Oral priming of mice by recombinant spores of Bacillus subtilis

Vaccine 22 (2004) 4139–4143

Short communication

Oral priming of mice by recombinant spores of Bacillus subtilis Annalisa Ciabattinia , Riccardo Parigia , Rachele Isticatob , Marco R. Oggionia , Gianni Pozzia,∗ a

b

Laboratorio di Microbiologia Molecolare e Biotecnologia (LAMMB), Dipartimento di Biologia Molecolare, Universit`a di Siena, Policlinico Le Scotte V lotto, piano 1, 53100 Siena, Italy Dipartimento di Fisiologia Generale ed Ambientale, Sezione di Microbiologia, Universit`a Federico II, Napoli, Italy Received 27 August 2003; accepted 4 May 2004

Abstract Recombinant Bacillus subtilis spores were employed as a vaccine delivery system in a heterologous mucosal priming-parenteral boosting vaccination strategy in the mouse model. BALB/c and C57BL/6 mice were orally immunised with recombinant spores expressing tetanus toxin fragment C (TTFC) fused to the spore outer coat protein CotB, and then subcutaneously boosted with soluble TTFC (without adjuvant). Two weeks after boosting, a significantly higher serum TTFC-specific IgG response was stimulated in mice primed with recombinant spores (antibody concentration of 2600 ± 915 in C57BL/6 and 1200 ± 370 ng/ml in BALB/c) compared to mice inoculated with wild type spores (650 ± 250 and 250 ± 130 ng/ml, respectively). IgG subclass analysis showed a prevalence of IgG1 and IgG2b, indicative of a Th2 type of immune response. Oral administration of recombinant spores stimulated also a significant local TTFC-specific IgA response. These data show that recombinant spores of B. subtilis are able to prime the immune system by the oral route, and that a combined mucosal/parenteral strategy can stimulate both local and systemic antigen-specific immune responses. © 2004 Elsevier Ltd. All rights reserved. Keywords: Mucosal vaccines; Vaccine vectors; Prime-boost

1. Introduction Bacillus subtilis is a non-pathogenic aerobic Grampositive bacterium that differentiates into an endospore (spore), a metabolically quiescent and extremely resistant cell type, in response to adverse environmental conditions. The peculiar structure, including a proteinaceous bilayer coat and the low content of water, make spores extremely resistant to desiccation, high temperature and penetration by chemicals [1–3]. Spores from different Bacillus spp. have been used in several fields. A toxigenic live attenuated variant of B. anthracis spores is currently used worldwide as veterinary vaccine and has been used in humans in the former Soviet Union and China [4]. Spores of different Bacillus species are used as probiotics in animals and humans [5–7], and in some regions of Asia and Africa there is a widespread consumption of spore-based foods [8]. ∗

Corresponding author. Tel.: +39 0577 233299; fax: +39 0577 233334. E-mail address: [email protected] (G. Pozzi).

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.05.001

Physical features of spores, together with biological properties, such as safety record in humans and animals [5,6,8,9], and their ability to interact with antigen presenting cells and to stimulate cytokine release [10], make Bacillus spores extremely interesting candidates as vaccine vectors [10,11]. Furthermore, large-scale production is inexpensive, and genetic tools as well as complete genomic data are available [12]. A genetic system for the construction of recombinant B. subtilis spores expressing heterologous antigens on the spore surface was recently developed [11]. The spore coat protein CotB was used as fusion partner for the surface display of tetanus toxin fragment C (TTFC), a well-characterized and highly immunogenic model antigen [13–15]. The immunogenicity of TTFC expressed on the spore surface was demonstrated in mice immunized by the subcutaneous route [11], as well as by the oral route [16]. Recombinant B. subtilis spores are the first evidence that a heterologous protein can be expressed on the surface of a bacterial spore and points to this peculiar cell form as a novel and potentially powerful system to display bioactive molecules.

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Fig. 1. Schematic representation of recombinant spore structure. Recombinant B. subtilis spores expressing TTFC fused to the outer coat protein CotB. The genetic strategies employed [10] allows the expression of both CotB alone (grey) and CotB–TTFC fusion protein (grey–black) on the spore surface. Different layers of the spore structure are reported; the figure is not on scale.

Herein, a combined mucosal/parenteral prime-boost vaccination strategy using recombinant spores and soluble antigen, was investigated. We previously demonstrated that recombinant spores of B. subtilis strain RH103 display on their surface the CotB–TTFC fusion protein [11]. In fact, both the endogenous CotB and the CotB–TTFC fusion are actually expressed on the spore surface (Fig. 1), since CotB–TTFC is inserted into a chromosomal nonessential gene (amyE) generating a merodiploid strain for cotB [10]. Strain RH103, which was used in previous studies by the subcutaneous and the oral routes [11,16], was here tested in a prime-boost immunization strategy, where recombinant spores were used for oral priming and soluble recombinant protein as the parenteral boost.

2. Methods 2.1. Bacterial growth and spore preparation Recombinant spores of B. subtilis expressing TTFC (RH103) were obtained as previously described [11]. Sporulation of RH103 and the isogenic wild type strain PY79 was induced by modified nutrient exhaustion method [17]. Bacteria were cultured in Luria-Bertani (LB) medium for 5 h at 37 ◦ C and then inoculated (1:400) into Difco Sporulation Medium (DSM). After incubation for 24 h at 37 ◦ C in agitation, spores were collected and treated as previously described [11]. Spores were finally counted using a B¨urker chamber under an inverted light microscope (400× Nikon eclipse, TS100). 2.2. Immunization of mice and sample collection Female C57BL/6 and BALB/c mice (5 weeks old) were obtained from Charles River (Lecco, Italy) and maintained

in our animal facilities for the duration of the experiments. All animal procedures were in accordance with institutional guidelines. Mice (eight per group) were inoculated intragastrically (i.g.) with RH103 or PY79 in a volume of 200 ␮l per mouse. The i.g. inoculum was carried out using 1 ml syringe (26GA, BD Plastipak, Italy) fitted with a 0.7 mm-diameter polyethylene cannula (Intradermic Polyethylene Tubing, BD). Spores were resuspended in sterile water containing 3% skimmed milk. The i.g. immunization was performed twice (4 weeks apart) and each immunization consisted of three doses (24 h apart) of 1.5 × 1010 spores per dose; a final single dose was administered at week 5 [14]. Mice were then subcutaneously boosted with 1 ␮g per mouse of soluble TTFC (Boehringer Mannheim) without adjuvant, at week 8. Blood samples were taken at weeks 0, 7, 9, 10 and 11 and faecal samples at weeks 0 and 7. Specimens were collected as previously described [13,18] and stored at −70 ◦ C. 2.3. Antibody detection 2.3.1. Serum IgG Determination of TTFC-specific serum IgG was performed by ELISA as previously described [13]. Briefly, microtitre plates (high-binding capacity; Greiner, Frickenhausen, Germany) were coated with recombinant TTFC (1 ␮g/ml, Boehringer Mannheim), blocked with PBS and 1% BSA and then added with serum samples initially diluted 1:20 and titrated in two-fold dilutions, in duplicate. Plates were then incubated with alkaline-phosphatase-conjugate goat anti-mouse IgG1, IgG2a or IgG2b (diluted 1:1000; Southern Biotechnology Associates, Birmingham, AL) or a mix of them for assessing total IgG and developed adding p-nitrophenyl phosphate (Sigma). Absorbance was recorded at 405 nm using a 340 ATC reader (SLT Labinstrument, Austria). The TTFC-specific IgG concentration in each sample was calculated using a standard curve of anti-TTFC monoclonal antibody (Boehringer, Mannheim) starting from a concentration of 8 ng/ml and titrated in two-fold dilution in duplicate. 2.3.2. Mucosal IgA The concentration of TTFC-specific IgA was detected in faecal samples of individual animals and normalized to the total IgA concentration detected in each sample. The concentration of total and TTFC-specific IgA was determined on flat-bottomed microtitre plates (medium binding capacity, Greiner) coated with anti-mouse IgA (1 ␮g/ml, Southern Biotechnology Associates), and TTFC (1 ␮g/ml, Boehringer Mannheim), respectively. After blocking, samples were diluted 1:300 in duplicate and titrate in three-fold dilutions for total IgA, and diluted 1:2 in duplicate for TTFC-specific IgA. Plates were incubated overnight at 4 ◦ C, washed three times and alkaline-phosphatase-conjugate goat anti-mouse IgA was added (1:1000, Southern Biotechnology Associates) for 2 h at 37 ◦ C. Plates were developed by adding the substrate

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and recording the optical density at 405 nm. The concentrations of total and TTFC-specific IgA were calculated against a standard curve of mouse myeloma IgA determined on the same plate. Results were expressed as ␮g of TTFC-specific IgA per mg of total IgA detected in each sample. 2.4. Data analysis Samples were tested individually, and values were expressed as the arithmetic mean ± S.E.M. (standard error of the mean). The statistical significance of the difference between group means was assessed by unpaired Student’s t-test, and the threshold was set at ∗ P ≤ 0.05 or ∗∗ P ≤ 0.01. Statistical analysis was performed with Prism 4 software (GraphPad Software Inc., San Diego, USA).

3. Results and discussion Groups of C57BL/6 and BALB/c mice were immunized by the oral route with recombinant spores of TTFCexpressing strain RH103, and with isogenic control strain PY79. With the exception of one individual, animals immunized with recombinant spores did not show a significant TTFC-specific IgG response in serum, whereas a significant TTFC-specific IgA response in the gut was shown both for C57BL/6 (P = 0.04), and BALB/c (P = 0.01) mice (Fig. 2). Three weeks after the last dose of spores, mice were boosted by the subcutaneous route with soluble TTFC without adjuvant, and the serum IgG response was followed over 3 weeks. Two weeks after boosting, a significantly higher serum TTFC-specific IgG response was stimulated in mice primed with recombinant spores (antibody concentration of 2600 ± 915 in C57BL/6 and 1200 ± 370 ng/ml in BALB/c) compared to mice inoculated with wild type spores (650 ± 250 and 250 ± 130 ng/ml, respectively). The antibody concentration was indeed four and five times higher than control in C57BL/6 and BALB/c mice (P = 0.022 in BALB/c mice) (Fig. 3A). In the following week, TTFC-specific IgG levels were still higher

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in mice primed with recombinant spores (2971 ± 918 ng/ml in C57BL/6, and 2485 ± 518 ng/ml in BALB/c) compared to control mice (1070 ± 465 and 580 ± 144 ng/ml, respectively), and the difference between primed and unprimed mice was still statistically significant for BALB/c mice (P = 0.005) (Fig. 3A). The IgG subclass analysis, performed on pooled sera collected 3 weeks after TTFC injection (week 11), showed IgG1 and IgG2b prevalence in C57BL/6 and IgG1 in BALB/c mice suggesting a Th2 type of immune response (Fig. 3B). The use of heterologous priming/boosting vaccination is an attractive strategy to generate stronger immune responses than either route alone [19,20]. Parenteral prime-mucosal boost [21,22], or mucosal prime-parenteral boost [23,24] have been assessed in order to stimulate the best immunity against different pathogens. The mucosal prime-parenteral boost strategy is generally obtained with live replicating delivery systems [23] or with soluble antigens co-administered with potent mucosal adjuvant, such as cholera toxin (CT) or heat-labile enterotoxin of Escherichia coli (LT), or their derivatives [20,24,25]. Nevertheless, these adjuvant toxins are not licensed in humans because of their toxicity, and the use of soluble antigens alone tends to establish immunotolerance rather than immunity. In our experiment we have demonstrated the induction of both local and systemic immune responses by employing a metabolically quiescent delivery system – the recombinant spore – for priming the immune system, and a soluble antigen without adjuvant for boosting. Previously, we demonstrated that spores do not germinate after oral administration, but transit through the murine intestine and are excreted in the faeces [9]. Furthermore, the recombinant vaccine expresses the heterologous antigen only in the spore form (Fig. 1), since the genetic construct is obtained by fusing the heterologous gene to the gene encoding for CotB spore-coat protein. Thus, this novel vaccine vector is able to prime the immune system and stimulate a local response, although it does not replicate at the mucosal site like live delivery systems. By combining the oral administration of recombinant spores, with the subcutaneous injection of

Fig. 2. Local TTFC-specific IgA. C57BL/6 and BALB/c mice were orally immunized with recombinant spores RH103 expressing TTFC (filled histograms) or wild type PY79 spores (open histograms). The concentration of TTFC-specific IgA, normalized to the total IgA assessed in each faecal sample, was determined by ELISA. Values are referred to faecal sample collected 2 weeks after the last i.g. inoculum (week 7). Statistical significance was set at ∗ P ≤ 0.05 and ∗∗ P ≤ 0.01.

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Fig. 3. Serum TTFC-specific IgG immune response. (A) C57BL/6 and BALB/c mice were orally immunized with recombinant spores RH103 (filled circles) or wild type PY79 spores (open circles) and then parenterally injected with soluble TTFC (as indicated by arrows). Serum TTFC-specific IgG concentrations, reported in the y-axis, were assessed by ELISA at different times (x-axis). Statistical significance was set at ∗ P ≤ 0.05 and ∗∗ P ≤ 0.01. Values are reported as arithmetic means, and error bars are S.E.M. (B) Sera collected at week 11 were pooled and assessed for IgG subclasses (black, IgG1; white, IgG2a; grey, IgG2b). Antibody titres were expressed as the reciprocal of the highest serum dilution with an optical density (OD) value > 0.2 after subtraction of the background value.

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