Vaccines against protozoan parasites

Abstract

In a first aspect, the present invention relates to vaccines for preventing or treating protozoan parasite infections in an individual, whereby the vaccine contains multiple protozoan variable surface antigens (VSAs) of said protozoan parasite microorganism. In particular, protective vaccines are disclosed. In a further aspect, a method for the preparation of the vaccine, particularly the protective vaccine, is described. The vaccine comprises multiple distinguishable VSAs, preferably comprising the entire repertoire of the different VSAs encoded by the gene family in said protozoan parasite. In a second aspect, the present invention relates to vaccines for preventing or treating microbial infections in an individual, whereby the vaccine contains microvesicles induced by confronting protozoan microorganisms with antibodies directed to their VSAs. In particular, protective vaccines are disclosed. The vaccine is composed of multiple antigens of said protozoan parasite. In a further aspect, methods for the preparation and administration of these vaccines, particularly the protective vaccines, are described.

Description

[0001] Vaccines against protozoan parasites

[0002] In a first aspect, the present invention relates to vaccines for preventing or treating protozoan parasite infections in an individual, whereby the vaccine contains multiple protozoan variable surface antigens (VSAs) of said protozoan parasite and / or released microvesicles of said parasites produced by treating a parasite clone with an antibody directed to its expressed single type VSA. In particular, protective vaccines are disclosed. In a further aspect, a method for the preparation of the vaccines, particularly the protective vaccines, is described. The vaccines involve parasite populations expressing multiple distinguishable VSAs, preferably comprising the entire repertoire of the different VSAs encoded by a family of homologous genes in said protozoan parasite. The parasite clone is induced to switch its VSA by confronting the protozoan microorganism with antibodies directed to their expressed VSA, resulting in a population in which each parasite expresses a different VSA other than the initial or original VSA of said clone. In a second aspect, the present invention relates to vaccines for preventing or treating microbial infections in an individual, whereby the vaccine contains microvesicles induced by confronting protozoan microorganisms with antibodies directed to their expressed VSA. In particular, protective vaccines are disclosed. The vaccines may further contain multiple antigens of said protozoan parasites apart from the VSAs. In a further aspect, methods for the preparation and administration of these vaccines, particularly the protective vaccines, are described.

[0003] Prior Art

[0004] A characteristic developed by parasitic organisms is their great capacity to adapt to environmental changes. Most parasites, whether unicellular, such as protozoa, or multicellular, such as helminths, occupy different niches during their passage through vectors and hosts and have evolved extraordinary defense mechanisms that allow them to survive in environmental conditions that would otherwise destroy them. The complexity of these defense mechanisms is a fascinating challenge for researchers in the field. Thus, the survival of pathogenic microorganisms depends not only on their ability to colonize a host but also on their ability to counteract its defense mechanisms. Therefore, the pathogenicity or virulence of parasites reflects the dynamic interaction between them and the host and their capacity to respond to the defensive systems of the infected individual, a necessary condition for parasitic survival and the maintenance and / or transmission of the infection. Remarkably, some pathogens, including parasites, have developed mechanisms of evasion of the immune response (i.e., antigenic variation), which allows them to survive and complete their life cycle within the host (Zambrano-Vila S et aL, Trends Parasitol. 2002;18(6):272-8).

[0005] Antigenic variation is a clonal phenotypic variation developed by some pathogenic microorganisms involving surface-exposed antigenic determinants. These organisms use different mechanisms to change the expression of their surface antigens to maintain chronic infection under continuous immune pressure generated by their host. Antigenic variation in unicellular microorganisms involves three essential requirements: (i) a large family of homologous genes encoding immunodominant surface antigens, (ii) a mechanism that allows the exclusive expression of a single member of that family on individual cells, and (iii) a molecular process that allows the change of the initially expressed antigen to another of the same family (Deitsch KW et aL, Nat Rev Microbiol. 2009; 7:493- 503. DOI: 10.1038 / nrmicro2145).

[0006] Concerning antigenic variation, many parasitic microorganisms are protected by a tight coat of molecules called variable surface proteins / glycoproteins / adhesins (VSAs, also identified as VSPs or VSGs, in the following generally referred to as VSAs), which cover the entire surface of the parasite and towards which the host immune response is generated. Each individual microorganism expresses only one VSA from a repertoire of dozens to thousands of VSAs at any given time, and, for reasons not well known, this antigen is changed to another antigenically different. Thus, the host immune response is evaded, and chronic and / or recurrent infections are generated.

[0007] To date, the only possibility of controlling most parasitic diseases is to use drugs whose active ingredients are not efficient, present many side effects, and have been reported to have resistant strains. In addition, for parasites that manifest antigenic variation, re-infection is frequent after treatment because these individuals do not generate efficient defense mechanisms to eliminate the parasite. In developing countries, the continued mortality and morbidity caused by infections are an indication that ongoing treatments are inadequate. In the genome of the protozoan parasites, there is a multiplicity of genes encoding variable surface antigens. Still, only one is expressed at any given time on the surface of each cell. From this, it is easy to conclude that to counteract this mechanism of parasite evasion, the host should generate antibodies directed against each of these potentially expressed antigens. Naturally, this is impossible, but what can be done is to stimulate the mechanism of antigenic variation of the parasites so that they can be "forced" to express the entire repertoire of their variable antigens in a population. Thus, immunizing individuals using these cells as a vaccine would be possible and generate a specific and protective immune response in the host against the entire repertoire of potential surface antigens. In the event of subsequent reinfections, the immune system will respond rapidly to these parasites thanks to the memory immune response.

[0008] It was recently determined that low concentrations of antibodies directed against variable surface antigens of known isolates of Giardia lamblia (Variantspecific Surface proteins or VSPs), Trypanosoma brucei (Variant Surface Glycoproteins or VSGs), and even of the free-living protozoan Tetrahymena thermophila (i-Antigens) induce antigenic switching. Indeed, the incubation of clones of these protozoa with antibodies directed against their corresponding variable surface antigen generated parasite populations expressing multiple variants, and extracellular microvesicles containing the antibody target antigen were generated during this process (Tenaglia AH et al. https: / / www.biorxiv.org / content / 10.1101 / 2022.06.21 .497077v1 ; Tenaglia AH et al. Nature Communications. 2023. 14(1 ):2537. DOI: 10.1038 / s41467-023-38317-8). Therefore, these populations could become a fundamental tool to generate vaccines against protozoan parasites for which antigenic variation has been demonstrated but also for others in which this phenomenon has not been yet described (Trypanosoma cruzi / Trichomonas vaginalis / Tritrichomonas foetus).

[0009] The reports on stimulating antigenic variation in protozoa were only based on defining the molecular and structural processes of the antibody / variable surface antigen interaction. They were not considered that they could be used in formulations that induce a protective response against infections by these microorganisms, given that, at present, there are still no vaccines against parasites that manifest antigenic variation. The flagellate protozoan Giardia lamblia (syn. G. duodenalis or G. intestinalis) is one of the most common causes of human intestinal disease worldwide and one of the most frequent enteric pathogens of domestic and farm animals, as it is possibly the only microorganism capable of colonizing both the duodenum and jejunum, a highly hostile environment where nutrient digestion takes place. Infection is initiated by ingesting parasite cysts in contaminated water or food. Giardia has been reported as the cause of outbreaks of waterborne diarrhea in developed countries and in cases of diarrhea in daycare centers, institutionalized people, and travelers. In Asia, Africa, and Latin America, about 200 million people suffer from symptomatic giardiasis, and about 500,000 new cases are reported each year. Giardia infections are almost universal in regions with poor sanitation by two years of age. Giardia has been recognized as a re- emerging infection and included in the "Neglected Diseases Initiative" by WHO. In addition, clinical manifestations of giardiasis, such as diarrhea, anorexia, weight loss, and lethargy, have been associated with giardiasis in both domestic and farm animals. Some antiparasitic drugs are commonly used to treat giardiasis in animals and humans, but reinfections are frequent, and drug-resistant strains have already been described.

[0010] Giardia has a simple life cycle consisting of an infective cyst and a vegetative trophozoite. Infection is transmitted by ingesting cysts, which are excreted in the feces. After excystation in the upper small intestine, flagellated trophozoites are released. Trophozoites are non-invasive, proliferate attached to the surface of intestinal epithelial cells by a sucking disc, but do not invade the mucosa and cause little or no mucosal inflammation. Trophozoites are responsible for the clinical manifestations associated with the disease, which range from asymptomatic infections to severe acute or chronic diarrhea. Some individuals are chronically infected without symptoms of diarrhea but suffer malabsorption and, predominantly in young individuals, long-term detrimental effects on growth and development. The host's immune status influences its susceptibility to infection and the severity of clinical signs. Young or elderly individuals are especially susceptible to Giardia infections. Symptoms usually appear one to two weeks after infection and last 2 to 5 days. Giardiasis appears to be limited, but in some cases, chronic infections occur without any apparent immunodeficiency. The prevalence of Giardia has been studied, and isolates obtained from different hosts were characterized at the molecular level to determine their zoonotic potential (Sprong H et aL, PLoS Negl Trop Dis. 2009;3(12):1 -12. DOI: 10.1371 / journaLpntd.0000558). Phylogenetic analyses identified eight assemblages (genetic groups) in G. lamblia: A to H. It was observed that assemblages A and B could infect several mammals and become reservoirs for human infections, just as humans may also be a potential reservoir of Giardia for domestic and production animals. G. cam's (assemblage C / D), G. cati (assemblage F), and G. bovis (assemblage E) would be limited to certain species or host types. In contrast, other Giardia assemblages have a broad host range, including assemblages A and B that infect humans and can be transmitted to livestock and domestic animals (Thompson RCA et aL, Parasitol Today. 2000;16(5):210-3 (11 -13). DOI: 10.1016 / s0169-4758(99)01624-5).

[0011] Antigenic variation in Giardia involves Variant-specific Surface Proteins (VSPs). VSPs are integral cysteine-rich membrane proteins that possess a variable extracellular N-terminal region (ectodomain), a conserved C-terminal region that includes a single transmembrane domain, and a short cytoplasmic tail of only five amino acids. The ectodomain of VSPs allows the parasite to survive in the upper portion of the small intestine. These proteins are highly resistant to pH changes (between pH 1 and 10) and to degradation by trypsin and other proteases in the gastrointestinal tract (Serradell MC et al. Nature Communications.

[0012] 10(1 ):361 . DOI: 10.1038 / s41467-018-08265-9). VSPs form a thick layer over the parasite that represents the host-parasite interface. Only one VSP, out of a repertoire of approximately 140 homologous genes present in the parasite genome, is expressed on the surface of each trophozoite at any given time (Nash TE., Philos Trans R Soc B Biol Sci. 1997;352: 1369-75. DOI:

[0013] 10.1098 / rstb.1997.0122), but the switch to the expression of an antigenically distinct VSP has been reported to occur even in the absence of any immune pressure, albeit with a very low turnover frequency. Post-transcriptional and epigenetic mechanisms have been reported to regulate the expression of a single variant in each trophozoite (Prucca CG et aL, Nature. 2008; 456(7223):750-4. DOI: 10.1038 / nature07585). Since disruption of the mechanism of antigenic variation has previously been shown to be crucial for generating effective vaccines (Rivero FD et aL, Nature Medicine. 2010;16(5) :551 -7. DOI: 10.1038 / nm.2141 ), the utilization of Giardia populations containing trophozoites expressing the complete repertoire of VSPs resistant to proteolytic degradation would allow their use as a vaccine formulation to be administered orally to generate a mucosal immune response that protects against subsequent infections. The anti- Giardia vaccine developed by disrupting the RNAi pathway by knocking down the expression of Dicer and / or RdRP allowed the expression of the entire repertoire of VSP in individual trophozoites (W02010064204A2; Rivero FD et aL, Nature Medicine. 2010;16(5):551 -7. DOI: 10.1038 / nm.2141 ; Serradell MC et al. npj Vaccines.

[0014] 1 :16018. DOI: 10.1038 / npjvaccines.2016.18). An oral vaccine was generated after purifying these antigens from these genetically modified parasites (Rivero FD et aL, Nature Medicine. 2010;16(5):551 -7. DOI: 10.1038 / nm.2141 ).

[0015] Recently, antibodies against VSP-expressing clonal populations have been demonstrated to induce antigenic variation in wild-type parasites, replacing the original VSP with a multiplicity of VSPs in different trophozoites of the resulting population. In addition, it was verified that the original VSP was released to the extracellular medium into microvesicles while switching to other VSPs was taking place (Tenaglia AH et al. Nature Communications. 2023, 14(1 ):2537. DOI: 10.1038 / s41467-023-38317-8). This occurs without disrupting the RNAi pathway in the trophozoites, and the entire repertoire of VSP is represented in the resulting population and not in individual cells.

[0016] Human African Trypanosomiasis, or Sleeping Sickness, is a vector-borne parasitosis caused by Trypanosoma brucei, a protozoan transmitted by the bite of the tsetse fly. It is a lethal infection without treatment. This parasitosis is endemic in 36 countries in sub-Saharan Africa, where tsetse flies, specifically of the Glossina spp. transmitting the disease is present.

[0017] Human African Trypanosomiasis caused devastating epidemics during the 20th century. However, the number of reported cases has declined thanks to consistent and coordinated efforts in recent years ("WHO Human African Trypanosomiasis" 2022). The disease remains endemic in sub-Saharan Africa, representing a major problem for rural communities, particularly in central Africa. As cases have also been reported in patients from non-endemic countries, this disease should be considered in the differential diagnosis of travelers, tourists, emigrants, and expatriates who have visited or lived in endemic areas. Population displacement, war and poverty are important factors facilitating transmission. The drugs available for treatment are not optimal; without a vaccine, disease control relies on case detection and treatment and vector control.

[0018] Trypanosoma brucei is a hemoflagellate protozoan of the genus Trypanosoma. Two subspecies that are morphologically indistinguishable cause distinct disease patterns in humans: T. b. gambiense, which causes chronic African Trypanosomiasis, and T. b. rhodesiense, which causes the acute form. The third subspecies, T. b. brucei, is a parasite mainly of cattle and occasionally other animals and, under normal conditions, does not infect humans, although the presence of trypanosomiasis in domestic animals, particularly cattle, is a major obstacle to the economic development of affected rural areas (https: / / www.cdc.gov / dpdx / trypanosomiasisafrican / index.html).

[0019] T. brucei is an extracellular parasite that undergoes several developmental transformations both in the blood of the mammalian host and in different compartments of the female tsetse fly. When the parasite is ingested by the tsetse fly from an infected host, the intestinal conditions of the insect allow its morphological change to the procyclic form, followed by the epimastigote form and, finally, the infective metacyclic form in the salivary glands of the vector.

[0020] An infected tsetse fly injects metacyclic trypomastigotes into the skin tissue of a mammalian host. The parasites enter the lymphatic system and pass into the bloodstream. Within the host's bloodstream, they transform into trypomastigotes, which are transported to other sites in the body. Thus, they reach other body fluids (lymph, cerebrospinal fluid) and continue replication by binary fission.

[0021] Initially, the parasites multiply in the subcutaneous tissues, blood, and lymph, known as the endolymphatic phase, characterized by episodes of fever, headache, lymphadenopathy, joint pain, and pruritus. When the parasites cross the blood-brain barrier and infect the central nervous system, this is known as the neurological or meningoencephalic phase. This is usually when the disease's most obvious signs and symptoms (behavioral changes, confusion, sensory disturbances and lack of coordination) occur. Sleep cycle disorders, which name the disease, are evident at this stage. If untreated, the disease is considered fatal, although cases of healthy carriers have been reported.

[0022] The type of treatment depends on the stage of the disease, as the earlier it is diagnosed, the better the chances of a cure. The success of second-stage treatment depends on a drug that crosses the blood-brain barrier to reach the parasite. In the first stage of the disease, pentamidine and suramin are recommended for T. b. gambiense and T. b. rhodesiense infections, respectively. Melarsoprol, a highly toxic arsenic derivative, is used for advanced stages of T. b. rhodesiense infections.

[0023] Other treatments, such as eflornithine or the combination of nifurtimox- eflornithine (NECT) and, more recently, fexinidazole (first oral treatment), are more commonly used in chronic disease. Unfortunately, these drugs are highly toxic, and resistant strains have been found (Fall F et aL, Metabolomics. 2022;18(4). DOI: 10.1007 / sl 1306-022-01880-0).

[0024] The presence of regularly changing environments requires microbial pathogens to adapt rapidly to fluctuating conditions. Like other parasites, Trypanosoma brucei encounters different environments as it transitions between its mammalian host and vector. The parasite has had to evolve in these different environments to optimize its survival and ensure transmission to the next host (Silvester E et aL, PLoS Negl Trop Dis. 2018;12(10):1- 25).

[0025] Trypanosoma brucei regularly changes its main surface antigen, variable surface glycoprotein (VSG), to evade the host immune response. Antigenic variation is a key pathogenic mechanism that allows T. brucei to establish longterm infections under continuous immune pressure generated by its host. VSGs are a family of highly immunogenic proteins (Silva-barrios S et aL, Trends Parasitol. 2017;34(2):1-17. DOI: 10.1016 / j.pt.2O17.10.001 ). However, T. brucei undergoes antigenic variations and sequentially expresses immunologically distinct VSGs, effectively evading the host response. VSG expressions are essential for parasite virulence.

[0026] Trichomoniasis is caused by Tritrichomonas foetus, a sexually transmitted disease listed by the World Organization for Animal Health (OIE). This disease is one of the main causes of early reproductive failure in cattle, causing important economic losses. It is currently considered an endemic disease affecting herds managed under grazing conditions with natural service breeding and where artificial insemination is included as a method for reproduction.

[0027] Recently, T. foetus has been shown to colonize portions of the large intestine of cats and other felids producing chronic, recurrent diarrhea with mucus and fresh blood, often unresponsive to common drugs. Without proper treatment, the disease may resolve spontaneously in months or years, but cats may continue to carry the parasite.

[0028] These microorganisms reside in the epithelium of the preputial cavity of infected bulls; the epithelial crypts of the foreskin provide a microaerophilic environment that favors the replication of these parasites. Consequently, bulls can develop a lifelong infection without showing disease symptoms.

[0029] Trichomonas have been shown to adhere to sperm, causing decreased sperm motility, agglutination, and phagocytosis, and they can survive even during semen cryopreservation. Transmission of T. foetus to the female can cause vaginitis, cervicitis, endometritis, infertility, delayed return to estrus, low pregnancy rate, early embryonic death and even abortions. It has been demonstrated that T. foetus can be invasive for the fetus, found in the placenta, fetal lung, intestine, and lymph nodes.

[0030] Tritrichomonas foetus is perhaps the non-human Trichomonas that is more similar to Trichomonas vaginalis. Besides having three anterior flagella compared to the four of T. vaginalis, there are few morphological differences between the two parasites. Both T. fetus and T. vaginalis contain gene families in their genomes that code for adhesins and variable surface antigens (Burgess DE et aL, Infect Immun. 1990;58(11 ):3627-32. DOI: 10.1128 / iai.58.11 .3627-3632.1990).

[0031] T. foetus is pyriform in shape, has a single nucleus, an undulating membrane with three to five waves, and a characteristic vibratory motion. These features make it a distinctive diagnostic feature when compared with most other protozoa found in preputial samples contaminated with fecal material.

[0032] The life cycle is simple since it only presents the trophozoite stage, although in recent years, a form called pseudocyst has been described. Some authors argue that, under well-controlled experimental conditions in vitro and in vivo in infected bulls, the parasite acquires a spherical or elliptical shape, and the flagella are internalized, but the cells do not show an encysted wall. This form, known as endo flagellar or pseudo cystic, is viable and can revert to pear-shaped trophozoites.

[0033] The drug of choice for the treatment of bovine trichomoniasis is Metronidazole. The therapy has been associated with temporarily resolving clinical signs but cannot control the disease. In recent years, the emergence of aerobic and anaerobic resistance in vivo and in vitro has been described as leading to ineffective treatments (Rivero MB et aL, Acta Parasitol. 2019;64(2):232-5. DOI: 10.2478 / sl 1686-019-00031 -1 ).

[0034] The cellular mechanisms by which T. foetus colonizes mucosal surfaces and causes tissue damage are poorly defined. Little is known about the mechanisms involved in the host-host relationship and how the parasite evades the immune pressure generated by the host. For a better understanding of the biology and biochemistry of this protozoan, several authors have proposed various animal models that have helped to understand this disease better.

[0035] Some studies using Balb / c mice demonstrated that a vaginal infection with T. foetus could be established with only 100 trophozoites and that it could persist for more than 30 days, and even no pretreatment prior to inoculation was necessary for the establishment of the disease. In studies of T. foetus infection during early pregnancy using animal models, changes were found in the local immune response at the maternal-fetal interface and in the expression of uterine epithelial carbohydrates. Some studies in these models concluded that induction of apoptosis is an additional mechanism involved in the pathogenesis of early embryonic death that occurs during infection with T. foetus, with increased cell proliferation being only a compensatory host response.

[0036] As the above demonstrates, developing effective vaccines against protozoan parasites that undergo antigenic variation would avoid infection in hosts and limit the spread of the disease. However, despite the many years researchers have been studying the possibility of implementing new vaccines, there are few commercial vaccines against parasites.

[0037] Brief description of the present invention

[0038] The present inventors aimed to provide suitable vaccines against protozoan parasites that undergo antigenic variation, protecting individuals from infections.

[0039] In a first aspect, the present invention relates to vaccines for preventing or treating protozoan parasite infections in an individual; said vaccine comprising an active agent, and excipients, diluents or carriers and, optionally, adjuvants, wherein the active agent comprises multiple variable surface antigens (VSAs) and / or microvesicles generated on the parasite by confronting said parasite clone with an antibody to its expressed VSA to induce the switching of VSA and the expression of multiple distinguishable VSAs in the obtained population, including the released microvesicles containing the original VSA. In particular, protective vaccines are provided.

[0040] In a further aspect, the present invention relates to a method for producing a vaccine according to the present invention comprising the steps:

[0041] - Providing a protozoan parasite clone expressing a single type of variable surface antigen (VSA) encoded in the genome of said protozoan parasite clone;

[0042] - Culturing said protozoan parasite clone in the presence of a specific concentration of an antibody against the single type of VSA for inducing antigenic variation;

[0043] - Propagation of the cultured and switched protozoan parasites population expressing multiple VSAs other than the single type of VSA the specific antibody is directed against;

[0044] - Obtaining multiple VSAs in a form applicable in a vaccine from the propagated protozoan parasite population and / or obtaining microvesicles from the culture of the propagated protozoan parasite population in a form of an applicable vaccine; and

[0045] - Preparing the vaccine, including mixing the obtained multiple VSAs and / or the obtained microvesicles with excipients, diluents, or carriers.

[0046] Brief description of the drawings

[0047] Fig. 1. Examples of VSPs expression in RNAseq experiments in Giardia

[0048] Sequencing experiments of total mRNA extracted from Giardia trophozoites of all parasite genes (grey dots) except VSPs mRNAs (black dots): Left: Experiment of individual trophozoites (Single Cell) expressing VSP417. Center: clonal population (Clone) of trophozoites expressing VSP417; Right: A Giardia population of a clone expressing VSP417 treated with the anti-VSP417 mAb 7C2 (Population). Individual cells and clones expressing VSP417 show high levels of VSP417 mRNA expression / accumulation (TPM values), which are then expressed on the surface of trophozoites. In contrast, in a non-clonal population obtained by treating for 6 h a clone expressing VSP417, most VSPs are expressed at the level of most housekeeping genes (Population). Fig. 2. Giardia single cell switching in culture in the presence of anti-VSP417 mAb.

[0049] Proliferation and clonality of VSP417 single cell cultures by limiting dilution with an unrelated mAb as control (mAb 8F12) and mAb anti-VSP417 (mAb 7C2). The percentage of wells where parasites grew and the percentage of positivity of the original VSP from the preceding figure is shown. While proliferation remains constant, most cells have switched their VSPs to populations expressing a variety of VSPs.

[0050] Fig. 3. Formation of microvesicles containing the original antigen in Giardia (left) TEM with gold particle-bound antibody labeling of a trophozoite expressing VSP417 incubated for 30 min with mAb 7C2 (black dots), (right) SEM of a VSP417-expressing trophozoite incubated with gold-labeled mAb 7C2 for 30 min showing an image obtained by backscattered electrons (light coloured dots).

[0051] Fig. 4. Protection from infection during vaccination against Giardia of gerbils and hamsters

[0052] Cysts per gram of fecal material collected from gerbils and hamsters vaccinated vs. non-vaccinated with assemblage A (isolate WB, cells plus microvesicles) and assemblage B (isolate GS, cells plus microvesicles).

[0053] Fig. 5. Protection from infections during vaccination against Giardia of dogs and cats

[0054] Cysts per gram of fecal material collected from vaccinated vs. nonvaccinated dogs and cats with assemblage A (isolate WB cells plus microvesicles) and assemblage B (isolate GS cells plus microvesicles).

[0055] Fig. 6. Protection from infections in vaccinated animals against T. brucei

[0056] Parasites per milliliter of blood were collected from Balb / c mice vaccinated with microvesicles or inactivated parasites and unvaccinated animals (Control). Fig. 7. Protection from infection in vaccinated animals against T. foetus

[0057] The number of infected and non-infected animals according to control and vaccinated groups (cells plus microvesicles).

[0058] Detailed description of the present invention

[0059] In a first aspect, the present invention relates to a vaccine for preventing or treating protozoan parasite infections in an individual said vaccine comprising i) an active agent, and ii) excipients, diluents or carriers and, iii) optionally, adjuvants, wherein the active agent comprises a multitude of variable surface antigens (VSAs) and / or microvesicles of the parasite to be treated or to be protected against whereby the multitude of VSAs and microvesicles are obtained by inducing the switching of the VSA of a parasite clone expressing a single type of VSA in the presence of a specific antibody against the single VSA present on the surface of said clone, producing the expression of multiple distinguishable VSAs in the obtained population, wherein a single protozoan parasite of the obtained population expresses one type of VSA and the release of extracellular microvesicles containing the single, original VSA and protective antigens other than VSA.

[0060] As used herein, the term "multiple variable surface antigens" refers to a multitude of proteins / glycoproteins that can be expressed on the surface of parasites such as Giardia lamblia (VSP, variable surface proteins); Trypanosoma brucei (VSG, variant surface glycoproteins; Trypanosoma cruzi (transialidases), Trichomonas vaginalis and T. foetus (adhesins), etc. In an embodiment of the present invention, the multiple VSAs represent the entire repertoire of VSAs encoded in the genome of the protozoan parasite.

[0061] The term "switching the variant of surface antigen" refers to the activation and process of the antigenic variation machinery, as described above.

[0062] The term “microvesicles” refers to extracellular vesicles whose size ranges between 60 and 200 nm that are released by incubating a parasite clone with antibodies directed to its VSA. Typically, these microvesicles are isolated from the supernatant of the culture by known methods. In an embodiment, these microvesicles comprise the VSA initially expressed by the cultured clone before switching. The terms initially or originally are used herein interchangeably unless otherwise indicated. Further, other antigens stemming from the parasite are present.

[0063] The term "protective vaccine" refers to a vaccine that avoids infections in the host.

[0064] By starting the switching process of the VSA in a single clone of the protozoan parasite, it is possible to obtain a population of descendants of protozoan parasite from the single clone expressing multiple different VSAs. Namely, the switching results in the expression of one different type of VSA other than the VSA initially expressed before switching. In addition, microvesicles containing multiple protective antigens can be obtained. This population of protozoan parasites stemming from the single clone can be obtained by incubating the clone with a specific concentration of an antibody against the single VSA present on the surface of the single clone.

[0065] This methodology allows the generation of populations of any parasite that can be cloned and induced to express multiple different VSAs, preferably the entire repertoire of potential VSAs other than the VSA originally expressed, and the release of microvesicles containing original VSA and, in addition, containing additional protective antigens other than VSA. This, in turn, allows the provision of active vaccine agents suitable for treating protozoan parasite infection and for protective vaccination against subsequent infection with the protozoan parasite.

[0066] The protozoan parasites present in the population obtained by switching the single clone do express only a single, different VSA per parasite. In other words, the single protozoan parasite does not express two or more VSA at the same time and does not have different VSA on its surface. Further, the protozoan parasite present in the population are not genetically engineered protozoan parasite containing DNA present in plasmids or vectors or integrated into the genome enabling the expression of different VSAs at the same time.

[0067] In an embodiment, the vaccine, according to the present invention, protects against subsequent infection with the protozoan parasite. As demonstrated, protection against subsequent infection can be achieved. It was not obvious from the prior art whether the vaccine comprising populations of parasites plus microvesicles or microvesicles alone produced by treating the parasites with anti- VSA antibodies protects the vaccinated individual according to the present invention.

[0068] Further, in an embodiment of the present invention, the vaccine against a protozoan parasite is selected from the Trypanosoma, Tritrichomonas, or Giardia genus.

[0069] Moreover, in an embodiment of the present invention, the vaccine is against the protozoan parasite selected from Trypanosoma brucei, Tritrichomonas foetus, and Giardia lamblia.

[0070] According to the present invention, other protozoan parasites with VSAs can be used to generate a protective vaccination with the vaccine as described. In an embodiment, the individual to be vaccinated is a mammal, including a domestic or farm animal or a human. The individual is typically the natural host of the protozoan parasite.

[0071] According to the present invention, the active agent included in the vaccine may be an active agent provided in a form selected from attenuated parasites, dead parasites, parts of the parasites including the plasma membrane, purified VSAs, microvesicles containing parasite antigens, or combinations thereof. The skilled person is aware of suitable methods for preparing the active agent with known techniques. Appropriate procedures are described in the examples below.

[0072] In an embodiment, the vaccine comprises multiple distinguishable VSAs and microvesicles as active agents. The components may be a mixture or separate, allowing administration simultaneously, separately, or sequentially.

[0073] The vaccines may be adapted for mucosal and / or systemic administration. That is, the skilled person may provide the vaccine in a form suitable for administration depending on the protozoan parasite and its location in the body of the individual who can be infected with it.

[0074] Further, the vaccines may be adapted for administration by the mucosal route, like the intranasal route, or orally. Alternatively, the administration may be conducted by intravenous, intramuscular, or subcutaneous administration. For example, administration may be orally or topically.

[0075] The vaccines may contain additional suitable diluents, carriers, excipients, or effluents. The skilled person is aware of this, particularly depending on the way of administration.

[0076] The vaccines may contain additionally one or more types of adjuvants. Adjuvants in the vaccine are known to the skilled person. The term "adjuvant" means substances that are added and / or co-formulated in a vaccine or immunization to an active agent, i.e., the substance that provokes the desired immune response to enhance or elicit or modulate the humoral and cell-mediated (cellular) immune response against the active agent. Preferably, the adjuvant can enhance or elicit the immune response. The skilled person is aware of suitable adjuvants for administration in vaccination strategies.

[0077] According to the present invention, vaccines contain the active agent in a therapeutically or pharmaceutically adequate amount. That is, the amount is sufficient to induce the desired result, such as protective vaccination. The term "administered" means administering a therapeutically effective dose of the vaccine to an individual, that is, a dose that produces the effects for which it is administered.

[0078] The exact dose will depend on the purpose of the treatment and will be ascertainable by one skilled in the art.

[0079] In an embodiment, the vaccine according to the present invention, comprises administering, e.g. orally, two doses of VSAs in an amount of 50 to 500, like 200 pg per g body weight, within an interval of 10 to 20 days, like 15 days. Of course, the exact dosage depends on various parameters of the individual to be vaccinated.

[0080] In an embodiment of the present invention, the vaccine is formulated as a topical creme for administering multiple times, particularly 15 days for two months. In another embodiment, the vaccine is a protective vaccine for administering food or drinking water to treat the mammal.

[0081] The induction of antigenic variation by antibodies against surface antigens may be used to generate novel vaccines that differ from the previous anti- Giardia vaccine (Rivero FD et aL, Nature Medicine. 2010;16(5) :551 -7. DOI: 10.1038 / nm.2141 ) because they are less expensive to produce and more effective since they may use fixed parasites and microvesicles (particles) instead of purified VSAs (soluble proteins / glycoproteins) and do not use genetically modified parasites. Besides, microvesicles provide additional protective antigens (i.e., annexins) and are more effective in generating long-lasting mucosal and systemic immunity.

[0082] Antibodies against VSGs induce antigenic variation, which can be used to generate novel, inexpensive, and effective vaccines. These vaccines may use microvesicles to provide protective antigens and be more effective in generating long-lasting systemic immunity.

[0083] Antibodies against i-Antigens may induce antigenic variation in T. foetus, which can be used to generate novel, inexpensive, and effective vaccines. These vaccines include microvesicles to provide additional protective antigens and be more effective in generating long-lasting mucosal immunity.

[0084] In another aspect of the present invention, a method for producing a vaccine according to the present invention is provided comprising the steps of: - Providing a protozoan parasite clone expressing a single type of multiple variable surface antigens (VSAs) encoded in the genome of said protozoan parasite clone;

[0085] - Culturing said protozoan parasite clone in the presence of a specific antibody against the single type of VSA to induce the switching of VSA;

[0086] - Propagation of the cultured and switched protozoan parasite population expressing switched multiple VSAs other than the single type of VSA the specific antibody is directed against;

[0087] - Obtaining multiple VSAs in a form applicable in a vaccine from the propagated protozoan parasite population and / or obtaining microvesicles from the culture of the propagated protozoan parasite population in a form applicable in a vaccine; and

[0088] Preparing the vaccine including mixing the obtained multiple VSAs and or obtained microvesicles with excipients, diluents, or carriers.

[0089] The obtained protozoan parasite population is composed of different parasites expressing each a single type of VSA only.

[0090] In an embodiment, the specific antibody against the single type of VSA is a monoclonal antibody. Of course, the antibody may be a polyclonal antibody. Any molecule binding specifically to the single type of VSA expressed initially by the protozoan parasite clone may be applied unless the molecule induces a switching resulting in antigenic variation and the release of microvesicles.

[0091] Further, the skilled person can easily determine the specific antibody concentration by routine measures. That is, the skilled person can determine the specific antibody concentration in pre-experiments.

[0092] In an embodiment, in the method for producing the vaccine according to the present invention, the step of obtaining the multiple VSA in a form applicable to a vaccine includes the purification and / or the inactivation of the protozoan parasite after cultivation and propagation. Further, microvesicles, if present in the vaccine according to the present invention and obtained in the method according to the present invention whereby the microvesicles are released into the culture medium during cultivation and propagation, can be purified by centrifugation and filtration, e.g., as described in the Examples. For instance, parasites that have modified their surface antigen and microvesicles can be inactivated by treatment with [3- propiolactone (1 / 1000 v / v), gamma irradiated (Furuya Y et al., J Virol. 2010;84(9):4212-21. DOI: 10.1128 / J VI.02508-09) or killed by sonication, or treated with 10 cycles of freezing at -180°C and rapid thawing at 37°C.

[0093] In an embodiment, the present invention relates to vaccines against Trypanosoma brucei and related species and methods of manufacture for use in mammals.

[0094] The vaccines can be administered intramuscularly or subcutaneously to protect at the systemic level.

[0095] The vaccines consist of an effective immunostimulatory mixture containing parasites induced by expressing their entire repertoire of variable surface glycoproteins or microvesicles containing parasite antigens.

[0096] One method that has been invented involves administering a suspension of dead or inactivated parasites previously subjected to the process of inducing the turnover of their surface proteins and the release of microvesicles.

[0097] Another method of the invention involves administering a suspension of microvesicles generated when the parasites were previously subjected to the process of inducing switching of their VSAs.

[0098] In an embodiment, blood pleomorphic forms from a T. brucei clone are grown in a culture medium and in the presence of a specific antibody against their corresponding variable surface glycoprotein (VSG), which induces VSG exchange and the expression of multiple distinct VSGs in parasites of the resulting population, in addition to the release of microvesicles containing vital parasite antigens. Trypanosoma brucei clones that have modified their surface antigen and the released microvesicles can be collected by centrifugation and inactivated by treatment with [3-propiolactone (1 / 1000 v / v), gamma irradiated (Furuya Y et al., J Virol. 2010;84(9):4212-21. DOI: 10.1128 / J VL02508-09), killed by sonication, or treated with 10 cycles of freezing at -180°C and rapid thawing at 37°C.

[0099] In an embodiment, the present invention relates to a vaccine against Giardia lamblia and a method of manufacture for domestic and farm animals. In another embodiment, the vaccine is for use in human. The vaccine can be administered orally, preferably in domestic and farm mammalian animals, to provide systemic and mucosal protection.

[0100] The vaccine consists of an effective amount of an immunostimulant mixture containing parasites induced to express their entire repertoire of variable surface proteins and microvesicles containing other parasite antigens than VSA. The method of the invention consists of administering a suspension of dead or inactivated parasites previously subjected to inducing switching of their surface proteins and a suspension of microvesicles generated when the parasites were induced to antigenic exchange.

[0101] Trophozoites from a G. lamblia clone are grown in a culture medium with a specific antibody against a variable surface protein (VSP). This induces the turnover of this VSP and the expression of multiple distinct VSPs in parasites of the resulting population, in addition to the release of microvesicles containing parasite antigens.

[0102] Trophozoites that have modified their surface antigen and the released microvesicles can be collected by centrifugation and can be inactivated by treatment with [3-propiolactone (1 / 1000 v / v), gamma ray-irradiated (Furuya Y et al., J Virol. 2010;84(9):4212-21. DOI: 10.1 128 / J VI.02508-09), killed by sonication, or treated with 10 cycles of freezing at -180°C and rapid thawing at 37°C.

[0103] It is administered orally 2x106trophozoites per kilogram of body weight resuspended in 1 ml of sterile saline solution in two doses with an interval of 15 days to achieve systemic and mucosal protection in animals. This formulation can also be included in capsules that can be degraded in the intestine.

[0104] In an embodiment, the present invention relates to a vaccine against Tritrichomonas foetus and a method of manufacture for use on farm and domestic animals.

[0105] The vaccine can be administered orally or topically, preferably in farm and domestic mammalian animals, to protect at the mucosal level.

[0106] The vaccine consists of an effective amount of an immunostimulatory mixture containing parasites induced by expressing their entire repertoire of VSA adhesins, variable surface antigens, and antigens present on the released microvesicles.

[0107] The method of the invention consists of administering a suspension of killed or inactivated parasites previously subjected to the induction of switching of their surface proteins. Optionally, microvesicles, as described herein, are administered simultaneously, separately, or sequentially.

[0108] Trophozoites from a T. foetus clone are grown in a culture medium and in the presence of a polyclonal antibody which induces switching for the expression of multiple different surface proteins. Trophozoites of Tritrichomonas foetus that have modified their surface antigen and the released microvesicles can be collected by centrifugation and inactivated by treatment with [3-propiolactone (1 / 1000 v / v), gamma irradiated (Furuya Y et aL, J Virol. 2010;84(9):4212-21 . DOI: 10.1 128 / JVL02508-09), killed by sonication, or treated with 10 cycles of freezing at -180°C and rapid thawing at 37°C.

[0109] To achieve systemic and mucosal protection in animals, two doses of 2x106trophozoites per kilogram of body weight resuspended in 1 ml of sterile saline solution are administered orally every 15 days. The formulation can also be included in topical creams administered every 15 days for two months.

[0110] Further, in an embodiment, the protection against Trypanosoma brucei 'xs obtained by administering a vaccine composed of microvesicles according to the present invention.

[0111] Finally, the present invention relates to a method of prophylactic or therapeutic treatment of protozoan parasite infection in an individual. Said method includes administering the vaccine according to the present invention at least once to the individual. Administration may be affected once or several times.

[0112] The present invention will be described further by way of examples without limiting the same thereto.

[0113] Examples

[0114] Example 1 : Giardia lamblia

[0115] 1. Giardia culture

[0116] G. lamblia trophozoites assemblage A1 isolate WB (ATCC® 50803) and clone GS / M-83 (ATCC® 50581 ) (assemblage B) were cultured in borosilicate glass tubes containing TYI-S-33 medium supplemented with 0.5 mg / ml bovine bile (Sigma-Aldrich, Cat. #B3883) and 10% adult bovine serum (Natocor) at 37°C. Clones expressing different VSPs were obtained by limiting dilution in 96-well culture plates placed in anaerobic chambers (Anaerogen™ Compact, Thermo Scientific® Oxoid®, Cat. no. AN0010C) at 37°C for 5 days. Then, positive clones were selected using anti-VSPs specific mAb by immunofluorescence assay (IFA). Reactive clones were expanded in a culture medium overnight and checked for homogeneity before use. For antigenic variation induction studies, cultures expressing a given VSP were re-cloned in the presence of 50 nM of their specific anti-VSP mAb.

[0117] 2. Laboratory animals

[0118] Balb / c mice, gerbils, and Syrian hamsters (6-8 weeks old) of both sexes were used and maintained under specific pathogen-free (SPF) conditions in boxes connected to microisolators, following NIH guidelines for laboratory animals.

[0119] 3. Production of monoclonal antibodies

[0120] Monoclonal antibodies (mAbs) against individual VSPs were generated in 6- week-old Balb / c mice that were immunized subcutaneously with 200 pl of an HPLC-purified preparation of antigen emulsified with adjuvant (Sigma™ Adjuvant System MPL+TDM+CWS) according to the manufacturer's recommendation. Mice were vaccinated again after 21 days with 200 pl of the same preparation and 20 days later immunized intravenously with 100 pl with the same preparation. After three days, the mice were sacrificed, and the spleen cells were used for fusion to NSO myeloma cells. ELISA assays and immunofluorescence on fixed and permeabilized trophozoites tested the antibody-secreting hybridomas. Purification of monoclonal antibodies was performed by FPLC using the AKTA Pure 25 apparatus (GE Healthcare Life Sciences) coupled to a HiTrapTM Protein G HP (GE Healthcare®, Cat. # 29-0485-181 ), following the manufacturer's protocol. After purification, a buffer change to PBS was performed using the same equipment coupled to the HiPrep™ 26 / 10 desalting column (GE Healthcare, Cat. # 17-5087- 01 ). Subsequently, protein concentration was measured using the BCA Protein Assay Kit (Pierce®, Cat. # 23225). The concentrations, expressed in molarity, are derived from the concentration of protein present in the purified antibodies, considering a molecular weight of 150 kDa (for IgG). The purified antibodies were stored in small aliquots at -20°C.

[0121] 4. RNAseq analysis in populations

[0122] Total RNA was purified from the trophozoite population of assemblage A1 isolate WB (ATCC® 50803) by extraction with Trizol following the manufacturer's protocol. Ribosomal RNA was removed from all samples, and sequencing gen libraries were constructed using Illumina strand-specific sequencing. RNA sequencing was performed using a paid service from Genehub.com. The gen libraries were sequenced as 2x150 bp paired-end reads on an Illumina MiSeq, and 3.1 -14.3 million reads were subsequently generated for each library. The genome index was generated using RSubread with default parameters. Quality control of the raw reads was performed with FastQC v0.11 .5. If necessary, low-quality bases (below 30) and adapter sequences were removed with TrimGalore vO.6.4 and Cutadapt v1 .15 to improve mapping efficiency. Sequences shorter than 70 bp were removed. Reads that passed the quality criteria were aligned to the most recent RSubread reference genome with default parameters except for (i) phredOffset=33, (ii) unique = FALSE, (iii) nBestLocations = 2, which was set to 2 to account for duplicate genes. On average, -80% of all sequenced reads could be mapped to a single location in the genome. After mapping, reads were assigned to features using FeatureCounts in RSubread. Reads with multiple overlaps or those that did not overlap with any feature in the annotation file were not counted (Rodrfguez-Walker M et al. Genomics. 2022;114(5). DOI:

[0123] 10.1016 / j.ygeno.2022.110462), Liao Y et al. Nucleic Acids Res. 2019;47(8). DOI: 10.1093 / nar / gkz114).

[0124] 5. Single-cell antigenic variation assay

[0125] Fifty trophozoites expressing a specific VSP (VSP417) were diluted in 20 ml of TYI-S-33 medium containing 50 nM of the corresponding anti-VSP mAb and distributed in 96-well plates. The same procedure was followed in the TYI-S-33 medium with an unrelated mAb as a control (mAb 8F12; anti-CWP2). The plates were incubated for 5 days; the number of clones obtained was then counted, and IFA measured the percentage of the original VSPs.

[0126] 6. Purification of microvesicles

[0127] Exponentially growing trophozoites (1.5x108) of clone VSP417 were washed twice with filtered PBS at 37°C and incubated for 4 h at 37°C in the presence of mAb 7C2 (50 nM) in microvesicle purification medium (ultrafiltered TYI-S-33 medium, 100 kDa MWCO, supplemented with 3% adult bovine serum previously ultracentrifuged overnight at 250,000 g). The tubes were cooled on ice and centrifuged in an SW41Ti rotor (Beckman) at 4°C for 10 min. The supernatants were collected in 50 ml centrifuge tubes and centrifuged at 3,000 g at 4°C for 40 min; this step was repeated once. The supernatants were then concentrated 10X in a centrifugal filtration device (Centricon™ Plus-70-100K, Millipore®, Cat. # UFC710008) and ultracentrifuged in a Beckman® SW41Ti rotor (25,000 rpm, 2 h, at 4°C). The pellets were washed in an equal amount of ice-cold filtered PBS and ultracentrifuged again to recover the microvesicles. IFA validated the purification of microvesicles.

[0128] 7. Scanning electron microscopy (SEM) and helium ion microscopy (HM)

[0129] Glutaraldehyde-fixed cells were post-fixed in 1 % OsCh for 15 min. The samples were then dehydrated in increasing series of ethanol up to 100 %, dried at the critical point with liquid CO2, and coated with charcoal to observe the cell surface in detail. The samples were examined on a Quanta™ SEM (FEI Co., The Netherlands) equipped with FEG filament. Images were obtained by secondary electron (SE) and backscattered electron (BSE) detection at an accelerating voltage of 15 kV. For high-resolution scanning microscopy analysis, the sputter coating step was performed using a thin (2 nm) platinum layer, and the cells were observed on an Auriga™ high-resolution SEM (Zeiss). For HM, cells were processed as described above and observed, without any subsequent coating, under a Zeiss Orion helium ion microscope.

[0130] 8. Transmission electron microscopy (TEM).

[0131] Cells fixed with glutaraldehyde were subsequently fixed with 1 % OsO4 and 0.8 % potassium ferrocyanide for 40 min. Samples were dehydrated in increasing degrees of acetone to 100% and embedded in epoxy resin. Ultra-thin sections (50- 60 nm thick) were cut, collected, and stained with uranyl acetate and lead citrate. Finally, the samples were analyzed with a Tecnai™ Spirit TEM (FEI Co.).

[0132] 9. Infections with G. lamblia in gerbils and hamsters

[0133] Before infection, ELISA tested 6-week-old gerbils (Meriones unguiculatus) and Golden Hamsters (Mesocricetus aerates) for the negativity of serum antibodies against Giardia antigens. Ten animals of each species were inoculated for the trophozoite- and microvesicle-infected groups and 10 animals were used for each control group. Animals were immunized by orogastric administration of 2x105trophozoites and their microvesicles, which were resuspended in 300 pL of sterile PBS, with an interval of 15 days. Trophozoites were previously induced to switch by culturing in the presence of the specific mAb against the VSP expressed on the surface of the WB clone VSP417 (assemblage A) and GS VSPH7 (assemblage B) against which the microvesicles containing the membrane antigen of the original clone were released. These parasites were inactivated by sonication on ice (Al-Sabi MNS et aL, J Appl Microbiol. 2015;119(3):894-903. DOI: 10.1111 / jam.12898). The absence of live parasites in the preparation was verified by culture in Giardia basic medium for up to one week after inoculation and by negativity to fluorescein diacetate staining (Saruyama N, et aL, Anal Biochem. 2013 ;441 (1 ):58-62. DOI: 10.1016 / j.ab.2O13.06.005). Control animals were inoculated with 0.5 ml of PBS / cysteine by the same route. In all cases, vaccination did not trigger any signs of disease. After 2 months, the gerbil and hamster groups were inoculated with 1 x102cysts resuspended in 0.5 ml PBS with 5 mM cysteine. Fecal samples were collected from both groups of animals every three days between days 0 and 60 post-infection. Excreted cysts were visually identified by immunofluorescence assays using mAbs that recognize specific proteins in the Giardia cyst wall.

[0134] 10. Protection for dogs and cats

[0135] Assays 6-8-week-old mongrel puppies and 5-8-week-old kittens were used. The animals were free of Giardia, which was determined by analysis of their feces by immunofluorescence assays using specific mAbs against cyst wall proteins, as well as of any other detectable infectious disease (all animals were fully vaccinated against common bacterial and viral infections before the start of the experimental period: Nobivac DP and Nobivac HHPPI from MSD Animal Health, Bagovac Rabies from Biogenesis-Bago). During the first months of the challenge experiments, the animals were kept isolated in individual roofed compartments with access to an outdoor concrete-floored runway for immunizations and infections. They were offered autoclaved food and sterile water supplemented with a vitamin mixture (Vigorex, Labyes) ad libitum. All animals were housed following the standard operating procedures of the International Association for Laboratory Animal Care.

[0136] Dogs and cats were tested for the presence of the parasite and treated with antiparasitic drugs before participation in the trial. Each animal was identified with a unique tag. Before infection, serum from all animals was tested for antibodies to Giardia antigens by enzyme-linked immunosorbent assay using a preparation of total proteins extracted from trophozoites and cysts. Animals with anti- Giardia antibodies were omitted. Some animals were treated orally with 25 mg metronidazole and 25 mg albendazole per kg for 3 days, 10 days before testing, to rule out any possible presence of Giardia. Three days before day 0, the animals were weighed and randomly assigned to experimental (n=10) or control (n=10) groups, although the animals were housed in isolation. The animals were clinically examined weekly throughout the trial. After the animals were infected, fecal samples were collected to detect the presence of Giardia cysts.

[0137] Animals were immunized with two oral administrations of 1 ml of parasites expressing the complete repertoire of VSPs (2x106trophozoites per kilogram weight). Their microvesicles resuspended in 0.5 ml of PBS, with an interval of 15 days that were previously induced to switch when cultured in the presence of the specific mAb against the VSP expressed on the surface of the original clone WB VSP417 (assemblage A). GS VSPH7 (assemblage B), including the microvesicles containing the membrane antigen of the original clone, was released. The parasites were inactivated by sonication on ice (Al-Sabi MNS et aL, J Appl Microbiol. 2015;119(3):894-903. DOI: 10.11 1 1 / jam.12898). The absence of live parasites in the preparation was verified by culture in Giardia basic medium for up to one week after inoculation and by negativity to fluorescein diacetate staining (Saruyama N, et aL, Anal Biochem. 2013 ;441 (1 ):58-62. DOI:

[0138] 10.1016 / j.ab.2013.06.005). Control animals were inoculated with 0.5 ml of PBS / cysteine by the same route.

[0139] Sixty days post-immunization, both groups were infected by orogastric inoculation of 1 x102cysts resuspended in 0.5 ml PBS with 5 mM cysteine. Freshly collected cysts from experimentally infected gerbils were used to prevent samples from rapidly losing viability and infectivity. Fecal samples were collected from both groups of animals every three days between days 0 and 60 post-infections and weekly until day 120. Excreted cysts were visually identified by immunofluorescence assays using mAbs that recognize specific proteins in the Giardia lamblia cyst wall.

[0140] Results

[0141] It was observed in a transcriptome of a Giardia lamblia clone of assembly A1 isolate WB (ATCC® 50803) that the entire VSP repertoire is expressed with some level of detection but that the transcript that is translated and expressed on the surface of trophozoites is expressed / accumulated several orders of magnitude above any other gene. As seen in clonal populations, the same was observed when individual cells were analyzed. On the other hand, clones expressing VSP417 that were incubated for six days with the anti-VSP417 antibody show the expression of multiple VSPs in that population (Table 1 , Figure 1 , and Table 2). Table 1 shows the expression of selected VSP and Housekeeping genes in different Giardia transcriptomic experiments. Three single cells, three clones expressing VSP417, and three populations obtained after 6 days of culture in the presence of mAb 7C2 of VSP417-expressing cells were sequenced and analyzed. The values are expressed in TPM. A massive level of expression / accumulation of a particular VSP (highlighted in bold) could be observed both in single cells and in clonal populations (VSP GL50803-50229 and GL50803-d50229 are duplicated VSP genes). In contrast, no VSP has mRNA levels (highlighted in bold) well above any other Giardia gene in populations expressing multiple VSPs. At the protein level, it was observed that when a clonal population of trophozoites expressing VSP417, VSP1267, or VSPH7 was cultured in 12-well plates in the continuous presence of their corresponding anti-VSP mAb (0.25-10 nM), after 6 days, in each well the surface antigen expression was modified, expressing in all cases VSPs different from the original, but not in the control experiment, where the rate of variation never exceeded 10% in that given period (Table 2).

[0142] Table 2. Induction of switchers in Giardia. Percentage of switchers after culturing a clonal population of VSP417, VSP1267, and VSPH7 trophozoites with their respective specific mAb and without antibodies.

[0143] The same was observed when trophozoites from a clonal population expressing VSP417 were distributed in 96-well plates by limiting dilution. In the plate where trophozoites were cultured with their corresponding mAb, after 6 days, in the wells where there was growth, the original VSP was not found. In contrast, in the control plate and the presence of an unrelated antibody, the trophozoites expressed the original VSP in a high proportion (Figure 2).

[0144] To determine the turnover of the original VSP after treatment with the antibody against the corresponding VSP, transmission (TEM) and scanning (SEM) microscopy analyses were performed using antibodies labeled with gold particles.

[0145] The results showed accumulations of microvesicles on their surface containing the VSP target of the corresponding antibody (Figure 3).

[0146] Immunizations and challenges were performed in various animal models to determine whether these trophozoites and their microvesicles stimulated the mucosal immune system, especially secretory immunoglobulin A (slgA).

[0147] Oral immunization with trophozoites that were previously induced to switch when cultured in the presence of the specific mAb against the corresponding VSP plus their released microvesicles induced the expression of anti-VSPs slgA for both the A and B assemblage-derived parasites, demonstrating a robust immune response that prevented infection in gerbils and hamsters. In contrast, uninoculated control animals were efficiently infected by cysts whose VSP was unknown (Figure 4). Similar results were observed when vaccinating dogs and cats (Figure 5).

[0148] Example 2: Trypanosoma brucei

[0149] 1. T. brucei culture

[0150] T. brucei in its pleomorphic forms was maintained in an HMI-9 medium supplemented with 10% fetal bovine serum and 10% antibiotic / antimycotic solution at the recommended concentration (Gibco®, Cat. # 15240062). Cell numbers were monitored, diluting them regularly to avoid densities greater than 1 .5x106 / mL Pleomorphic forms of the EATRO1125 clone expressing VSG AnTatl .1 were used for switching assays. Fifty parasites expressing a specific VSG (AnTatl .1 ) were diluted in 20 ml of HMI-9 medium containing 50 nM of the corresponding anti-VSG pAb. They were distributed in 96-well plates. The plates were incubated for 5 days; the number of clones obtained was then counted, and IFA was used to measure the percentage of the original VSGs. The different clones obtained were amplified in T75 flasks with 30 ml of HMI-9 medium supplemented with 10% fetal bovine serum and 10% antibiotic / antimycotic solution at the recommended concentration (Gibco®, Cat. # 15240062). Cells were labeled with 1 / 500 polyclonal antisera in a culture medium on ice for 40 min. Subsequently, after two washes with TDB (Trypanosome dilution buffer, KCI 5 mM, NaCI 80 mM, MgSO4 1 mM, Na2HPO4 20 mM, NaH2PO4 2 mM, Glucose 20 mM; pH 7.7), the parasites were fixed in suspension with 4% paraformaldehyde in PBS at room temperature for 1 h. The parasites were then adhered to coverslips using a poly-L-lysine solution. They were washed 3 times with PBS. Blocking was performed with 3% bovine serum albumin (BSA) solution in PBS for 1 h. Cells were incubated with Alexa Fluor™- 546 goat anti-mouse IgG (H+L) antibody (Invitrogen™, Cat. # A11030) or Alexa Fluor™-488 goat anti-mouse IgG (H+L) antibody (Invitrogen™, Cat. # A11003) at 1 / 1 ,000 dilutions. For all parasites, nuclei and kinetoplasts were stained with DAPI at a concentration of 1 pg / ml in PBS for 10 min. For IFA, images were acquired using an epifluorescence microscope.

[0151] 2. Laboratory animals

[0152] Balb / c mice (6-8 weeks old) of both sexes were used and maintained under specific pathogen-free (SPF) conditions in boxes connected to microisolators, following NIH guidelines for laboratory animals.

[0153] 3. Production of polyclonal antibodies

[0154] Polyclonal antisera against T. bruceiwere produced in 6-week-old Balb / c mice. Mice were infected intraperitoneally with 1 .5x106parasites from 4 different clones of pleomorphic forms of EATRO1125 expressing VSG-4156 also called AnTatl .1 , VSG-4710, VSG-353 or VSG-62). After 48 h, mice with detectable parasitemia were cured with two consecutive doses, 24 h apart, of 40 mg / Kg Berenil™ (MSD Animal Health®, SAGARPA Reg. Q-0273-121 ). The entire procedure was repeated 15 days later. Visualization and blood culture of the animals showed the absence of parasites after Berenil™ treatment. At 1 and 2 weeks after the booster (7 and 24 dpi), blood samples were taken from the retro- orbital plexus using heparinized capillaries. From the sera obtained, the presence of antibodies reactive against the surface of the parasites was confirmed by immunofluorescence microscopy (IFA). The serum obtained at 7 dpi was used at 1 / 1 ,000 dilution to stimulate switching, while the serum obtained at 24 dpi was used at 1 / 50 dilution to kill non-switchers.

[0155] 4. Purification of microvesicles

[0156] Parasites (2.5x107) were resuspended in 5 ml of HMI-9 medium supplemented with 3% adult bovine serum previously ultracentrifuged overnight at 250,000 g with a 1 / 50 dilution of polyclonal antibody against the corresponding VSG and cultured 37°C for 30 min in HMI-9 medium. The tubes were then cooled on ice and then centrifuged in an SW41Ti rotor (Beckman) at 4°C for 10 min. The supernatants were collected in 50 ml centrifuge tubes and centrifuged at 3,000 g at 4°C for 40 min; this step was repeated once. The supernatants were then concentrated 10X in a centrifugal filtration device (Centricon™ Plus-70-100K, Millipore®, Cat. # UFC710008) and ultracentrifuged in a Beckman® SW41Ti rotor (25,000 rpm, 2 h, at 4°C). The pellets were washed in an equal amount of ice-cold filtered PBS and ultracentrifuged again to recover the microvesicles. IFA validated purification of the microvesicles.

[0157] 5. Vaccine protection assays in Balb / c mice.

[0158] Balb / c mice (6 to 8 weeks old) of both sexes were maintained under specific pathogen-free (SPF) conditions in boxes connected to microisolators, following NIH guidelines for laboratory animals. The animals were free of T. brucei, which was determined by blood analysis by immunofluorescence assays using pAbs. Before infection, serum from all animals was tested for antibodies to T. brucei antigens by enzyme-linked immunosorbent assay using a preparation of total proteins extracted from the parasite. Three days before day 0, animals were weighed and randomly assigned to the experimental groups vaccinated with parasites (n=10), immunized with microvesicles (n=10), or unvaccinated control (n=10). Animals were clinically examined weekly throughout the trial. After the animals were infected, blood samples were collected to detect the presence of the parasite. One group of animals was immunized with two successive intraperitoneal administrations of 300 pL of microvesicles expressing parasite antigens resuspended in sterile PBS / 0.01 % Tween 20, with an interval of 15 days that were previously induced to switch VSGs by culturing in the presence of the specific pAb against VSG expressed on the surface of the original clone. Another group was immunized with 2x105parasites resuspended in 300 pL of sterile PBS at a 15-day interval. Parasites were previously induced to switch by culturing in the presence of the specific pAb against VSG that was expressed on the surface of the original clone. The parasites were inactivated by sonication on ice (Al-Sabi MNS et aL, J Appl Microbiol. 2015;119(3):894-903. DOI: 10.1 11 1 / jam.12898). The absence of live parasites in the preparation was verified by culture in HMI-9 medium for up to 1 week after inoculation and by negativity to fluorescein diacetate staining (Saruyama N et aL, Anal Biochem 2013 ;441 (1 ):58-62. DOI:

[0159] 10.1016 / j.ab.2O13.06.005). Control animals were inoculated with 300 pL of PBS by the same route.

[0160] Sixty days post-immunization, all groups were infected via the intraperitoneal route with 103parasites resuspended in 300 pL of HMI-9 medium. Blood samples were taken from both groups of animals every 2 days between days 0 and 3.5. The blood sample was treated with 0.8% w / v CINH4 for better visualization under microscopy. Pleomorphic parasites were counted in a Neubauer chamber.

[0161] Results

[0162] Immunization with microvesicles released after culturing the parasites in the presence of the specific pAb against the initially expressed VSG generated a robust immune response that prevented infection in Balb / c mice. Immunization with inactivated parasites that were previously induced to switch when cultured in the presence of the specific anti-VSG pAb expressed on the surface of the original clone was partially effective, with infected animals exhibiting low levels of parasitemia in the blood. In contrast, uninoculated control animals were readily infected (Figure 6).

[0163] Example 3: Tritrichomonas foetus

[0164] 1. T. foetus culture

[0165] Trichomonas parasites were grown axenically at 37°C in TYM medium supplemented with 10% heat-inactivated horse serum (Invitrogen), 10 U penicillin (Invitrogen), 10 g streptomycin (Invitrogen), adjusted to pH 7.0 (Petrin D et aL, Clin Microbiol Rev. 1998;1 1 (2):300-17). Clones expressing different surface antigens were obtained by limiting dilution in 96-well culture plates at 37°C in TYM medium supplemented with 10% heat-inactivated horse serum (Invitrogen), 10 U penicillin (Invitrogen), 10 mg streptomycin (Invitrogen), adjusted to pH 7.0 for 5 days. Clones were expanded in culture medium overnight. For antigenic switching induction studies, cultures expressing a given surface antigen were cultured in the presence of their specific pAb at the dilution that does not bind the parasites for more than 2 days in 12-well plates. After 5 days of culture, the number of Trichomonas was quantified in a Neubauer chamber, and IFA characterized the surface antigen expression with the pAb corresponding to the original clone.

[0166] 2. Laboratory animals

[0167] Balb / c mice (6 to 8 weeks old) of both sexes were used and maintained under specific pathogen-free (SPF) conditions in boxes connected to microisolators, following NIH guidelines for laboratory animals.

[0168] 3. Production of polyclonal antibodies

[0169] Polyclonal antisera (pAb) were produced against clones derived from a local isolate of T. foetus in 6-week-old Balb / c mice. Mice were infected intraperitoneally with 1 .5x106parasites emulsified with adjuvant (Sigma™ Adjuvant System MPL+TDM+CWS) according to the manufacturer's recommendation. The entire procedure was repeated 21 days later. Ten days after the last immunization, blood samples were taken from the retro-orbital plexus using heparinized capillaries. From the sera obtained, the presence of antibodies reactive against the surface of the parasites was confirmed by immunofluorescence microscopy (IFA). The sera obtained were used at a dilution of 1 / 1 ,000 to stimulate switching.

[0170] 4. Vaccine protection assays in Balb / c mice.

[0171] Female Balb / c mice (6-8 weeks old) were used and maintained under specific pathogen-free (SPF) conditions in boxes connected to microisolators, following NIH guidelines for laboratory animals. The animals were free of Tritrichomonas foetus, which was determined by blood analysis by immunofluorescence assays using pAbs.

[0172] Before infection, serum from all animals was tested for antibodies to T. foetus antigens by enzyme-linked immunosorbent assay using a preparation of total proteins extracted from the parasite. Three days before day 0, animals were weighed and randomly assigned to the experimental parasite-vaccinated (n=10) or control (n=10) groups. Animals were clinically examined weekly throughout the trial. After the animals were infected, blood samples were collected to detect the presence of the parasite.

[0173] One group of animals was immunized with two successive oral administrations of 300 pL of Tritrichomonas foetus (2x106trophozoites per kilogram body weight) expressing different parasite antigens resuspended in sterile PBS / 0.01% Tween 20, with an interval of 15 days. T. foetus cells were from a clonal population and were induced to switch their surface antigen when cultured in the presence of the original clone-specific pAb. Tritrichomonas were inactivated by sonication on ice (Al-Sabi MNS et aL, J Appl Microbiol. 2015;119(3):894-903. DOI: 10.1111 / jam.12898). The absence of live parasites in the preparation was verified by culture in TYM medium supplemented with 10% heat-inactivated horse serum (Invitrogen), 10 U penicillin (Invitrogen), 10 mg streptomycin (Invitrogen), adjusted to pH 7.0 for up to one-week post inoculation and by negativity to fluorescein diacetate staining (Saruyama N et aL, Anal Biochem. 2013 ;441 (1 ):58- 62. DOI: 10.1016 / j.ab.2O13.06.005). Control animals were inoculated with 300 pL of PBS by the same route.

[0174] Twelve days after inoculation, vaginal cytology was analyzed in both infected and control groups to determine the efficacy of immunization.

[0175] Results

[0176] It was observed that when a clonal population of Tritrichomonas was cultured in 12-well plates in the continuous presence of its corresponding pAb (50 nM), after 5 days, each well's surface antigen expression was modified, expressing antigens different from the original in all cases, but not in the control experiment, where the rate of variation never exceeded 15% in that given period (Table 3).

[0177] Incubation with T. foetus after culturing them in the presence of the specific pAb against the initially expressed surface antigen generated a strong immune response that prevented infection in Balb / c mice. In contrast, non-inoculated control animals were easily infected (90%) (Figure 7).

[0178] Table 3. Antibody-induced antigenic variation in T. foetus. Percentage of switchers after culturing a clonal population of Tritrichomonas with their respective specific pAb and without antibody.

Claims

Claims:1 ) A vaccine for preventing or treating protozoan parasite infections in an individual said vaccine comprising i) an active agent, and ii) excipients, diluents or carriers and, iii) optionally, adjuvants, wherein the active agent comprises a multitude of variable surface antigens (VSAs) and / or microvesicles of the parasite to be treated or to be protected against whereby the multitude of VSAs and microvesicles are obtained by inducing the switching of the VSA of a parasite clone expressing a single type of VSA in the presence of a specific antibody against the single VSA present on the surface of said clone, producing the expression of multiple distinguishable VSAs in the obtained population, wherein a single protozoan parasite of the obtained population expresses one type of VSA and the release of extracellular microvesicles containing the single, original VSA and protective antigens other than VSA.2) The vaccine according to claim 1 , wherein the vaccine includes the entire repertoire of VSA and further contains microvesicles released by said treatment containing a variety of protozoan antigens other than VSA.3) The vaccine according to claim 1 , containing only microvesicles released by said treatment containing a variety of protozoan antigens other than VSA.4) The vaccine according to claims 1 to 3, being a protective vaccine against subsequent infection with the protozoan parasite.5) The vaccine according to any one of the preceding claims, wherein the protozoan parasite undergoes antigenic variation.6) The vaccine according to claim 5 wherein the protozoan parasite is selected from the genus of Trypanosoma, Tritrichomonas, and Giardia.7) The vaccine according to claim 6 wherein the protozoan parasite is selected from the genus of Trypanosoma, and Tritrichomonas.8) The vaccine according to claim 6 or 7, wherein the protozoan parasite is selected from Trypanosoma brucei, Tritrichomonas foetus, Giardia lamblia.9) The vaccine according to claim 8, wherein the protozoan parasite is selected from Trypanosoma brucei, or Tritrichomonas foetus.10) The vaccine according to any one of the preceding claims, wherein the individual is a mammal like a domestic or farm animal.11 ) The vaccine according to claim 10, wherein the individual is a human.12) The vaccine according to any one of the preceding claims, wherein the active agent is provided in a form selected from attenuated parasites, dead parasites, parts of the parasites including the plasma membrane, purified VSAs, microvesicles containing parasite antigens, or combinations thereof.13) The vaccine according to any one of the preceding claims for mucosal and / or systemic administration.14) The vaccine according to claim 13 wherein the vaccine is administered subcutaneously or intramuscularly.15) The vaccine according to claim 13 wherein the vaccine is administered orally.16) The vaccine according to claim 15 comprising the oral administration of two doses of antigens in an amount of 50 to 500, like 200 pg per Kg of body weight, within an interval of 10 to 20 days, like 15 days.17) The vaccine according to any one of claims 1 to 14, wherein the vaccine is provided in a formulation of a topical creme for administering multiple times, in particular, 15 days for two months.18) The vaccine according to any one of the preceding claims being a protective vaccine adapted for administering in food or in drinking water to treat the mammal.19) A method for producing a vaccine according to any one of claims 1 to 18 comprising the steps of:- Providing a protozoan parasite clone expressing a single type of multiple variable surface antigens (VSAs) encoded in the genome of said protozoan parasite clone;- Culturing said protozoan parasite clone in the presence of a specific antibody against the single type of VSA to induce the switching of VSA;- Propagation of the cultured and switched protozoan parasite population expressing switched multiple VSAs other than the single type of VSA the specific antibody is directed against;- Obtaining multiple VSAs in a form applicable in a vaccine from the propagated protozoan parasite population and / or obtaining microvesicles from the culture of the propagated protozoan parasite population in a form applicable in a vaccine; and- Preparing the vaccine including mixing the obtained multiple VSAs and or obtained microvesicles with excipients, diluents, or carriers.20) The method for producing the vaccine according to claim 19 wherein the specific antibody against the single type of VSA is a monoclonal antibody.21 ) The method for producing the vaccine according to 19 or 20 wherein the propagation is conducted in the presence of the specific antibody directed to the expressed VSA, preferably, the specific antibody is a monoclonal antibody or polyclonal antibody.22) The method for producing the vaccine according to claim 19 to 21 wherein the monoclonal or polyclonal antibody being generated in mice, rats, rabbits, or goats.23) The method for producing the vaccine according to any one of claims 19 to 22 wherein the step of obtaining the multiple VSAs in a form applicable in a vaccine includes the purification and / or the inactivation of the protozoan parasite after cultivation and propagation.24) The method for producing the vaccine according to any one of claims 19 to 23 wherein the microvesicles containing a variety of protozoan antigens other than VSAs, when obtained through release by said treatment, are purified from the culture supernatant by centrifugation and filtration.

Images