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. 2001 Dec;126(3):482–487. doi: 10.1046/j.1365-2249.2001.01708.x

Comparative protective effects of recombinant DNA and Mycobacterium bovis bacille Calmette–Guérin vaccines against M. avium infection

E Martin *,†, J A Triccas *, A T Kamath *, N Winter ‡, W J Britton *,§
PMCID: PMC1906218  PMID: 11737066

Abstract

A range of strategies are being explored to develop more effective vaccines against mycobacterial infection, including immunization with DNA plasmids encoding single mycobacterial bacterial genes and the use of recombinant live vectors based on the current vaccine, Mycobacterium bovis bacille Calmette–Guérin (BCG). We have compared these two approaches using a model of virulent M. avium infection, and the gene for the immunodominant 35 kDa protein which is shared by M. avium and M. leprae, but absent from BCG. Recombinant BCG over-expressing the M. avium 35 kDa protein (BCG-35) induced strong antigen-specific proliferative and interferon-γ (IFN-γ)-secreting T cell responses. These were comparable to those induced by a single immunization with a plasmid expressing the same antigen (DNA-35); however, repeat DNA-35 immunization evoked the strongest IFN-γ release. Immunization with BCG-35 significantly reduced the growth of virulent M. avium, although this effect was similar to that induced by wild-type BCG. Immunization with DNA-35 resulted in significantly greater (2 × log10) reduction in the growth of M. avium. Prime-boost strategies combining DNA-35 and BCG-35 increased the protective effect above that achieved by BCG-35, but they were not more protective than DNA-35 alone. Therefore, recombinant BCG-35 and BCG induced similar levels of protection in this model, and maximal protection against M. avium infection was attained by immunization with DNA encoding the 35 kDa protein.

Keywords: DNA vaccination, 35 kD antigen, Mycobacterium avium, protection, recombinant BCG

Introduction

Mycobacterium avium complex (MAC) is the most common bacterial infection of patients with AIDS in the United States, leading to substantial morbidity and mortality [1]. The HIV/AIDS epidemic has contributed dramatically to the increase in MAC disease in recent years; however, the number of non-AIDS-related MAC infection has also been increasing, particularly in the older female population [2]. Treatment for M. avium infection requires multiple drug therapy (MDT) [3] but is becoming increasingly difficult to treat because of its natural resistance to many antibiotics. BCG reduces the incidence of M. avium infection in humans [4]. However, BCG offers only moderate levels of protection in animal models [5,6]. A more effective vaccine combined with MDT may contribute to the control of M. avium infections.

Recent new vaccine strategies against mycobacterial infection include subunit vaccines such as DNA vaccines [6–9] and recombinant or attenuated mycobacterial vectors [10,11]. DNA vaccination against M. tuberculosis infections has provided encouraging results. However, the level of protection ranged from 0·5 to 1·0 log10 and was less than that achieved with BCG alone [7,8,12]. BCG-based vaccine delivery systems share the advantages of BCG, including low incidence of serious side effects, safety for immunization of infants, unique adjuvant potential [13] and in particular production of long-lasting immunity following a single dose immunization [10]. A variety of viral, bacterial and parasitic antigens have been now successfully expressed in BCG [14–18]. In experimental models, recombinant BCG (rBCG) expressing foreign antigens elicited protective immunity against Lyme's disease [19], pneumococcal infection [20] and cutaneous leishmaniasis [21,22]. Recombinant BCG expressing mycobacterial antigens induced protective immunity against a heterologous mycobacterial infection [23]. Immunization schedules based on priming with DNA vaccines and boosting with recombinant viral vectors enhanced protective immune responses to viral and protozoal antigens [24]. Therefore we investigated immunization strategies with BCG (BCG-35) and plasmid DNA (DNA-35) expressing the 35 kD protein of M. avium, a major antigen component of both M. leprae and M. avium which is absent from BCG [25,26].

Materials and methods

Bacteria

The M. avium isolate used was a virulent strain of serotype 8 isolated from an AIDS patient [27], kindly provided by Dr C. Cheers (University of Melbourne, Victoria, Australia). It was grown in Middlebrook 7H9 broth with ADC supplement (Difco Laboratories, Detroit, MI, USA). For manipulation of plasmids, Escherichia coli strain MC1061 was utilized and grown in LB broth or agar supplemented with ampicillin (100 µg/ml) as required. For large-scale plasmid purification, the transformed bacteria were grown in Circlegrow broth (BIO 101, Vista, CA, USA) supplemented with ampicillin [7].

Construction of recombinant BCG strains and DNA vaccines

Plasmid pAJ11 [25] was used to transform BCG CSL by electroporation (Biorad Gene-Pulser, Richmond, USA) to produce BCG expressing the M. avium 35 kD antigen. Individual kanamycin (Km)-resistant colonies were inoculated into 7H9 medium and grown for 3 weeks with shaking at 37°C. Cell-free extracts were obtained by sonication of bacterial cells at 50 W for 5 min five times on ice. Twenty µg of sonicate was separated by 12% SDS-PAGE, stained with 0·5% Coomassie brilliant blue G-250 (Bio-Rad) and following immunoblotting, performed with murine anti-M. leprae 35 kDa protein MoAb CS-38, a kind gift of Professor P. J. Brennan (Colorado State University, Fort Collins, CO, USA).

The vector pJW4303, kindly provided by Dr J. I. Mullins, University of Washington, Seattle, WA, USA, contains the cytomegalovirus (CMV) early immediate promoter with intron A upstream of the gene of interest, and a bovine growth hormone polyadenylation sequence downstream. The gene for the M. avium 35 kDa protein was amplified from the plasmid pAJ11 [25] and cloned into pJW4303 as described previously [29].

Immunization of animals

C57BL/6 female mice were supplied as SPF mice by ARC (Perth, Australia) and were maintained in SPF conditions. Mice were immunized between 8 and 12 weeks of age with 50 µg of each plasmid by intramuscular injection (i.m.) into the tibialis anterior muscle of each hindleg. Mice were immunized at 2-weekly intervals with one or three doses of DNA-35. For the prime-boost studies mice were immunized with DNA-35 followed by BCG-35 2 weeks later, administered i.v. as 1 × 105 CFU (DNA35-BCG35) or vice versa (BCG35-DNA35). Control mice were immunized with DNA-Neg, BCG-35 alone (1 × 105 CFU) or BCG alone (1 × 105 CFU i.v.). All injections were timed such that groups received the last injection at the same time.

Antibody determination

Mice were bled sequentially following immunization and the presence of antigen-specific MoAbs determined by enzyme-linked immunosorbent assay (ELISA) as described previously [25] using recombinant mycobacterial proteins (at 10 µg/ml) and alkaline phosphatase-conjugated goat antimurine immunoglobulin G (IgG) (Sigma). The recombinant M. avium 35 kD protein was purified by MoAb affinity chromatography as described previously [25].

Lymphocyte proliferation and cytokine assays

The inguinal, axillary and para-aortic lymph nodes (LN) and the spleen were collected from immunized mice and single cell suspensions prepared. T lymphocytes were enriched by passing leucocytes through a nylon wool column. Cells were incubated in triplicate with varying concentrations of antigen, concancavalin A (Sigma) or media alone as described previously [29]. Specific [3H]thymidine incorporation was calculated by subtracting the mean counts per minute (cpm) in control wells (media) from the mean cpm of test samples. In similar cultures, supernatants were collected after 48 h and stored at −20°C for later cytokine analysis. IFN-γ was detected by capture ELISA with the MoAbs R46A2 and biotinylated XMG 1·2 (Endogen, Woburn, MA, USA) using murine rIFN-γ as standard (5·08 × 106 U/mg; Genzyme, Cambridge, MA, USA). The limit of detection of the assay was 0·4 U/ml.

Mycobacterium avium challenge

Twelve weeks after the last boost mice were infected by an intravenous (i.v.) challenge with 1 × 106 CFU M. avium. Mice were sacrificed at 4 weeks after the infection and the number of bacteria in the spleen homogenates were enumerated on Middlebrook 7H11 Bacto Agar.

Data analysis

Statistical analysis of the experimental data and controls was conducted by analysis of variance (anova). Fisher's protected least significant difference anova post hoc test was used for pair-wise comparison of multi-grouped data sets.

Results

Expression of the M. avium 35 kD protein in M. bovis BCG

To permit expression of gene encoding the M. avium 35 kD protein in BCG, plasmid pAJ11 was introduced into the BCG CSL strain to yield BCG-35. Plasmid pAJ11 expresses the M. avium 35 kD protein-encoding gene under the control of the strong M. fortuitum pBlaF* promoter [25]. In sonic extracts of BCG/pAJ11, a band with Mr of 35 kD was detectable by SDS-PAGE and was reactive with MoAb CS-38 following immunoblotting (Fig. 1).

Fig. 1.

Fig. 1

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Expression of the 35 kD protein in BCG. To permit expression of the gene encoding the M. avium 35 kD antigen in BCG, the plasmid pAJ11 was electroporated into BCG CSL. Sonic extracts of BCG, 10 µg (lane 1), and 5, 10, 15 and 20 µg of rBCG-35 (lanes 2–5) and r35 kD antigen, 5 µg (lane 6) were analysed by SDS-PAGE and immunoblotting with anti-35 kD MoAb (CS-38).

Immune responses after vaccination with BCG-35 or DNA-35

Mice were immunized i.v. with BCG-35 and 3 weeks later anti-35 kDa antigen IgG antibody responses measured. All mice mounted high antigen-specific IgG titres when compared to BCG alone (Fig. 2a). Splenocytes from BCG-35 immunized mice proliferated (Fig. 2b) and produced IFN-γ (Fig. 2c) in response to the 35 kDa antigen. Similar responses were observed within LN cells (data not shown). These antibody and cellular responses were similar to those when mice were vaccinated once with DNA expressing the 35 kD antigen (Fig. 2). Mice vaccinated with three doses of DNA-35 mounted the highest antibody titres, and strongest T cell proliferation and IFN-γ responses (Fig. 2). These results indicate that the 35 kD protein is recognized by the immune system when expressed in BCG, and the magnitude of the response is equivalent to that achieved when delivered in the form of a DNA vaccine.

Fig. 2.

Fig. 2

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Induction of antigen-specific immune responses by immunization with DNA or rBCG vaccines expressing the M. avium 35 kDa antigen. Data represent (a) the mean IgG antibody titres (n = 5), and the proliferative (b) and IFN-γ (c) responses of spleen-derived lymphocytes from mice (n = 5) immunized with either DNA-35 or BCG-35 constructs or control DNA or BCG vaccine as shown. These data are representative of three separate experiments.

Protection afforded by BCG expressing the 35 kDa antigen

The protective efficacy of BCG expressing the 35 kD antigen was assessed by measuring the bacterial load in immunized mice 4 weeks after M. avium challenge. BCG-35 vaccinated mice contained significantly fewer bacteria in their spleens than control animals; however, this level of protection was similar to that achieved with BCG lacking the 35 kD antigen (Fig. 3). This effect was significantly less than afforded by DNA immunization, as recipients of DNA-35 displayed a 2 log10 reduction in M. avium in the spleen (Fig. 3). Therefore the expression of the 35 kD protein did not improve the protective efficacy of BCG against M. avium despite the presence of a robust immune response directed against the antigen.

Fig. 3.

Fig. 3

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The effect of immunization with DNA-35 or BCG-35 on the protective efficacy against challenge with virulent M. avium. Twelve weeks following immunization with the schedule as shown, mice (n = 5) were challenged with M. avium 1 × 106 CFU i.v. Four weeks post-infection, bacteria in the spleen were enumerated (mean ± s.e.m.). The significance of differences between DNA-Neg immunized animals and other vaccination protocols (*P < 0·01, **P < 0·001) and between 3 × DNA-35 and BCG-immunized groups (†P < 0·05) were determined by anova Fisher test. Data are representative of three separate experiments.

Prime-boost strategies using DNA and BCG expressing the 35 kD antigen

Immunization schedules involving priming with DNA vaccines and boosting with recombinant viral vectors have been successful for enhancing protective immunity against pathogens [24]. Therefore we investigated if priming with DNA-35, boosting with BCG-35, or the reverse combination could comprise a more effective immunization schedule for protection against M. avium infection. Priming with DNA-35 and boosting with BCG-35 provided no increase in immunogenicity compared to DNA-35 or BCG-35 immunization alone, as determined by specific IgG production (Fig. 2a), T cell proliferation (Fig. 2b) or IFN-γ release (Fig. 2c). Thus there appeared to be no additive effect of priming with DNA-35 and boosting with BCG-35 (or vice versa) on anti-35 kD antigen antibody and T cell responses. To determine whether prime-boost strategies have the potential for conferring greater levels of protection mice were immunized with the same protocols as for immunogenicity studies and challenged with virulent M. avium 12 weeks after the last boost. The combination of DNA-35 with BCG-35 significantly increased the protective effect above that of BCG-35 alone (Fig. 3). This effect, however, was not greater than that of DNA-35 alone (Fig. 3). The order of immunization in regard to DNA-35 and BCG-35 was not important for optimizing protection against M. avium infection.

IFN-γ production following DNA or rBCG-35 immunization and M. avium infection

T cell-derived IFN-γ is vital for the activation of macrophage killing mechanisms. To determine whether immunization with 35 kD antigen delivery vectors confers greater protection through the enhanced release of IFN-γ, the immune responses following vaccination and subsequent M. avium infection was examined. Immunization with either DNA-35 or BCG-35 resulted in a more rapid antigen-specific recall proliferative response (data not shown) and stimulated antigen specific production of IFN-γ (Table 1). There was no significant difference between mice receiving the 35 kD antigen via DNA or BCG vectors. Consistent with the protection results there was also no significant difference between mice immunized with DNA-35 followed by BCG-35 or vice versa (Table 1). Mice which received BCG not expressing the 35 kD antigen were not primed for enhanced anti-35 kD antigen T cell responses after M. avium infection.

Table 1.

Immunization with DNA-35 or BCG-35 primes for specific IFN-γ T-cell responses following challenge with M. avium

In vitro restimulation with r35 kDa antigen
Group IFN-γ (U/ml) ± s.e.
DNA-35 227·8 ± 18·58*
BCG-35 199 ± 9·62*
BCG35-DNA35 215·6 ± 16·53*
DNA35-BCG35 219·4 ± 10·06*
DNA-Neg 109·8 ± 18·76
BCG 62·8 ± 13·06
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*

Splenocytes from mice immunized and challenged as described in Fig. 3 were restimulated with r35 kD antigen (10 µg/ml) for 48 h and the level of IFN-γ produced measured by ELISA. Data represent the mean IFN-γ production (U/ml) ± s.e. for five mice per group, each tested separately, from one of three separate experiments. The levels of statistical significance of the difference between DNA-35, BCG-35 or combinations of both and the DNA-Neg groups were determined by anova Fisher test where P ≤ 0·001.

Discussion

Several approaches to more effective antimycobacterial vaccines are currently being explored, including subunit vaccines utilizing secreted proteins of M. tuberculosis [30], DNA vectors [6–8,12] viable vectors such as auxotroph mutants of M. tuberculosis and BCG [11], live recombinant BCG [23] and viral vectors [31]. The immunogenicity and protective efficacy of these vaccines must be compared and new strategies for their utilization examined in order to develop more effective vaccines than BCG. We selected the 35 kD protein as a model antigen to compare recombinant BCG and DNA plasmids as vectors for three reasons. First, the M. leprae 35 kD protein is immunodominant with > 90% of leprosy patients displaying T cell or antibody responses to the protein [26], while its closely related M. avium homologue is recognized during experimental M. avium infection [25]. Secondly, the gene for 35 kD protein is absent from the BCG and M. tuberculosis genomes [26]. Therefore, comparison of recombinant BCG-35 with wild-type BCG will determine whether the expression of an immunodominant mycobacterial gene by BCG can increase protective immunity against infection with another mycobacterium. Thirdly, we have demonstrated recently that DNA vaccines expressing M. avium or M. leprae 35 kD gene are protective against M. avium and M. leprae infection in mice [29,32]. Therefore the relative protection conferred by BCG and DNA vaccines could be compared directly and combination vaccine strategies evaluated.

When the M. avium 35 kD gene was expressed under the control of pBlaF* promoter, relatively high levels of 35 kD protein were produced (Fig. 1). The BCG-35 was immunogenic leading to the rapid development of anti-35 kD antibodies and strong antigen-specific IFN-γ-secreting T cell responses, similar to those achieved by a single dose of DNA vaccine expressing the same antigen (Fig. 2). The protective effect against progressive M. avium infection, however, was similar for BCG-35 and wild-type BCG (Fig. 3). Therefore, the induction of T cell responses to the immunodominant 35 kD protein by BCG-35 did not confer greater protection than the T cell responses induced by BCG alone. Although BCG has been an efficient vehicle for the delivery of a variety of heterologous antigens [19–22], T cell responses to the antigens shared by BCG and M. avium appeared to stimulate the maximal protection that the mycobacterial vector could provide.

Priming with DNA-35 and boosting with BCG expressing the same antigen, or the reverse combination, significantly increased the protective effect of BCG-35 against M. avium challenge, but this did not exceed the efficacy achieved with DNA-35 alone. The initial DNA-35 priming was intended to focus the cellular immune response to the 35 kD antigen with the BCG-35 booster immunization enhancing the response by both the expression of higher levels of 35 kD protein and the immunostimulatory effects of live mycobacterial infection. However, the maximal protective effect was obtained with DNA-35 either alone or in combination with BCG-35 (Fig. 3). Several studies have demonstrated that priming with DNA vaccine followed by boosting with recombinant virus generates increased protective immunity in models of HIV [33] and malaria [24] infection. Boosting with recombinant viral vaccine is likely to cause a rapid production of antigen in the cytoplasmic compartment, which boosts the antigen-specific CD8+ T cell population resulting in the protective response required to control HIV and malaria. By contrast, boosting with recombinant BCG may result in a slow production of antigen in the phago-lysosomal compartment which stimulates antigen-specific CD4+ T cell response more slowly. This gradual effect did not lead to greater protection than the pool of antigen-specific CD4+ T cells generated by immunization with the DNA-35 vaccine. This may be influenced by the site of antigen expression. For example, the immune response elicited by rBCG was greater for some antigens when they were expressed as secreted rather than cytoplasmic proteins [19,34].

It was striking that immunization with DNA-35 conferred greater protection than BCG or BCG-35 (Fig. 3) which bear multiple cross-reactive antigens. Moreover, the level of protection against M. avium infection achieved with DNA-35 is substantially greater than the protective efficacy of DNA vaccines expressing other M. avium antigens and the efficacy of DNA vaccines against M. tuberculosis infection, which ranges from 0·5 to 1·0 log10 [7,8,12]. One explanation is that the 35 kDa protein is a strongly immunodominant antigen in M. avium infection. Following priming with either DNA-35 or BCG-35 and challenge with M. avium, 35 kD antigen-specific IFN-γ responses were significantly increased, compared to mice primed with BCG alone (Table 1). In addition, immunization with DNA-35 alone may avoid antigen antigenic competition from other components of the BCG vector. The adjuvant properties of DNA plasmid as a vehicle may also contribute to the greater efficacy of DNA-35. CpG motifs within the DNA construct stimulate production of IL-12 and other pro-inflammatory cytokines by antigen presenting cells [35] and the expansion of antigen-specific Th1-like T cells. Consequently, the pool of antigen-specific T cells induced by DNA-35 immunization may be larger and respond more rapidly to M. avium challenge than that stimulated by BCG vaccine.

Finally, the marked protective effect of DNA-35 immunization may relate to characteristics of M. avium infection, which is less virulent and more slowly progressive than M. tuberculosis [27]. DNA immunization delivers antigen to both the phago-lysosomal and cytoplasmic compartments leading to the efficient induction of CD4+ and CD8+ T cells. CD8+ T cells are less important for protection against M. avium than for the more virulent M. tuberculosis [36]. Consequently, the protective effect of DNA-35 immunization against M. avium is probably dependant on the induction of specific CD4+ T cells [29], although we cannot rule out the possibility that CD8+ T-cells generated by DNA-35 immunization may play some role in the superiority of this vaccination regime.

In the case of M. tuberculosis infection, the protective efficacy of DNA vaccines expressing a variety of M. tuberculosis secreted or heat shock proteins has usually been less than, or at best equivalent to, that of BCG alone [7–9,12]. A prime-boost strategy using BCG and DNA vaccines may therefore provide a synergistic effect and be more effective against M. tuberculosis than M. avium infection. Recently, we have observed that priming with DNA vaccine expressing the M. tuberculosis antigen 85B prior to BCG immunization did confer greater protection than BCG alone against pulmonary and systemic M. tuberculosis infection [37]. Therefore, the optimal vaccine strategy must be defined for each individual pathogen. Clearly, in the case of M. avium infection DNA immunization with a potent single antigen is an efficient means of inducing protective immunity.

Acknowledgments

This work was supported financially by the National Health and Medical Research Council of Australia and the Cooperative Research Centre for Vaccine Technology, Queensland. The support of the NSW Health Department through its research and infrastructure grants programme is gratefully acknowledged. E. Martin and A. T. Kamath are recipients of Australian Postgraduate Research Awards and E. Martin received support from the CRC-VT. J. Triccas is the recipient of a NHMRC Peter Doherty Fellowship. We thank Dr A. Bean and C. Feng for helpful discussions.

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