ T cells to the lungs of infected mice boosted with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs. Recruitment was also more pronounced in this group than in unvaccinated or nonboosted mice or mice that had been boosted with adjuvant alone (data not shown). Interestingly, Ag85B-ESAT-6-specific IFN-γ-secreting T cells were recruited to the lungs in significantly greater numbers than to the spleen. This suggests that vaccination via the mucosal route does indeed target effector cells to the mucosal surfaces. Notably, very few antigen-specific IL-4-secreting T cells were recruited, further indicating that the vaccine induced a Th1-dominated immune response. No difference in the numbers of IFN-γ-secreting TB10.4-specific T cells was recorded between the groups, and very few IL-4-specific Ag85B-ESAT-6- or TB10.4-specific T cells were recruited. Importantly, only boosting with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs promoted a significant protective effect over that afforded by BCG alone, and the difference in protection observed between the CTA1-DD/ISCOMs or Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs boosted groups was significant. A similar pattern was observed in the spleen. As a prophylactic vaccine, Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs only induced a minor protective effect against infection with M. tuberculosis (data not shown), indicating that the optimal use for this vaccine is as a BCG booster vaccine.

Thus, in this study we have demonstrated that i.n. administration of the antigen Ag85B-ESAT-6 mixed with CTA1-DD-ISCOMs as adjuvant invokes a Th1 immune response and enhances the preferential recruitment of antigen-specific IFN-γ-secreting T cells to the lungs following a live M. tuberculosis challenge. When used to boost prior BCG-induced immunity, Ag85B-ESAT-6 mixed with CTA1-DD-ISCOMs induced significantly greater protection than that afforded by BCG alone and therefore represents a suitable vaccine candidate for i.n. boosting of BCG. Furthermore, given that a Th1 response is crucial for the optimal response against TB and that CTA1-DD-ISCOMs can induce such a response in even a Th2-biased mouse strain, this indicates that the combined CTA1-DD/ISCOMs adjuvant may be effective as a mucosal adjuvant in TB vaccines for use in a mixed human population (21).

Acknowledgments

This work was supported in part by the Swedish Research Council, the Swedish Cancer Foundation, the Sahlgrenska University Hospital Foundation, and EU grants QLK2-CT-2001-01702, QLK2-CT-199-00228, and LSHP-CT-2003-503240.

We thank Charlotte Fjordager, Lars Pedersen, Birgitte Smedegaard, Lene Rasmussen, Tina Lerche, and Karin Schön for excellent technical assistance.

Editor: F. C. Fang

Footnotes

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Published ahead of print on 30 October 2006.

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