NO343949B1 - Formulation with immunostimulant / adjuvant activity and use as vaccines for vertebrates - Google Patents

Description

FIELD OF THE INVENTION

The purpose of the present invention is to provide a formulation containing an active principle with immunostimulating/adjuvant activities obtained from the resinous exudate of the plant species Heliotropium huascoense, and which is useful in salmonid species to increase the immunogenicity of an antigen and/or the components of a vaccine.

BACKGROUND OF THE INVENTION

Salmon industry and health challenges:

In recent years, Chile has developed a very important salmon aquaculture industry. In 1994, exports of salmon products increased to US$ 349 million. After a decade, this figure was seven times higher, and in 2007 it reached US$ 2207 billion. Today, salmon is one of the most important export industries in Chile, with a quota of 10.8% for exports. This industry constitutes an important element for the diversification of the national economy, and is one of the cornerstones of the strategy to make Chile a significant food producer (SalmonChile, 2010).

In general, fish farming is constantly exposed to attacks from several infectious diseases due to the extreme production conditions, which cause the growth of microorganisms to spread easily and represent a problem that is difficult to control. There are a number of infectious diseases that affect fish farming and represent the most important cause of death. Diseases can be caused by viruses, bacteria, parasites and fungi, causing significant production losses each year. The decline in salmon exports in 2009 was caused by an outbreak of viral diseases. According to the monthly report on foreign trade (Informe Mensual de Comercio Exterior) prepared by the National Customs Service (Servicio Nacional de Aduanas), shipments that accounted for 55% of fishing and aquaculture decreased by 17% in 2009 (SalmonChile, 2010). For Chile, a country that for many years has been free of many diseases affecting animal production, the globalization of world markets has meant that the country no longer has this advantage, and the country's ability to achieve high competitiveness has been jeopardized.

The many diseases that have affected and spread in farmed salmon in southern Chile, which in some cases have become endemic pathologies, include bacterial kidney disease (BKD), salmonid rickettsial syndrome (SRS) and enteric red mouth disease (ERM). These pathologies probably arrived in this country through egg imports, on which Chile was almost exclusively dependent in the past, when the requirements for admission were not so strict (Pinto, 2003). In recent years, other diseases have emerged mainly caused by bacteria, such as vibriosis, streptococcal infection and atypical furunculosis, and by parasites such as Diphyllobothrium sp., which causes amoebic gall disease (Torres, 2000) and Caligus, a parasite that causes wounds and stress in the fish, weakens their defenses and makes them susceptible to attack by other pathogenic microorganisms such as bacteria and viruses. Among the viral infectious agents affecting salmonids is infectious pancreatic necrosis virus (IPNV), which is a birnavirus (Birnaviridae family) (Dobos et al., 1979), and the virus with the most cases worldwide (Wolf, 1988) . The virus has been detected in the majority of salmon production centers worldwide, including Chile (Wolf, 1988; Hill, and Way, 1995). IPNV mainly affects young fish (fry and small fish) and causes a high degree of mortality (Wolf, 1988, Imajoh et al., 2005), although it also infects more developed fish. The outbreak of infectious diseases in salmon is one of the main problems of fish farming centers, since they do not have closed, controlled rooms that make it possible to prevent pathogens from easily spreading in lakes and the sea.

Because of the environmental and economic importance, much effort has been put into studies associated with the infectious pancreatic necrosis virus (IPNV), and the search for antiviral compounds (Saint-Jean et al., 2003). There are now vaccines that help prevent infection by the IPN virus (Christie, 2004; Salgado Miranda, 2006), but the inoculation of small fish does not give the expected results since their immune systems are immature. On the other hand, there are some antiviral compounds that inhibit the replication of cell cultures, such as ribavirin and 5-ethynyl-1-β-D-ribofuransulimidazole-4-carboxamide (EICAR) (Migus and Dobos, 1980; Jashes et al., 1996 ; Jashes et al., 2000). However, ribavirin has not had good results in experiments carried out in vivo (Somogyi and Dobos, 1980), and fish mortality is reduced by EICAR, but it is not stable in water (Moya et al., 2000).

Another infectious disease that affects salmon farms to a significant extent is caused by the infectious salmon anemia virus (ISA virus), discovered in Norway in 1984 (Thorud and Djupvik, 1988). This agent has been a problem for salmon aquaculture in the Northern Hemisphere, including Norway, Canada, Scotland, the Faroe Islands and the USA, and the high mortality rates included almost the entire production in the case of Norway (Mjaaland et al., 1997). In Chile, ISA virus was first recorded in 2001, but unlike the case in the countries of the Northern Hemisphere, it was not associated with high mortality and losses immediately (Kibenge et al., 2001). However, in 2007 the ISA virus began to devastate the Chilean salmon up to a loss of 40% of the national production.

Today, diseases affecting salmon are prevented, mainly through health management practices, the use of authorized vaccines and/or the selection of healthy breeding fish. Treatments used must be prescribed by a veterinarian, and only the drugs registered for use in aquatic species can be used (Sandoval, 2004). The great losses caused by this type of infectious diseases lead to the search for solutions that can regulate their development in one way or another. On the other hand, a prophylactic treatment with antibiotics is used with the aim of preventing bacterial infections in highly susceptible fish. Treatment with antibiotics in salmon aquaculture is carried out by immersion with drugs and medicated food. In both cases, there is the possibility that the antibiotics are released into the environment and cause effects in wildlife. There are several concerns regarding the widespread use of antibiotics in salmon aquaculture, and among them are the growth of antibiotic-resistant bacteria in the flora and normal pathogens in pools, the effects caused by the persistence of these drugs, and the antibiotic residues found in sediments and in the water column . These antibiotics promote the growth of free antibiotic-resistant bacteria, a process that changes the composition of normal bacterial flora in fresh and salt water. Data indicate that these antibiotic-resistant organisms in the marine environment will in turn transfer their antibiotic-resistant genes to other bacteria, including human and animal pathogens. Antibiotics can also affect the composition of the phytoplankton community, the zooplankton community, and even the diversity of larger animal populations. Consequently, the possible changes in the marine microbiota diversity caused by the antibiotics may alter the homeostasis of marine environments and affect complex life forms, including fish, molluscs, marine mammals and humans. In the case of oxytetracycline, for example, low absorption has been shown in the intestinal tract of fish, therefore much of the drug is excreted unchanged into the aquatic environment, where it is distributed between the sediment and the water column, or consumed by marine wildlife (Paone, 2001). In summary, treatments that are used now to control infectious diseases in salmonids are based on the use of chemicals and vaccines that do not have a good percentage of effectiveness. On the other hand, many of these chemicals cause side effects or are banned substances in other countries.

In addition, the salmon's life cycle is affected by several factors induced by human intervention, such as fishing pressure, pollution, geographical barriers and aquaculture farms. These factors have a major impact on their survival, especially on their ability to withstand stress, such as being exposed to a confined culture system. This has created a significant decrease in their defense system that can be exploited by the infectious microorganisms that cause disease and death in salmon populations, or asymptomatic infections that lead to the entry of asymptomatic carriers that can transmit microorganisms over long periods of time and long distances. Unfortunate scenarios in culture, and mainly the exposure to pathogenic micro-organisms, require a good immunological state of the salmon for it to face its life cycle in a healthy way.

The immune system of teleosts, such as salmon, shares similarities with that of higher vertebrates. Mammals and teleosts are known to have well-defined lymphoid tissues, such as the thymus, spleen, kidney, and tissues associated with skin and gills (Press, Evensen et al. However, fish do not have bone marrow, lymphatic systems or Peyer's patches as mammals do (Rombout , Huttenhuis et al. 2005).The salmon immune system is characteristic of teleosts (Penagos et al., 2008) in that it has been identified (i) immune system with innate response, which corresponds to the phylogenetic or inherited mechanism that can remove any foreign body, mainly by to activate a set of pathogen-associated molecular pattern (PAMP) recognition receptors, and (ii) the adaptive immune response which, depending on the lymphocytes, can recognize and eliminate external invaders. Despite this classification, the mechanisms of innate and adaptive immunity interact and operate together against an external attack. Leukocytes involved in fish immunity are B lymphocytes (IgM+ and IgT+ cells) and T lymphocytes. The existence of T helper lymphocytes phocytes (CD4+ T cells) have been demonstrated indirectly in fish, including salmon and trout, due to the presence of genes homologous to CD4, TCR and CD3e, among others (Suetake, Araki et al 2004; Laing, Zou et al 2006; Nakanishi et al., 2011), and more recently, using immunomagnetic isolation and characterization of CD4 cells (Imarai et al., unpublished). In addition, cytotoxic T lymphocytes (CD8+ T) have been isolated, and their function in vitro has been demonstrated (Fischer, Utke et al 2006; Sato and Okamoto 2008; Utke, Kock et al 2008). Similarly, antigen entry appears to occur as in mammals due to the presence of MHC class I, TAP and LMP genes.

Antigen-presenting cells, such as macrophages, are abundant in fish, and cells resembling dendritic cells have recently been identified in fish (Ohta, Landis et al. 2004; Yoder and Litman, 2000). Together, the evidence supports the existence of an adaptive immune response in teleosts, including salmonids.

The immune response is influenced by several factors that depend on the host, including age, physiological state or stress, characteristics of the aquatic environment, such as water temperatures or the presence of toxins therein, and thirdly, by the organism involved (Penagos et al., 2009). . The high mortality caused by infectious diseases now observed in the salmon industry indicates the urgency of developing new pharmacological tools to stimulate the protective immune response in salmonids.

Genus Heliotropium:

Researchers' interest in medicinal plants as a natural source of many active components has increased considerably in the last two decades. In that sense, secondary metabolites obtained from the resinous exudates of the plants of the genus Heliotropium (family Heliotropiaceae) growing in the north of Chile have been isolated and characterized for far too long. The species Heliotropium grows in arid regions under extreme environmental conditions, and is characterized by producing a resinous exudate through glandular trichomes that cover their leaf surface and stem. Phytochemical research revealed that the resin mainly consists of flavonoids and aromatic geranylated derivatives, in smaller amounts (Torres et al., 1994, 1996; Urzúa et al., 1993, 1998, 2000, 2001;

Modak et al., 2003, 2007, 2009). In search of an explanation for the role of resinous exudates, it has been suggested that they may constitute the first protective barrier against predators. This protection may be due to a mechanical effect associated with its sticky nature that causes predators to stick (Eigenbrode et al., 1996), as well as a chemical protection due to the presence of secondary metabolites with antimicrobial, antioxidant and cytotoxic properties (Hoffman et al. ., 1983). Furthermore, the predominant presence of resinous exudate in plants growing in arid and semi-arid zones has been explained by the extreme environmental conditions (Downum et al., 1988), and especially the increased oxidative stress to which they are exposed (González-Coloma et al., 1987). It has also been suggested that the presence of antioxidant flavonoids in the exudates may prevent oxidative degradation of the resin's other components, which may lead to the loss of its chemophysical properties (Urzúa and Mendoza, 1993).

There are studies of the composition of resin from various species of the genus Heliotropium, as well as certain biological and chemical activities of such exudates and their components (Modak et al., 2004 and 2010). In particular, pinocembrin, 3-O-methylgalangine, 3,7-O-dimethylgalangine, alpinone and carrizal acid (eng.: carrizaloic acid) have been isolated from Heliotropium huascoense (Urzúa et al., 2000; Villarroel et al., 2001 ). In general, studies conducted with flavonoids isolated from the species Heliotropium show that they present mainly antioxidant activity (Lissi et al., 1999; Modak et al., 2003; Modak et al., 2007, Modak et al., 2009; Choudhary et al., 2008 ).

Vaccines for fish:

Under natural conditions, disease is a phenomenon that is inherent to all living beings, and which is strengthened when the individual is exposed to stressful conditions, including confined culture. The intensive animal production creates the conditions for a change in the balance between environment, pathogen and host, which leads to disease and mortality. Therefore, the submitted invention solves this technical problem in a very different way from what is described in the prior art.

To prevent infectious diseases, vaccination is one of the most valuable tools in aquaculture as it ensures a clean product that does not change the environment. Despite the fact that some authors believe that in aquaculture only one vaccination is sufficient to induce protection until the fish are collected (Heppell and Davis, 2000; Bowden et al., 2003), and this may be valid after the immunizations intraperitoneally, vaccination of populations of hundreds of thousands of individuals massive immunization methods (immersion and oral), which in most cases require revaccination (Romalde et al., 2004; Vandenberg, 2004). In some fish, suitable levels of protection are only achieved when modified live vaccines are used, as is the case with the vaccine against Edwardsiella ictaluri in catfish aquaculture in the USA. Despite the apparent safety of bacterins, fish vaccinations received a warning in 2001, when the appearance of an isotype with higher virulence, with modifications in the membrane proteins, which could infect and kill vaccinated fish, was introduced in the application of a bacterin against streptococci (Bachrach et al., 2001) in a rainbow trout (Onchorhynkus mykiss) farm in Israel. In addition, some extracellular products (ECPs) from bacteria have been tested with questionable results in the vaccination of fish against streptococci. Klesius et al., 2000, and Evans et al., 2004 compared in tilapia (Oreochromisniloticus) the protective effect of conventional bacterins and bacterins added to ECPs against Streptococcus iniae and Streptococcus agalactiae, and suitable levels of protection were found only when concentrated ECPs was added to the vaccines. DNA vaccines are in turn considered the most effective in inducing protection and response from the immune system of the fish by triggering innate and adaptive immune mechanisms), which also ensures effective humoral and cellular responses (Heppell and Davis, 2000). On the other hand, to date there is no inactivated vaccine available that is highly effective against the IPNV virus (Salgado-Miranda, 2006). The virus treatment with formalin or β-propiolactone for use in vaccines completely inactivated the virus but reduced antigenicity up to 50% (Dixon, 1983). An active vaccine which included a non-pathogenic strain of the IPNV virus, sensitive to normal trout serum, also did not transfer protection to the experimental challenge (Dorson, 1977). Inactivated and recombinant vaccines are also used. Therefore, the recombinant vaccine, the first licensed to be administered to fish, contains the structural protein VP2 produced in Escherichia coli, and induces the production of specific antibodies against the IPNV virus (Christie, 2004).

Veterinary grade adjuvants:

Conventional veterinary vaccines consist mainly of attenuated live pathogens, inactivated organisms or inactivated bacterial toxins (Chang et al., 1998). Although attenuated forms of pathogens are used as veterinary vaccines, there is concern regarding this, as it occasionally reverts to the virulent form or adverse effects are observed in immunocompromised animals. The use of dead organisms or parts thereof is an alternative, although the lower effect is evident.

As a result of these limitations, new vaccines have been developed with significant advantages over the previous ones. They include subunits of recombinant proteins and DNA (Rankin et al., 2002). Although these new alternatives may be beneficial, a common problem is that, in veterinary medicine, they are often immunogenically weak (Bahenmann et al., 1987; Loehr et al., 2001). Traditional vaccines often contain components that can contribute to the maturation of T cells and act as adjuvants, for example the bacterial DNA, or LPS. However, these components have been eliminated from the new vaccine generations, therefore increasing the need to identify adjuvants that will strengthen and drive the necessary immune response.

The immune adjuvants were originally described by Ramon (1924) as "substances used in combination with a specific antigen which produce a more robust immune response than the antigen alone". This definition includes a variety of materials. However, a comprehensive assessment of adjuvants used by the Food and Drug Administration in humans limited the use of aluminum salts. These salts show a good safety result, but comparative animal studies show that it is a good adjuvant for inducing antibodies in vaccines with recombinant proteins, and induces a type Th2 response, more than the desired type Th1 response (Gupta, 1998), which is more effective in protecting against intracellular pathogens. In addition, toxicity is a key element in the study of adjuvants. Consequently, during the first 50 years, permitted adjuvants referred to the aluminum salts.

For prophylactic immunization in animals, only adjuvants that produce minimal adverse effects, locally and systemically, can be accepted. Important issues in the development of adjuvants are the reaction at the injection site, the elimination or biological degradation of the adjuvant, and the duration and retention at the injection site. The types of adjuvants used in animals are summarized below (Singh and O'Hagan, 2003):

● Mineral salts: Aluminum hydroxide, aluminum phosphate.

● Immunostimulating adjuvants: Cytokines, saponins, bacterial DNA, LPS (lipopolysaccharide), lipopeptides.

● Lipid particles: Emulsions by Freud, ISA 25, 51, 206, SAF, MF59, liposome. ● Adjuvant mucus: Mutant toxins, including LTK63 and LTR72, microparticles, polymerized liposomes, chitosan.

Mineral salts, especially those of aluminium, stimulate the production of antibodies in a Th2 response. However, they have the disadvantage that they produce a low impact in inducing a cellular immune response. These adjuvants are also known to induce immunity in the short term, implying the use of multi-prime injections. In addition, it is not unusual to observe granulosis at the injection site (Nicklas, 1992).

The Freud adjuvant has been used for more than 50 years, especially when limited amounts of antigen are available. However, its toxicity has been known for a long time, and with the growing interest in the welfare of laboratory animals, there is considerable pressure to limit their use (Morris et al., 1999). In the veterinary industry, the more widely used adjuvants in veterinary vaccines are mineral oil emulsions (oil-in-water or water-in-oil), such as ISA 25,51, 206, MF59. MF59 is the oil-in-water emulsion that has been most evaluated for its use as an adjuvant. It is a microfluidized emulsion containing a terpenic derivative, squalene, together with two surfactants: Tween 80, and Span-85, in sodium citrate buffer. Squalene is a natural compound obtained from plants and animals. It is not an adjuvant per se, but the squalene emulsions with surfactants improve the immune response of humans and animals. An example is MF59 (US patent 6,299,884 B) which is an adjuvant produced by Novartis Lab, and is added in

the flu vaccine.

Saponins are another type of adjuvant used. These come from the Chilean tree Quillaja saponaria Molina, and consist of terpenoid derivatives, which have been used on humans and animals. The raw extract from this tree is called saponin. Quil A, and spicoside are partially purified mixtures, and QS21 is a fraction. Quil A is widely used in veterinary medicine, and has been used in farm animal, pig, horse, dog and cat vaccines, including the FeLV vaccine for the equine influenza virus, parvovirus. QS21 is used in the FeLV vaccines, and the dog vaccine for Lyme disease (Kim et al., 2006).

WO 2012178118 A1 describes pharmaceutical formulations comprising a combination of selected carriers, vitamins, tannins and flavonoids as antigen-specific immunomodulators.

WO 2010078556 A1 describes adjuvant formulations and uses thereof.

WO 0010600 A2 describes the activation and protection of T-cells (CD4+ and CD8+) using an H2-receptor agonist and other T-cell activating agents.

DETAILED DESCRIPTION OF THE INVENTION

The purpose of the present invention is to provide a formulation containing an active principle with immunostimulating/adjuvant activities obtained from the resinous exudate of the plant species Heliotropium huascoense, and which is useful in salmonid species to increase the immunogenicity of an antigen and/or the components of a vaccine.

The present invention produces a formulation with immunostimulating/adjuvant activity, comprising:

a) (-)-alpinone, whose structural formula is:

where R1 is OH, and R2 is OCH3, with an optical activity of [ α]25ºD= -28.1 (c.0.215,CHCl3), and an S configuration of carbons 2 and 3;

b) dimethyl sulfoxide; and

c) salt solution.

The formulation preferably comprises a) (-)-alpinone: 0.05% w/v to 0.5% w/v

b) DMSO: 0.8 to 8.0% w/v, and

c) Saline: Amount required to complete 100% volume.

The present invention further produces a formulation with immunostimulating/adjuvant activity according to the invention, for use as a vaccine for vertebrates.

Preferably, said vertebrate is a salmonid species.

The formulation's active principle is the flavonoid (-)-alpinone, and it shows no toxicity in treated individuals, on the contrary, it provides an improvement in general appearance, probably due to its antioxidant and stress-controlling properties in individuals undergoing treatment.

This active principle has the property of inducing maturation in dendritic cells, which corresponds to the first stages of induction of the immune response in higher vertebrates.

The active principle in the formulation according to the present invention is the flavonoid 3,5-dihydroxy-7-O-methoxyflavanone (Alpinone). The formulation according to the present invention differs from other previously patented ones in that it is a formulation in which the active agent is a natural flavonoid obtained from vegetable species, and is therefore biodegradable, since it does not affect the environment. In the present invention, a formulation has been developed according to which certain amounts of an active agent, i.e. a phenolic compound (flavonoid), are available, where positions 3 and 5 are hydroxylated, position 7 is methoxylated, and in position 4 there is a carbonyl. It has two stereogenic centers, where the configuration of carbons 2 and 3 must be "S". In the formulation according to the present application, the flavonoid is in association with pharmacologically accepted diluents, including, but not limited to, dimethyl sulfoxide, and saline.

Specifically, the formulation according to the present immunostimulating/adjuvant invention for fish consists of:

DMSO: 8% w/v

(-)-alpinone: 0.5% w/v (alpinone must present an "S" configuration in its stereogenic centers).

Salt solution: 91.5% weight/volume

The formulation has as an active ingredient the flavonoid with general formula:

where R1 must be OH, and R2 must be OCH3. In addition, it must have an optical activity of [ α]25ºD= -28.1 (c.0.215,CHCl3), and an "S" configuration of carbons 2 and 3.

The present invention provides an immunostimulatory formulation that is not specifically directed against a selected pathogen, or a grouping of selected pathogenic agents.

The purpose of the present invention is to provide an immunostimulating/adjuvant formulation that can be administered intramuscularly to trigger the immune response in salmonid species and/or improve the response to the existing vaccines.

The present invention has the advantage over other adjuvants of market quality, including, but not limited to, Montanide, where the active principle of the formulation is used in low doses, and in the zone where it is injected, no necrosis or damage is observed around the tissue. Instead, the market-grade adjuvant, Montanide, generates granulomas at the injection site (Mutoloki et al., 2004). Furthermore, the injectable solution according to the present invention requires a smaller amount of active principle, in contrast to the adjuvants of market quality.

DESCRIPTION OF THE FIGURES

Figure 1. Analysis of the expression of MHCII in mouse dendritic cells by flow cytometry.

Figure 2. Levels of cytokine mRNA in SHK-1 cells stimulated with potential immunostimulatory compounds.

Figure 3. Levels of cytokine transcripts in the kidney of fish treated with immunostimulating compounds.

Figure 4. Analysis of the humoral response in fish immunized with ovalbumin.

Figure 5. Detection of viral RNA by RT-PCR.

Example

Example 1: The effect of the active principle on the maturation of mouse dendritic cells

The immunostimulatory ability of the active principle of the formulation of the present invention in the primary culture of mouse dendritic cells (DC) was tested. For this experiment, dendritic cells obtained from mouse bone marrow were cultured for 6 days in an RPMI-1640 medium supplemented with 10% fetal calf serum (SFB), 4 mM L-Glutamine (Hyclone), penicillin/streptomycin (Hyclone), and 10 ng/ ml GM-CSF. After this time, the cells were incubated with 5 µg of the formulation's active principle for 6 h at 37 ºC. The cell triggering state was evaluated by the expression of class II MHC on the surface of mouse DC by using a specific antibody (FITC-antimouse IAd, BD, USA) to detect that molecule by flow cytometry. The observed results in this experiment (figure 1) indicate that the active principle increases the expression of class II MHC molecule in dendritic cells. This indicates that the compound induces maturation of the dendritic cells, which is a property of compounds used as immunostimulants or adjuvants. In conclusion, the active principle of the formulation has immunostimulatory activity, more precisely produces the triggering of mouse dentritic cells.

Example 2: The effect of the active principle on cytokine expression in the SHK-1 cell line

The evaluation of the immunostimulatory ability was also performed in the SHK-1 cell line which corresponds to cells derived from an enriched culture of macrophages obtained from the hind kidney of Atlantic salmon. How the immunostimulants will trigger the expression of cytokines that are essential for triggering the immune response in the individual, and this happens by activating the gene transcription; the increase in the expression of pro-inflammatory cytokine genes, including IL-1, IL-8, IL-10, IL12 and TNFα; anti-inflammatory cytokines, including TGFβ1; and the antiviral cytokine IFNα was quantified. The cells were cultured in 6-well plates during 3 days until a confluence of about 90% was reached; then they were incubated with 5 ug of the formulation's active principle for 24 h at 15 ºC in an L-15 medium supplemented with 10% SFB, 4 mM L-Glutamine, 40 uM 2-mercaptoethanol, 50 ug/ml Gentamicin. To obtain RNA, cells were detached by mechanical activation of the wells, concentrated by centrifugation, and homogenized in 1 ml of Trisure (Bioline, USA). Then 200 µl of chloroform was added to each sample and they were mixed in a vortexer for 1 min. The suspension was centrifuged at 13200 x g for 15 min at 4 ºC. The aqueous phase was collected and transferred to another tube containing 500 µl of cold isopropanol (Merck). The mixture was incubated for 20 min at -20 ºC, and was centrifuged again at 13200 x g for 10 min at 4 ºC; the supernatant was removed, and 1 mL of 75% cold ethanol was added to each tube. The tubes were centrifuged at 6000 x g for 5 min at 4 ºC, the supernatant was discarded, and the RNA pellet was dissolved in 20 µl of nuclease-free water (Invitrogen). RNA was incubated at 65 ºC for 10 min, and was stored at -20 ºC. The contaminating DNA cleavage in the sample was carried out by a treatment with DNAsa RQ1 (Promega). 2 µg of RNA was processed according to the conditions stated by the manufacturer. cDNA synthesis was performed using 200 U/ul reverse transcriptase enzyme M-MLV (Promega), 500 ng/ul oligo dT (Promega), 1 mM dNTPs (Bioline), and enzyme 5X (Promega) buffer in a final volume of 25 ul. The mixture was incubated for 60 min at 42 ºC, and then at 70 ºC for 15 min. Generated cDNA was stored at -20 ºC.

The quantitative real-time (qRT-PCR) reaction was performed in 96-well plates (AXIGEN) covered with optical lids on the STRATAGENE 7300 instrument. Each reaction was performed in a final volume of 25 µl, using 12.5 µl of Quantace SYBR Green /ROX qPCR master mix (2X) (Bioline), 1.25 µl sense primer (10 µM), 1.25 µl antisense primer (10 µM), 8 µl ultrapure distilled water (Invitrogen), 2 µl cDNA 1: 2 dilution. In all reactions, controls were performed without quenching. The results show that the formulation's active principle induces an increase in the expression of pro-inflammatory cytokines, including IL-1, IL-8, TNF-α; the antiviral cytokine IFNα, as well as cytokines important in this T-helper 1 (Th1)-type response, such as IL-12 (Fig. 2). The R compound used as a positive control increases the expression of cytokines, mainly of the pro-inflammatory type, including IL-1, IL-8 and IL-12 (Figure 2). These results show that the formulation's active principle can induce high levels of proinflammatory, antiviral and Th1 response-inducing cytokines in the SHK-1 cell line of salmon.

In conclusion, it has been established that the formulation's active principle can be used as an immunostimulant/adjuvant to trigger an early antiviral response and a type-Th1 response.

Example 3: The effect of the active principle as an immunostimulant in Atlantic salmon

For the in vivo analysis, 15 Atlantic salmon of 40–50 g were used, which were injected intramuscularly (IM) with doses of 100 µg of the formulation's active principle in a final volume of 100 µl. As controls, fish were injected with 8% DMSO (formulation component) dissolved in 100 µl saline, and with 5 mg of the control compound Ribomunyl dissolved in 100 µl saline. As a control group, one group of fish was only injected with 100 µl saline, while the other was not injected. After 48 h, the fish were euthanized with a benzocaine overdose, and their kidney and spleen were removed for subsequent analysis. To obtain RNA, organs were disintegrated in 1 ml of Trisure using a tissue homogenizer. Then, the same protocol used in the RNA extraction experiment from SHK-1 cells was performed. As a result, it was observed that in the kidney, the active principle of the formulation increases the expression levels of pro-inflammatory cytokines IL-1 and IL-8, and of IL-12 (type Th1 cytokine). In addition, it increases the levels of the anti-inflammatory cytokine TGF-β1 and of the antiviral cytokine IFNα (Figure 3).

The compound Ribomunyl, used as a control for its known effects on mouse cell activation (Spisek et al., 2004), increased the expression of IL-1, TNF-α, IL-12 and IFNα (Fig. 3). Furthermore, the kidney from the control, DMSO, saline and untreated fish presents no changes in the expression of such cytokines.

In conclusion, according to the present application, the formulation's active principle has immunostimulating properties in salmonid fish.

Example 4: The effect of the active principle as an adjuvant on vaccines for salmonids

In order to evaluate the adjuvant ability of the formulation's active principle from the present invention, an antigen-dependent lymphocytic proliferation experiment was performed using ovalbumin as an immunogen. That experiment is performed in lymphocyte cultures that proliferate clonally when stimulated with the antigen. This only happens when a previous immunization with the antigen has occurred. A protein such as ovalbumin requires an adjuvant compound for immunization to occur. Accordingly, groups of 350 g fish (rainbow trout) received two doses of the formulation combined with the protein ovalbumin (Sigma). Groups in the experiment received the injectable solution (formulation according to the present application) consisting of 100 µg of combined compound with 10 µg of protein ovalbumin dissolved in 100 µl of saline (0.9% NaCl). A positive control, Montanide ISA 763 a VG (Seppic, Francia), an oily adjuvant used in veterinary vaccines, was used and it was mixed with 10 µg ovalbumin. The preparation of Montanide was carried out according to the manufacturer's recommendations. On the other hand, two control groups were used, which were divided into fish that received 10 µg of protein ovalbumin dissolved in saline, and fish that were injected with 100 µl of saline. The dose of ovalbumin was determined in a previous experiment according to a protocol carried out by Joosten et al., 1997. Fish were kept in aquariums with 30 l of fresh water, and a continuous supply of oxygen to a biomass of 10–12 kg/m3 at 15 ºC. A pellet of market quality was used for feeding, and was administered in the aquariums once per day.

Temperature, pH, oxygen and ammonium levels were monitored daily to maintain parameters within optimal ranges. In addition, the aquarium's water was changed daily.

After 5 days, the spleens of treated fish were disintegrated in 100-mesh mesh with RPMI-1640 (Gibco) medium supplemented with 10% fetal calf serum, 4 mM L-Glutamine (Hyclone), 40 uM 2-Mercaptoethanol (Gibco) and 50 ug/ml Gentamicin (USB Biological). The cell suspension was centrifuged at 2600 x g for 5 min at 4 ºC.

The cell pellets were resuspended in supplemented RPMI medium, and the number of cells was determined in a hemocytometer, and viability by Trypan blue staining on an inverted phase contrast microscope. For in vitro stimulation, 1x106 cells were seeded in 96-well plates with 100 µl RPMI/s medium plus 50 µg ovalbumin (OVA). After 3 days, 18 h before the completion of the experiment, 1 µl of tritiated thymidine (3H-Thymidine) was added, 5 µCi/ml (PerkinElmer) to each well. Then the cells were resuspended in 1 ml of ultrapure water, and the suspension was transferred to fiberglass filters that were vacuum dried. The filters were placed in scintillation fluid, and radioactivity was quantified in a scintillation counter (Tri-Carb 2100 TR; Packard). Experimental data were expressed in counts per minute (CPM).

As a result, it is observed that the cultures from fish immunized with ovalbumin and Montanide (OVA+Montanide) presented higher levels of incorporated thymidine than those from the cells of control fish injected with only ovalbumin (OVA) (Table 1), confirming the adjuvant effect of the control compound. Furthermore, an increase in thymidine incorporation is also observed when the formulation is used (table 1).

Example 5: Analysis of specific antibodies

Immunization with ovalbumin in a formula containing the active principle as an adjuvant also induces a better humoral response. Specific IgM was quantified for OVA by ELISA in serum after the immunization. In this analysis, sera from fish treated with the formulation (100 μg active principle, 8% DMSO and 100 μl saline solution) were used. In the experiment, Maxisorp (Nunc) 96-well plates activated with 10 μg of the ovalbumin protein were used at 37 ºC overnight. The next day, 200 μl of blocking solution (1% BSA, 5% sucrose in saline phosphate buffer) was added to each well, and the plates were incubated for 30 min at 37 ºC. Then the blocking solution was removed and 100 μl of serum from each treated fish was added to each well in triplicate, and the plates were incubated for 1 h at 25 ºC. After this time, the wells were washed with 0.05% Tween 20 in PBS. Then 100 ul of trout anti-IgM antibody was added to the wells, and then the plates were incubated for 1 h at 25 ºC. Again, the wells were washed and incubated for 30 min with 100 µl mouse anti-immunoglobulin antibody conjugated to alkaline phosphatase (Sigma) diluted 1:1000. Finally, the wells were washed, and 100 µl of developing solution (50 mM Na 2 CO 3 , 2 mM MgCl 2 , and 14 mg p-nitrophenyl phosphate) was added to each well. The plates were incubated for 15 min at 37 ºC and analyzed in an ELISA reader at 405 nm.

As a result, it was noted that there was a greater amount of specific antibodies against OVA in the group of fish treated with the adjuvant Montanide (Figure 4) compared to the rest of the control groups and fish treated with the formulation according to the present invention, and the formulation according to the present application also present a small increase in the levels of specific antibodies against OVA compared to the controls which is statistically significant (Figure 4).

Example 6: Antiviral effect of the formulation's active principle

The increase in the transcribed IFNα levels in SHK-1 cells treated with the active principle suggests that IFNα may also be secreted into the culture medium. In this case, the supernatant of the treated cells must also show an antiviral effect. To verify this hypothesis, the supernatant effect on ISA virus proliferation in ASK cell monolayers was evaluated.

To obtain conditioned supernatants, SHK-1 cells were grown in 6-well plates at 15 ºC in Leibovitz L-15 medium supplemented with 10% SFB, 4 mM L-Glutamine, 40 µM 2-mercaptoethanol and 50 µg/ml Gentamicin until a confluence of 90–100% was reached, and they were stimulated by adding 5 µg of the formulation's active principle or 20 µg/ml Poly I:C (Sigma) to each well, used as a control. The plates were incubated for 24 h, and medium from each well was collected afterwards, and was centrifuged at 3000 rpm for 5 min at 4 ºC. The supernatants were stored at -80 ºC. Furthermore, the ISA virus spread in monolayers of SHK-1 cells at a 60% confluence, which were inoculated with the virus in an infection medium (MOI 0.1)( Leibovitz L-15 medium without SFB, 4 mM L-Glutamine, 40 uM 2-Mercaptoethanol, 50 ug/ml Gentamicin). The protocol described by Jensen et al., 2002, was followed.

ASK cells were used for the antiviral activity experiment. (Rivas–Aravena et al., 2011). Cells were spread in 48-well plates until a confluence of 90–100% was reached, then incubated for 48 h with 1:100 dilutions of each supernatant from both stimulated and unstimulated SHK-1 cells. Then the cells were infected with ISAV at a dilution of 1/40 in L-15 medium. At days 2, 4 and 7 post-infection (PI), the supernatant was collected and viral RNA was extracted using a Kit according to the manufacturer's protocol (Omega Biotek, E.N.Z.A).

The inhibition of the viral replication was determined by RT-PCR, this method being more specific, more sensitive and faster in detecting and identifying the virus. For this, specific primers targeting fragment 8 of the ISA virus were used. 10 µl of the RNA obtained from the extraction was incubated with 1 µl sense starter (10 µM), 1 µl antisense starter (10 µM) (see Table 1), 5 µl master mix (Kit one step Syber Green), and 3 µl ultrapure water (Invitrogen). cDNA was synthesized, and PCR was performed in the Stratagene 7300 equipment.

Table I: Sequence of primers used for detection of the ISA virus present in ASK cell cultures.

As a result, we noted that the supernatant from SHK-1 cells, stimulated with the active principle of the formulation, delays the viral replication until day 4 (Figure 6), in contrast to the supernatant obtained by stimulation with Poly I:C. The results indicate that there is a compound with antiviral activity induced by the active principle of the present invention in the supernatant of stimulated cells, which probably corresponds to a type-I IFN. Therefore, the formulation's active principle will be useful in triggering an early antiviral response.

Figure 1 shows the mean fluorescence intensity (MFI) for expression of MHCII in untreated control DC, and DC treated with 0.5 or 5 µg of the active principle of the formulation. R is a compound used as a positive control. As a negative control, untreated DC was used, as well as DC treated with DMSO (0.02%) corresponding to the vehicle used to dissolve the studied compound. Results were analyzed by one-way ANOVA along with Bonferroni's multiple comparison post-test (*P<0.05).

Figure 2 shows the levels of cytokine mRNA in SHK-1 cells stimulated with potential immunostimulatory compounds. For that, SHK-1 cells were incubated with the control compound Ribomunil and with the formulation's active principle, as well as DMSO solvent for 24 h at 15 ºC in supplemented L-15 medium. The expression of cytokine transcripts was evaluated by qRT-PCR. The graph shows the normalized and relative expression. Normalization was performed relative to the untreated cultures and the constitutively expressed gene 18s. Statistical differences were determined by the non-parametric t-test (Mann-Whitney) among the untreated and experimental groups (* P<0.05).

Figure 3 shows the levels of cytokine transcripts in the kidney of fish treated with immunostimulating compounds. Groups of fish (Salmo salar) were injected IM with 100 µg active principle in 100 µl saline, with the control compound Ribomunil, 8% DMSO, or with saline. The expression of cytokine transcripts was evaluated by qRT-PCR. The graph shows the normalized and relative expression (NRE). Normalization was performed in relation to the levels of expression in untreated fish and in relation to the constitutive expression of gene 18s. Statistically significant differences were determined by the non-parametric t-test (Mann-Whitney) between untreated fish and experimental fish (* P<0.05).

Figure 4 shows the analysis of the humoral response in fish immunized with ovalbumin. ELISA plates were activated with 10 µg ovalbumin overnight in phosphate buffer. 100 µl sera from fish treated with the formulation plus OVA, ISA763 plus OVA, only OVA, and untreated were added to the plates and incubated for 1 h at room temperature. 100 µl of trout anti-IgM antibody was added to each well, and finally the plates were incubated with an IgG anti-mouse antibody. The results were analyzed in a BIORAD plate reader.

Figure 5 shows the detection of viral RNA by RT-PCR. ASK cell monolayers were infected with a 1:40 dilution of the ISA virus. On days 2, 4 and 7, the supernatant was collected and the extraction of viral RNA was performed. Samples were analyzed by RT-PCR. Ct indicates the number of cycles at which amplification starts. (The higher the Ct, the lower the viral RNA present in the sample).

Table 2. Proliferation of antigen specific for the rainbow trout splenocytes immunized with ovalbumin (OVA).

The ovalbumin (10 µg) was injected neat, with ISA 763 (Montanide OVA), and together with the formulation. After extracting the splenocytes from the spleens, they were cultured for 3 days with 50 µg ovalbumin. After that time period, tritiated thymidine was added, and the addition of thymidine was quantified 18 h later in a scintillation counter. Each value represents the mean of 1 experiment in triplicate with 3 fish in each experimental group. Statistically significant differences were determined by one-way ANOVA together with a non-parametric t-test (Mann-Whitney) (* P<0.05).

Table III: Comparative chart between the proposed invention and the competition (market quality adjuvant Montanide).

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Claims (4)

P a t e n t requirement 1. Formulation with immunostimulating/adjuvant activity, where it comprises: a) (-)-alpinone, whose structural formula is: where R1 is OH, and R2 is OCH3, with an optical activity of [ α]25ºD= -28.1 (c.0.215,CHCl3), and an S configuration of carbons 2 and 3; b) dimethyl sulfoxide; and c) salt solution. 2. Formulation according to claim 1, where it comprises: a) (-)-alpinone: 0.05% w/v to 0.5% w/v b) DMSO: 0.8 to 8.0% w/v, and c) Saline: Amount required to complete 100% volume. 3. Formulation with immunostimulatory/adjuvant activity according to claim 1 or 2, for use as a vaccine for vertebrates. 4. Formulation according to claim 3, wherein said vertebrate is a salmonid species.