European Journal of Pharmaceutics and Biopharmaceutics
Research paperDevelopment of a novel mucosal vaccine against strangles by supercritical enhanced atomization spray-drying of Streptococcus equi extracts and evaluation in a mouse model
Graphical abstract
SDS–PAGE and Western-blot analysis showing integrity and immunoreactivity of Streptococcus equi enzymatic extract antigen processed by the supercritical enhanced atomization spray-drying technique.
Introduction
Streptococcus equi subspecies equi (S. equi) is the causative agent of strangles, an acute, extremely contagious, and occasionally lethal disease that infects animals of Equidae family [1], [2], [3], [4]. S. equi infection is very debilitating and difficult to defeat. The use of antibiotics is not consensual as most of the treatments are ineffective when external signs of disease are already noticeable. Typically, the disease signs include pyrexia, suppurative, mucopurulent nasal discharge, lymphadenitis, and abscessation, often in the lymph nodes of head and neck. Occasionally, the infection develops into an acute stage, commonly referred as bastard strangles, reaching other lymph nodes and organs, with high mortality. In most cases, the animals recover from strangles in 4–6 weeks; however, 10% become S. equi carriers for several months. For this reason, isolation of areas affected by strangles is the best strategy to handle the epidemic, even though it causes considerable impact in economic and cultural activities associated with the horse trade [1], [2], [3], [4].
Despite extensive efforts regarding prevention of large outbreaks, S. equi infection remains spread worldwide. The development of an effective vaccine for strangles would represent a major asset to overcome S. equi. However, thus far, commercially available vaccines have generated unsatisfactory results. They are often described to induce poor and short immunity and frequently produce strong adverse effects [4]. This may be related to their low stimulation of the secretion of IgA and IgG in the nasopharynx of the animals – conversely to what it is normally observed in animals that acquired immunity after recovering from a natural infection [3].
Particulate carriers are currently one of the most promising strategies to fight infectious diseases [5]. Particles with antigens are recognized and taken-up by antigen presenting cells (APCs) that can set up a broad immune action involving lymph nodes and other immuno-competent organs [6], [7], [8], [9]. There are many examples of particulate vaccines that induce local and systemic immune responses, capable of generating protection against numerous pathogens including S. equi. [5], [7], [9], [10], [11]. Particularly, Florindo et al. [10], [11] have shown that the intranasal delivery of polymeric particles loaded with S. equi antigens was able to induce humoral and cellular immune responses as well as mucosal immunity in the mouse model. The same authors also showed that a balanced immune response could be obtained if S. equi extracts were loaded on particles of poly lactic acid (PLA), instead of purified SeM antigen (the most important S. equi antigen) [11]. In line with this approach, Murillo et al. [12] also produced a vaccine against brucellosis using extracts of Brucella ovis loaded in poly-lactide-co-glycolide acid, but in this case, the emulsion was processed by spray drying.
Spray-drying technology has shown potential for the production of particles smaller than 2 μm, thus including particle size ranges suitable for mucosal administration [12], [13], [14], [15], [16], [17]. However, in most examples of encapsulated antigens by spray drying, the solutions were previously emulsified with organic phases containing the encapsulating polymers or lipids [12], [13], [14], [15], [16], [17]; Murillo et al.’s [12] vaccine is one of these examples. Spray drying of emulsions is a relatively easier process compared to the multiple steps required by other solvent evaporation alternatives involving emulsions [12], [13], [14], [15], [16], [17]. However, it still causes considerable stress to antigens because emulsions require the use of organic solvents, which may compromise the integrity of proteins. Moreover, further purification steps are required to deal with contamination with organic solvents, which are toxic, such as, for example, methylene chloride used by Murillo et al. [12].
In this work, we develop an extract-based vaccine against S. equi without using organic solvents. Herein, an aqueous extract of S. equi enriched with chitosan (a polysaccharide with adjuvant properties [18]) is converted into nanoparticles by using a Supercritical Enhanced Atomization (SEA) spray-drying technique. The direct processing of an aqueous solution into nanoparticles avoids emulsions with organic solvents, as usually required for encapsulation with polymers. Yet, using only water-soluble substances raises an important concern, the nanoparticles might dissolve instantly upon rehydration, compromising therefore its immunogenity. However, due to the complex composition of the extract and the possible physicochemical interactions of the constituents (either between themselves or with chitosan) during intense atomization and spray drying, it is difficult to anticipate what will be the properties of the resulting particles. Therefore, this issue could only be clarified experimentally.
A spray-drying setup was assembled with a coaxial high-pressure nozzle to enhance the atomization with a supercritical fluid (either CO2 or N2). Spraying using dense fluids, such as supercritical CO2, is particularly effective for the production of submicron protein particles, as reviewed elsewhere [19], [20]. This process consists essentially in dissolving the supercritical CO2 in a liquid phase and depressurizing the mixture though a nozzle with a small orifice (of the order of 100 μm diameter). The significant pressure drop, together with the burst of the dissolved gas, causes the formation of microdroplets and microbubbles, described as an effervescent atomization [19]. This supercritical enhanced atomization was first developed and registered as the CO2-Assisted Nebulization with a Bubble Dryer (CAN-BD®) [19], [21], [22]. Several variants exist, each with its distinct design (reviewed elsewhere [20]), some of which are also under intellectual property protection [23]. Herein, we consider the term “Supercritical Enhanced Atomization” (SEA) as an adequate generalization; it includes all processes that exploit the atomization enhancement caused by dense fluids applied to spray drying – including processes that use high-pressure N2 [24], [25].
The results discussed below show that the extract nanoparticles produced by SEA caused a well-balanced immune response in a mouse model, constituting therefore a potential effective vaccine against strangles. Although immunization experiments performed with mice may have limited application to equidae – the natural host of S. equi [26], [27], the mouse is still the only preclinical model for strangles vaccines. On the other hand, our previous reports suggest that particulate carriers should be studied further as antigen delivery systems and immunological adjuvants for S. equi antigens [10], [11]. From a manufacturing viewpoint, the technique described here offers the advantage of being a single step, continuous operation process that can readily be scaled up—an advantage that it is usually claimed for basic spray-drying processes [14], [20].
Section snippets
Preparation of the S. equi extract
The extracts S. equi were obtained from the lyses of the bacteria cell wall using n-acetyl muramidase, lysozyme and mutanolysin (Sigma Aldrich, UK), as previously reported by Florindo et al. [10]. Briefly, inactivated S. equi subsp. equi cells (ATCC 53186) were washed with phosphate buffered saline and homogenized by two passages at 6.2 MPa, (EmulsiFlex-C5, Avestin Inc., Canada). Lysozyme (3 mg/ml), mutanolysin (93.6 Unit/ml), and sucrose (170 mg/ml), all from Sigma Aldrich Co., UK, were added to
Powder production by SEA
Fig. 3 shows images obtained by SEM of typical powders produced by SEA when the prime solutions were processed separately, that is, Fig. 3a refers to particles produced using extract, without chitosan, and Fig. 3b refers to chitosan particles without extract.
The pure extract originated always fused agglomerated particles. Several runs were carried under different conditions (results not reported here) to obtain discrete particles but without success. These difficulties may be related to the
Discussion
The results show that the nanoparticles of S. equi extract with chitosan produced by SEA vaccine generated a sustained, well-balanced and broad immune response in mouse model, thus confirming previous reports [10], [11]. The anti-S. equi specific IgG secretion was high with a well-balanced ratio (IgG2a/IgG1) between both isotypes (approximately 1.0) as Fig. 9 shows. This reveals that the SEA vaccine stimulated both Th1 and Th2 helper T cells, as IgG2a isotype is related to a Th1 response,
Conclusions
SEA spray-drying of aqueous solutions containing S. equi water-soluble extracts and chitosan resulted in partially insoluble nanospheres. The process consisted in a single-step atomization without the previous preparation of emulsions or use of organic solvents. The resulting nanospheres were therefore composed exclusively of aqueous soluble substances. Nonetheless, the release was relatively slow causing an effective immune response in a mouse model, characterized by a broad and well-balanced
Acknowledgements
This work was supported by Fundação para a Ciência e Tecnologia (Portugal) and FEDER (Projects POCI/BIO/59147/2004; PPCDT/BIO/59147/2004; strategic project PEst-OE/SAU/UI4013/2011; and PTDC/EQU-EQU/104318/2008).
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