Elsevier

Vaccine

Volume 30, Issue 22, 9 May 2012, Pages 3266-3277
Vaccine

Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine

https://doi.org/10.1016/j.vaccine.2012.03.016Get rights and content

Abstract

Heat killed spores of the Gram-positive bacterium Bacillus subtilis have been evaluated as a vaccine delivery system with mucosal adjuvant properties for influenza. Killed spores were able to bind H5N1 virions (NIBRG-14; clade 1) and, when intra-nasally administered to mice, resulting immune responses, both humoral and cell mediated, were enhanced compared to immunization with the virion alone. Levels of both systemic IgG and mucosal sIgA specific to the virion were elevated. Levels of IgG2a (a Th1 antibody type) were strongly enhanced when the virion was co-administered with killed spores. Cytokine induction in stimulated splenocytes was also apparent indicating balanced Th1 and Th2 responses. Evidence of cross-neutralization of clade 2.2 viruses was shown. In a challenge experiment mice dosed two times with spores adsorbed with just 20 ng HA (hemagglutinin) of inactivated NIBRG-14 were fully protected against challenge with 20 LD50 of H5N2 virus. Interestingly, partial protection (60%) was observed in animals dosed only with killed spores. Mice dosed only with killed spores were shown to be fully protected against H5N2 (5 LD50) infection indicating that innate immunity and its stimulation by spores may play an important role in protection. Supporting this killed spores were (i) shown to stimulate TLR-mediated expression of NF-κB, and (ii) able to recruit NK cells into lungs and induce maturation of DCs. This work demonstrates the potential and underlying mechanism for the use of bacterial spores as an adjuvant for H5N1 vaccination.

Highlights

► We examine the ability of killed bacterial endospores as influenza adjuvants. ► Killed spores adsorbed with H5N1 are shown to stimulate protective immunity to H5N2 challenge. ► Killed spores can stimulate innate immunity conferring protection to H5N2 challenge.

Introduction

Highly pathogenic H5N1 influenza infections are still relatively rare in humans yet the continued evolution of this virus makes it a potentially serious and looming public health threat [1]. A vaccine suitable for protection against a future pandemic caused by an H5N1 virus should provide cross-clade protective immunity since the evolution of H5N1 viruses in birds in south-east Asia is unprecedented while the strain that may cause a pandemic cannot be predicted and current parenteral seasonal influenza vaccines are all largely strain-specific [1]. In addition, such a vaccine should have three further attributes: be safe, require a low antigen content (antigen-sparing) to combat the low immunogenicity of H5N1 viruses and cater for production problems and finally, induce a long lasting immunity [2], [3], [4]. Regarding antigen-sparing, adjuvants such as AS03 or MF59, oil-in-water emulsions (based on squalene-like molecules) have been used parenterally in humans successfully [5]. A number of studies have shown that secretory IgA (sIgA) from the respiratory tract is more effective in cross-protection against heterologous influenza compared to IgG induced by parenteral vaccination [6], [7]. Accordingly, mucosal adjuvants such as modified cholera toxin and the related E. coli heat labile enterotoxin (LT) have been shown effective at augmenting responses to inactivated influenza virus [8], [9] but unfortunately, have been linked to adverse reactions including facial paralysis [10]. The nasal route of vaccination has been shown to induce more appropriate immune responses at the mucosal surface, the actual site of infection, and has been more effective to epidemics caused by a heterologous virus [11], [12].

Spores of the Gram-positive bacterium, Bacillus subtilis, have been used successfully for antigen delivery. Using genetically engineered spores that express heterologous antigens protection has been achieved against a number of pathogens using animal models, notably Clostridium perfringens [13], Clostridium difficile [14], Clostridium tetani [15], and Rotavirus [16]. In all these examples spores have been delivered by a mucosal route where they have been shown to efficiently stimulate both systemic and localized immune responses. The attraction of using spores as a delivery system is 2-fold, first, their heat stability, and second, their existing use, worldwide as probiotic dietary supplements. With regard to stability, while native spores are intrinsically heat stable [17], this property has also been shown to be transferred to heterologous antigens expressed on the spore surface. In particular, the tetanus protective antigen TTFC (tetanus toxin fragment C) expressed on the spore surface has been validated in a challenge model after storage of lyophilized spores for 12 months [18]. As a probiotic, spores of a number of Bacillus species are used in human (e.g., Enterogermina®) and animal feed products (e.g., Gallipro®) and as live organisms for human consumption [19].

More recently, a non-GM approach to using spores has been developed making use of a novel attribute of the spore surface in binding heterologous antigens [20]. Using a combination of electrostatic and hydrophobic interactions proteins have been shown to bind to the spore surface. This approach enables the spore to serve as an antigen carrier without the need for genetic modification. Remarkably, using such an approach antigen presentation appears equally as efficient whether the spores are live or inactivated (heat killed). This approach then resembles other microparticulate adjuvants, which, by virtue of their size and multimeric presentation, mimic the pathogens the immune system has evolved to control, and facilitates uptake by antigen presenting cells [21], [22]. Studies on the nature of antibody responses in mice have shown that spores co-administered with protein antigens at mucosal surfaces augment antigen-specific secretory IgA (sIgA) as well as inducing balanced Th1/Th2 responses [20], [23].

Many animal viruses carry an envelope comprised of phospholipid embedded with lipoproteins and/or glycoproteins. Such viruses have been shown to efficiently adsorb to hydrophobic surfaces where binding increases as the pH declines [24]. This phenomenon should enable a virion to adsorb to the spore surface and was the rationale for the present study. In this work, we have shown that spores have unique characteristics as both an adjuvant and for antigen delivery. Mucosal delivery of a low dose of virion-adsorbed spores is shown to elicit protective immunity to influenza in a murine model of infection. We also show that spores by themselves act as a potential immune stimulant able to confer protection to influenza and we provide a basic mechanism for how this might occur.

Section snippets

General methods

Methods for work with B. subtilis are described elsewhere [25]. B. subtilis strain PY79, is a standard, prototrophic, laboratory strain [26]. Sporulation was confirmed by phase-contrast microscopy and spore crops were harvested and purified as described elsewhere [27]. Spores were killed by autoclaving (121 °C, 15 p.s.i., 30 min).

Reagents

Inactivated H5N1 virus, A/Vietnam/1194/2004 (NIBRG-14; clade 1) and A/Turkey/1/2005 (clade 2.2) as well as reference antisera were obtained from the National Institute

Adsorption of virus to spores

Purified H5N1 (NIBRG-14) virions were labeled with TMR and mixed with a suspension of pure spores of B. subtilis strain PY79 that had been labeled with FITC. Spores were harvested, washed repeatedly and examined by confocal imaging (Fig. 1). FITC-labeled spores were found to be coated with TMR-labeled virions (Fig. 1B and C). Generally, NIBRG-14 was uniformly labeled on the spore surface although some clustering was apparent (visible as intense red patches, see Fig. 1B, C and F) which is

Discussion

Resistance to influenza virus infection and disease relies upon mucosal and systemic immunity with sIgA active primarily in the upper respiratory tract and serum IgG in the lower tract [4]. Cell-mediated immune responses, while important, are more focused on clearing virus-infected cells than in prevention. Parenteral influenza vaccines can induce neutralizing IgG but fail to produce sIgA with this being one of the major drawbacks with current vaccination strategies [2], [4], [38]. In this work

Acknowledgements

Man Ki Song was supported by the governments of the Republic of Korea, Sweden, and Kuwait, and by the Top Brand Project grant from KRIBB (KGM0821113). Nguyen Thi Van Anh was supported by a research grant (NAFOSTED project 106.06-2010.06) from the Vietnam Ministry of Science and Technology and the L’Oreal-UNESCO National Fellowship for Women in Science. Dinh Duy Khang was supported by a research grant from the Vietnam Ministry of Science and Technology.

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    These authors contributed equally to this paper.

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