Vaccine against strangles of the Equidae

Field of invention

5 The present invention relates to vaccines that comprise composite particles containing bacterial extracts aiming preferentially the administration by the mucosal routes, particularly with vaccines against Strangles of the Equidae. It also includes processes for the production of vaccines by means0 of processing total aqueous bacterial extracts by spray drying.

This invention is applied to the formulation and production of veterinary vaccines against Strangles of the Equidae.

State of the art 5 Strangles is an infection of the respiratory tract of the equidae, caused by Streptococcus equi subspecies egui (S. egui). It is an infectious disease, very contagious, that may be mortal in its acute form. The ease of contagion, high morbidity effects, and the inexistence of effective vaccines, may start large outbreaks with devastating effects. The emergence of cases of strangles may result, among others, the ban on exports of horses and equestrian sports events cancelled. Even if generally after 4-6 weeks the infected animals recover from disease, 10% constitute long-term carriers, harbouring the microorganism for months. [Chanter et al . , 2000, Newton et al., 1997, Newton et al. , 2000] .

The rapid propagation of the disease, the reduced efficacy of antibiotics when animals already present signs of the disease and mainly the lack of efficient vaccines, make a quarantine___p_exi-ad-,—e-i-t-he-—fox—arriirra s or affected areas, the most suited solution. Generally, current vaccines against strangles have shown to be efficient but unsafe or safe but not protective [Waller and Jolley, 2007]. Particularly, conventional vaccines administered by the intramuscular or subcutaneous routes (such as Equivac ^® S, Pfizer) do not induce a balanced immune response against S. equi infection. On the other hand, live vaccines (such as Pinnacle™ I.N., Fort Dodge) given by the intranasal route are more efficient but the immune response that is induced is too strong, causing severe side effect and owners tend to avoid its use. This is also due to the risk that attenuated live microorganisms may recover their original virulence, including the most severe form of infection (bastard strangles) , which is often fatal [Kemp-Symonds et al. 2007 and Newton et al. 2005].

As a respiratory tract infection, bacteria penetrate the oral and nasal mucosa, lodging at the pharyngeal and upper respiratory tract lymph nodes, forming painful abscesses. Therefore, intranasal (i.n.) vaccination emerges as the logical objective in order to reach protective immunity through the local secretion of antibodies to prevent bacterial colonisation (mucosal immunity) and production of systemic specific antibodies that will prevent propagation to other organs (systemic immunity) . Mucosal immunisation with non-viable antigens appears as the best vaccination strategy, although presenting the following technological challenges:

( 1 ) Antigen immobilisation using particulate systems

Many vaccines currently used in human medicine contain attenuated or inactivated microorganisms. The investigation of new vaccines based on synthesis, isolation and antigen purification, either proteins, polysaccharides or nucJLeic_ aerirds-,—aftrerr poor immunogens due to the loss of their intrinsic adj uvanticity [Almeida and Alpar, 1996; Bramwell and Perrie, 2006] . The use of immunological adjuvants able to potentiate, through several mechanisms, the immune response against these antigens is a crucial requirement in vaccine formulation .

Since the 1980' s several vaccine adjuvants have been associated to S. equi antigens for parenteral immunisation purposes, with low success. This is due to the inability of parenteral vaccines to elicit a strong mucosal response, expressed as secretory IgA (SIgA) , at the respiratory tract [Sweeney et al . 2005; Amidi et al. 2007] . For their ability to control antigen presentation and rate of release, several vaccine delivery systems, such as microspheres and nanopart icles , also act as immunological adjuvants. Such particulate carriers also protect the antigens against premature degradation in vivo, and release the antigens in a sustained manner thus prolonging the contact with the immune system and allowing a reduction in the number of administrations.

Currently most vaccines are administered (intramuscularly or subcutaneously) , thus eliciting systemic responses instead of mucosal responses, which are needed for protection from pathogen penetration. Mucosal vaccination (oral, nasal, genital or even pulmonary) may induce specific humoral and cellular responses, local or systemic, able to confer protection [Yuki and Kiyono, 2003]. Hence, mucosal administration of antigen-containing microspheres or nanoparticles will elicit systemic and local immune responses due to the dissemination of sensitised lymphocytes [Storni et al., 2005].

(2 ) Narrow particle size distribution

Antigen formulation for mucosal vaccination is a very complex rocess ^" Immunisation will depend on particle uptake and translocation across epithelial surface, which in turn will depend on particles physicochemical characteristics, such as particle size, composition, surface hydrophobicity and superficial electric charge.

Therefore, particle size is determinant. Particles of certain compositions with a particle size smaller than 5μιτι are taken up at mucosal epithelia but translocation occurs preferably for particles smaller than The literature is plenty of publications describing the production of nanoparticles for mucosal administration, including those intended for vaccination against strangles [Azevedo et al. 2006; Florindo et al., 2008]. For example, the processes for production of nanoparticles polymeric or lipidic. In these processes, the particles are produced by emulsion of organic solvents (containing dissolved lipids or polymers) in aqueous solutions. The particles are obtained by evaporation of the organic solvents. The so prepared particles are then suspended in antigen solutions for loading of the antigens by adsorption. Otherwise, antigens may be added to the excipients during the process of production of the nanoparticles. These are lengthy processes involving several dissolution, emulsification, evaporation, centrifugat ion and filtration steps, which makes them very difficult to scale-up and unattractive for industrial application, particularly when products are intended for veterinary use.

Drying by atomization can be used to obtain particles with the required sizes to be delivered by the mucosae. Yet recovery of the particles is a technological challenge. Cyclones that are typically used in the recovery of particles produced by spray drying are inefficient for particles smaller than 2 ym.

Therefore in most cases there are significant product losses.

This factor is obviously both unacceptable and limiting to a successfully industrial application of this process taking into consideration the high-cost associated to the purified antigens-.- (3) Adj uvanticity

Other important factors towards the success of a vaccine are its hydrophobicity, superficial electric charge, shape and elasticity, physico-chemical stability, target ligand specificity [Almeida AJ and Alpar, . 1996] . Towards this end, agents are added to the antigen-containing formulations - the adjuvants.

For Strangles vaccines some authors [Azevedo et al. 2006; Florindo et al. 2009] showed that vaccines formed from polylactic acid nanospheres with polylactic glicol (PLA/PLGA) containing the antigens ^' and the adjuvants trigger an immune response in BALB/c mice - the biological model for vaccines for Equidae [Chanter et al . 1995]. The same authors also showed that an improvement of the immune response was observed when extracts of the S. equi bacteria were used as antigens. The improvement in the performance of the vaccine-containing extracts is related to the higher level of antigens in the extracts and also to the higher content of adjuvant agents naturally produced by the S. equi bacteria themselves. Therefore, comparably to the vaccines with purified antigens, the extract vaccine keeps the microorganisms' intrinsic adjuvanticity and shows potential to trigger a response from the immune system that is closer than that triggered by live bacteria .

Towards this end, Murillo et al. [Murillo et al. 2002] produced a brucellosis vaccine by spray drying of Brucella ovis extracts. Yet in this case the aqueous solution of the extract was emulsified before the atomization step with a solution of an encapsulating polymer in an organic solvent. The polymer inhibits the immediate dissolution of the particles upon their intake,, enabl-i-n-g—th-e-r-e-f-ar-er-a ^~~~ coTTtrOTled release. Spray drying is a relative simpler technique than emulsion processes that typically require further steps such as solvent evaporation. However, the use of organic solvents is a critical and undesirable factor because they affect the antigens' integrity and stability. Moreover, it requires additional steps towards the purification of toxic organic solvents such as the methylene chloride, as used by Murillo et al.

Effective vaccines against Strangles

There are a few examples reported in the literature of vaccines that were successful used against Strangles. One parameter that may be used to quantify an effective immune response is the ratio between concentrations of specific antibodies class IgG2a and IgGl because the IgG2a quantities produced are determining for the protection of mucosae infection. Towards this end, Florindo et al. [Florindo et al . 2009] produced (by emulsion and solvent evaporation) a vaccine formed by PLA/PLGA particles with adsorbed components of the total extracts of S. equi. The vaccine described by Florindo et al. trigged the production of IgG2a and IgGl in BALB/c mice with a IgG2a/IgGl ratio of 0.5. This positive result is the consequence (according to the authors) of the adsorption of substances present in the total extract at the surface of the PLA/PLGA particles. Although this vaccine is potentially better than other previous tries, it contains some limitations under the therapeutic point of view and disadvantages from the point of view of its production at industrial capacity. For example, the vaccine's production technique requires the preparation of particles of insoluble polymers. This issue requires the use of toxic organic solvents contributing to a method that is laborious, costly and difficult to transpose to industrial scale and to validate the process. Moreover, the method proposed by Florindo et al. compromises the

adsorption, which is naturally selective process, not ensuring therefore that all substances present in the 5. equi extract be incorporated equally into the particles.

For a vaccine to keep the intrinsic adj uvanticity of the extract it is necessary that all substances of the extract be present in the particles and preferably with the same relative composition. Under this scope, ideally the particles should be formed by the extract only, or else that the eventual added excipients were soluble in the extract itself to avoid the use of toxic organic solvents and multiple processing operations.

Summary of the invention

The present invention relates to a vaccine against strangles of the Equidae preferentially aiming administration by mucosal routes, characterized in that it comprises composite particles with a size of 0.1 μιη to 2 μπι, which composition consists of substances which in their pure state are soluble in aqueous medium, comprising all the . substances present in an aqueous extract of a lysate of S. equi. and showing a partial solubility when re-dissolved in an aqueous medium. These features result in greater efficacy of this vaccine (compared to the current market alternatives) which is characterized in that it triggers a cellular and humoral immune response with production of specific antibodies with a unit ratio concentration of IgG2a and IgGl (between 0.6 and 1.4). It also comprises a process for preparing this vaccine characterized by comprising the spray drying of aqueous solutions that comprise total extracts of S. equi and capture of the particles in cyclones, electrostatic precipitators and / or filters. Detailed description of the invention

The present invention relates to a vaccine against strangles of the Equidae preferentially aiming administration by mucosal routes and a process for producing it. However, the use of this vaccine is not limited to administration by mucosal, it may also be administered by other routes, such as parenteral routes (intramuscular, subcutaneous, intradermal) . This vaccine is characterized in that it comprises particles with a size distribution between 0.1 μιτι and 2 μιτι - (Ferret diameter) . These particles are characterized in that they integrate the full range of solutes present in an aqueous extract of a lysate of S. equi in relative compositions similar to those that existed in the extract before it is processed. The use of bacterial extracts enables the preservation of the intrinsic immunogenicity of S. equi, which is a significant difference compared with other vaccines that use purified antigens. In this document, the intrinsic immunogenicity is defined as the attribute of a vaccine to contain all substances which are present in the aqueous extract of bacterial lysate and in the same relative compositions (taking into account the total mass of solutes present in the extract) .

The use of total extracts has two key advantages. First, it is susceptible of eliciting an immune response closer to that what it would be expected in case of an infection with the pathogen, rather than an immune reaction against a particular protein; second, it is considerably more economical to process directly bacterial extracts without proceeding to purification prior to any of its components, advantage that can be crucial to the industrial feasibility of a veterinary vaccine.

The extract of S. equi can be obtained, for example, by enzymatic lysis of thes_e__b^cjteji_a_u.s.ing £o-&~-tn-s-ta-n-ce—lysozyme ^" and mutanolysin. After lysis of the bacteria, the resulting suspension should be separated, for example by centrifugation, to obtain the aqueous total extract with no particles . in suspension. Although the composition of the extract is not fully known, it is assumed that it consists of many substances because it integrates all the soluble substances present in bacteria. The extract can be characterized by electrophoresis, as shown in Figure 1 where the bands of total protein extract are observed (lane A2 of Figure 1) .

The particles that constitute the vaccine can be obtained by a process of spray drying. In particular, by atomization of the extract through a nozzle, together with a fluid (gaseous or supercritical fluid) that assists the disintegration of the liquid jet into droplets in the interior of a dryer. The droplets are dried in the dryer by a stream of hot gas (typically nitrogen or air) . After spray drying the particles are captured in cyclones, electrostatic precipitators and / or filters. The fluid which assists the disintegration of the liquid jet through the orifice of the nozzle may be, for example, nitrogen, carbon dioxide or air. This fluid has the function to accelerate the jet of fluid and disintegrate it due to instabilities caused by share stress between the two phases (liquid and gaseous or supercritical fluid) . For this, the fluid that assists atomization is mixed with the liquid immediately before the nozzle. Since the mixing occurs just before the nozzle, the volume of the mixing chamber is small (typically less than 0.5 cm ³ ) . The total pressure that can be achieved before the nozzle varies between 1,0 and 10 Pa due to the pressure drop caused by the hole (typically 50 μ ι to 500 mm of diameter) .

Before the extract is converted into particles through spray drying, it can be supplemented with excipient substances,

to increase the mass of vaccine produced (acting as carrier excipient), encapsulating the constituents of the extract or acting as adjuvants. Examples of such excipients are soluble polymeric excipients such as polysaccharides like chitosan, alginate, hyaluronic acid and starch. Figure 2 shows an example of particles wherein a polysaccharide was added to the extract. In this invention, only water-soluble substances are used, which simplifies the production of the vaccine. Thus, particles can be produced by a single unit operation (spray drying) which can operate in a continuous regime. This feature is advantageous when compared to most other processes. For example, Florindo and coauthors [Florindo 2009; Florindo 2009b; Florindo 2010] prepared a vaccine using particles of water-insoluble polymers such as polylactic acid and polylactic glycolic acid ( PLA / PLGA) as the support, where the antigens are loaded by adsorption. Regarding the techniques which the work of Florindo et al. serve as examples [Florindo 2009; Florindo 2009b; Florindo 2010] , the present invention also has another important advantage which consists in the integration, in the vaccine, of all the substances which are present in the extract of S. equi; being conserved the corresponding intrinsic immunogenicit . Florindo et al. [Florindo 2009; Florindo 2009b; Florindo 2010], carry the antigen extract by adsorption, which means that part of the extract is not loaded in the particles, and that different substances have different partitions between the fraction that adsorbs to the particles and the fraction that remains in the extract at the end of the adsorption operation.

The intrinsic immunogenicity of this vaccine may be demonstrated by electrophoresis after the particles' suspension and redissolution in water, as shown in Figure 1. In Figures 1A and IB, the bands obtained in the well 2 (extract unprocessed) are similar to those obtained in the well 3 (relatively to the vaccine redissolution) . The presence of the protein bands shows

_^hat_ _^ jDrjDi^ (mo-s-—1-i-k-e-ty— ntigens ^" ) were not ^" lost during processing. It is also expected that all other non-protein substances that are present in the extract, be also included in the vaccine, because the particles result from the complete drying of the extract. This differs from all other publications in which extracts of S. equi were used to develop vaccines [Azevedo et al. 2006, Florindo et al . 2009, 2009b, 2010)]. For example, Florindo et al. (2009), showed that the protein adsorbed to the particles lacks some proteins that were present in the extract.

The intrinsic imrnunogenicity of this vaccine results in a greater efficiency, which can be confirmed by in-vivo tests. The results obtained in' vivo in BALB/c mice showed that the vaccine produces a humoral and cellular immune response characterized by a balanced unit ratio of ^' concentrations of specific IgG2a/IgGl, as shown in Figure 3. This finding overcomes the weak production of IgG2a which was obtained with the adsorbed antigen loaded polymer particles (IgG2a/IgGl less than 0.5) described by Florindo et al. [Florindo 2009; Florindo 2009b; Florindo 2010].

A key factor in this invention is the low dissolution rate of some of the substances present in the particles which were generated by this process. After processing, the particles of the extract, or the extract containing natural polysaccharides, do not dissolve completely. This feature is crucial for the functioning of the vaccine because, even if the substances in their pure state the substances are soluble in aqueous medium (typically more than 1 mg / ml) during their joint dehydration (by spray drying) interactions occur between the said substances that make the resulting particles (composite) partially insoluble. For this reason, the non-solubilized fraction retains part of the antigens which are then slowly released, extending the stimulation of the immune system and thereby potentiating the immune response as shown in Figure 4. Description of the Drawings

Figure 1 represents an analytic electrophoresis through the techniques of electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. SDS-PAGE (A) and the Western-Blot (B) of the S. equi enzymatic extract, evidencing the integrity and immunoreactivity of the vaccine. Wells: (1) molecular standards; (2) S. equi extract (non-processed); (3) solution with dissolved particles of the vaccine produced according to the Example 1.

Figure 2 represents an image of scanning electronic microscopy of particles produced according to the process described in Example 2, with a magnification of 10.000 times.

Figure 3 represents the concentration ratio of specific anti-5. equi IgG2a/IgGl in the BALB/c mice serum after priming (day 1), followed by boost (day 21), through the intranasal route with antigens processed according to Example 3 (n=5, average ± standard deviation) .

Figure 4 represents the cumulative curve in percentage of the total protein released (xx axis) in vitro. The release assays were carried out in triplicate at 37°C in phosphate saline buffer (PBS) 10 mM, pH 7.4, and 0.02% of sodium azide with continuous stirring. The total protein was quantified through the bicinconinic acid method (BCA) using a MicroBCA Kit (pierce) , after centrifugation 12 OOOxg during 10 minutes for particles separation . Examples

Example 1

Preparation of the S. egui extract:

The S. equi extract (strain LEX - ATCC 53186) was obtained by enzymatic lysis of the bacteria, after homogenization at high pressure, by addition of lysozyme (3 mg/ml) and mutanolizine 93.6 units per milliliter. The suspension was centrifuged for separation of the solids (8000xg) . The extract had a concentration of total protein of 5 mg/cm ³ .

Production of particles

The extract was atomized trough a nozzle with 100 um of diameter and 250 μτη of length. The solution flow-rate (extract) loaded in the nozzle was 0.5 g/min, using a liquid pump. The atomization was assisted by a flow-rate of 20 g/min of carbon dioxide that mixed with the solution (in a volume smaller than 0.5 cm ³ ) immediately before the nozzle. The jet was dried in a drier at 50 °C and the particles which constitute the vaccine were recovered in a filter.

Example 2

Enrichment of the extract with a polysaccharide

An extract of S. egui was prepared according to the description presented in the Example 1. It was added to the extract solution an equivalent volume of a chitosan solution of low molecular weight (<150 kDa) . The resulting solution had a final concentration of chitosan of 5 mg/cm ³ . Production of particles

The extract was atomized trough a nozzle with 100 μιη of diameter and 250 um of length. The solution flow-rate (extract) loaded in the nozzle was 0.5 g/min, using a liquid pump. The atomization was assisted by a flow-rate of 20 g/min of nitrogen that mixed with the solution (in a volume smaller than 0.5 cm ³ ) immediately before the nozzle, driven by a compressor. The final pressure of the mixture before the atomization was of 3 MPa. The jet was dried in a drier at 50 °C and the particles which constitute the vaccine were recovered in a cyclone.

Example 3

Enrichment of the extract with a polysaccharide

An extract of S. egui was prepared according to the description presented in the Example 1. It was added to the extract solution an equivalent volume of a chitosan solution of low molecular weight (<150 kDa) . The resulting solution had a final concentration of chitosan of 5 mg/cm ³ .

Production of particles

The extract was atomized trough a nozzle with 150 μτα of diameter and 250 μηα of length. The solution flow-rate (extract) loaded in the nozzle was 3 g/min, using a liquid pump. The atomization was assisted by a flow-rate of 30 g/min of carbon dioxide (driven by a compressor) that mixed with the solution immediately before the nozzle in a volume smaller than 0.5 cm ³ . The final pressure of the mixture before the atomization was of 8 MPa. The jet was dried in a drier with a flow of hot _air___ajL_8_0___lC a-n- —fehe-

—pa-rtd ^~ ci¾ ^~ 5 wftTcE constitute the vaccine were recovered in a cyclone and an electrostatic precipitator. Example 4

Immunization of BALB/c mice with the vaccine produced according to Example 2

Two groups of mice (females) BALB/c (25 g; 5/group) were immunized by the intranasal route (i.n.) on day 1 and a boost was given on day 21, using a micropipette to deliver 50 um of vaccine (25 μτα in each nostril) . All formulations were prepared by dispersion of the particles, aseptically and short time before administration.

Blood samples were collected from the tail vein at 2, 4, 6, 8, 10, 12, 14 and 16 weeks after immunization. The serum was separated by centrifugation (18 000 g, 5 min at 4°C, Allegra 64R, Beckman, USA) and tested by ELISA ( Enyme-Linked Immunosorbent Assay) for the classes of specific IgG antibodies namely: IgG subclass 1 (IgGl) and IgG subclass 2 (IgG2a) .

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Date: February 28 ^th , 2013