Patent Application
20240376161
The present invention relates to a recombinant chimeric protein comprising immunogenic regions of the trans-sialidase (TS) protein and of the Trypanosoma cruzi amastigote surface protein 2 (ASP-2) and a composition containing such protein which showed vaccine potential in a murine model. The invention further comprises the use of a chimeric protein for the manufacture of vaccines.
Chagas disease is caused by the intracellular protozoa Trypanosoma cruzi, belonging to the Trypanosomatidae family and which is currently a serious public health problem. Chagas disease is a neglected disease that mainly affects low-income populations, and, despite existing prophylaxis, the disease is still responsible for approximately 10 thousand deaths per year, with approximately 6 to 7 million people infected worldwide, especially in Latin America. Transmission occurs mainly by triatomine insect vectors belonging to the Triatoma, Panstrongylus and Rhodnius genera, although it can also occur through transfusion of infected blood, oral transmission through contaminated food, congenital transmission, organ transplantation or laboratory accidents.
The complex biological cycle of the parasite involves replication and extracellular differentiation of the parasite in the insect vector (triatomine) and intracellular differentiation in the vertebrate host. This favors its escape from immunological surveillance and persistence in the infected organism.
The existing medication for the treatment of Chagas disease in Brazil is benznidazole, which acts on both the trypomastigote and amastigote forms and is more effective in the acute phase of the disease. A study showed that the efficacy of benznidazole in the acute phase is 70-80% (assessed by negative serology), but in the chronic phase it is only 8%. A recent prospective study showed that the treatment of patients with established Chagas disease cardiomyopathy with benznidazole significatively reduced the detection of circulating parasites, but not disease progression. Therefore, the use of this drug in the chronic phase is still debatable. Furthermore, treatment can last up to 90 days and there are many adverse reactions, with allergic dermatitis occurring in around 30% of patients, nausea, vomiting, anorexia, weight loss, insomnia and loss of appetite. A major problem with existing treatments is that they are more effective in the acute phase, when symptoms are non-specific, which is one of the major causes of mortality caused by Chagas disease.
Additionally, despite the implementation of systems for controlling and the preventing Chagas disease, the challenges for its eradication are still big.
Due to this fact, the development of a vaccine is needed. However, despite important advances in knowledge about Chagas disease since its characterization more than a century ago, the complexity of the host-parasite interaction has not yet allowed the production of an effective vaccine. Several initiatives have investigated the possibility of prophylactic and therapeutic vaccines against Chagas disease.
Some prior art documents describe vaccine compositions containing different antigens and several vaccination protocols.
Two proteins expressed by T. cruzi have been studied as potential vaccine antigens: Trans-sialidase (TS) and Amastigote surface protein 2 (ASP-2). TS is present mainly in the membrane of trypomastigotes, the extracellular stage of the parasite, and has a repetitive region in its C-terminal portion (SAPA) which induces an intense antibody production by B lymphocytes (SCHENKMAN et al., 1992: AFFRANCHINO et al., 1989). ASP-2 is typically found in the membrane of amastigotes, intracellular form of T. cruzi. In murine model, the immunization with plasmids containing the ASP-2 coding sequence is capable of inducing a potent cellular immune response, leading to the production of IFN-γ by CD4+ and CD8+ T cells (VASCONCELOS et al., 2004).
The most effective vaccine protocol to date, tested on mice, uses an initial dose of plasmids containing the TS and ASP-2 genes and a booster dose of adenovirus type 5 (Ad5) which also encodes these proteins (PlAd-TS/ASP). It has been shown that this immunization is capable of protecting not only resistant mice (C57BL/6), but also the most susceptible (A/Sn) to infection with the Y strain of T. cruzi. In addition, the vaccine also obtained satisfactory results in chronic experimental models, in which the animals were challenged with the Brazil and Colombiana myotropic strains (VASCONCELOS et al., 2004: ALENCAR et al., 2009: ARAÚJO et al., 2014).
Despite being potentially immunogenic and strongly inducing a cellular response, the use of DNA vaccines (with plasmid and/or viral vectors) is still under debate. The US Food and Drug Administration (FDA) sets out in its guide “Points to Consider on Plasmid DNA Vaccines for Preventive Infectious Disease Indications” (1996, updated in 2007) some contingents that should be considered by vaccine developers. It was described the possibility of generating autoantibodies, which could lead to an autoimmune disease: the induction of immunological tolerance rather than immunogenicity: and also, the risk of plasmid DNA being integrated into the genome of host cells, increasing the chance of carcinogenesis. (KLINMAN et al., 1997: MOR et al., 1997; KLINMAN et al., 2000).
As regards viral vectors, there is concern about the possibility of virulence reversal, mainly through gene recombination between the wild virus and its attenuated version (CONDIT et al., 2016). Another factor to consider is the pre-existence of immunity to the vector, which nullifies the vaccine effect, as reported in the clinical trial of an HIV vaccine using Ad5 (“STEP” trial, 2005). In this study, it was observed that individuals with high antibody titers against Ad5 had a higher incidence of HIV infection when compared to the placebo group (GRAY et al., 2010).
Patent document U.S. Pat. No. 9,028,844, entitled “Vaccine against Trypanosoma cruzi infection” whose priority date is Mar. 17, 2006, describes a vaccine against Trypanosoma cruzi infection, useful in the treatment and/or prevention of Chagas disease. The vaccine of the aforementioned invention comprises a mutant and recombinant trans-sialidase.
Patent document U.S. Pat. No. 9,250,239, entitled “Compositions and methods for detecting microbial infections”, whose priority date is Feb. 7, 2014, describes a DNA vaccine for Chagas disease prophylaxis in which the DNA encoding the ASP-2 protein is used as a part of the glycoprotein-encoding plasmid described in the document.
Patent document AR100707, entitled “Polipéptido quimérico, vacunas contra la enfermedad de Chagas y método de inmunización” (in English, Chimeric polypeptide, vaccines against Chagas disease and immunization method), whose priority date is Jun. 2, 2015, describes a polymeric peptide obtained from the sequences of a catalytic domain of cruzipain from T. cruzi and a region of the surface antigen Amastigote Surface Protein 2 (ASP2) from T. cruzi, joined by a connector that can be, in a preferred embodiment of the invention, a helix of the T. cruzi trans-sialidase.
The present invention deals with a chimeric protein whose sequence derives from the prediction of epitopes made with TS and ASP-2, using bioinformatics tools, in order to recognize the immunogenic regions, also considering the variability of HLAs in the population. By combining the immunogenic regions of both proteins, a chimeric protein was developed that showed vaccine potential in a murine model.
In a first aspect, the present invention relates to a chimeric protein comprising a peptide sequence with at least 80% identity with the SEQ ID No 1.
In an embodiment, the chimeric protein comprises the peptide sequence of SEQ ID No 1.
In a second aspect, the present invention relates to the use of a chimeric protein, according to the first aspect of the invention, for the manufacture of a composition for the prevention and/or treatment of infection by Trypanosoma cruzi.
In an embodiment, the composition is a vaccine.
In a second embodiment, the infection by Trypanosoma cruzi is Chagas disease.
In a third aspect, the invention refers to a composition comprising a chimeric protein, according to the first aspect of the invention, and adjuvants.
In an embodiment, the composition is a vaccine.
In a second embodiment, the adjuvants are selected from the group comprising CpG, aluminum hydroxide: monophosphoryl lipid A (MPL); polyinosinic-polycytidylic acid; and compositions containing mineral oils, vegetable oils, animal oils, compounds derived therefrom and/or combinations thereof. Preferably, the adjuvants are selected from the group comprising CpG, aluminum hydroxide; monophosphoryl lipid A (MPL); polyinosinic-polycytidylic acid; and compositions containing an emulsion of mineral oils, peanut oil, olive oil, sesame oil, soybean oil, wheat germ oil, grape seed oil, sunflower oil, castor oil, linseed oil, soybean oil, corn oil, copra oil, palm oil, walnut oil, hazelnut oil, rapeseed oil, squalane or squalene from olives or extracted from fish livers, or a combination thereof.
In another embodiment, the composition comprises 1 to 500 μg of protein, 10 to 500 μg/mL of CpG and 20 a 40% v/v of aluminum hydroxide.
In another embodiment, the composition comprises 1 to 500 μg of protein, and 30 to 70 μg/mL of polyinosinic-polycytidylic acid.
As previously mentioned, the present invention relates to a protein for the prevention or treatment of Trypanosoma cruzi infections. Specifically, the present invention deals with a recombinant chimeric protein containing immunogenic regions of TS and ASP-2.
In a preferred embodiment, the recombinant chimeric protein of the present invention comprises a peptide sequence with at least 80% identity with SEQ ID No 1. In a more preferred embodiment, the protein comprises the peptide sequence SEQ ID No 1.
The chimeric protein according to the present invention can be combined with any suitable vehicle or excipient, as known by a person skilled in the art, in order to increase its stability or provide a release at a specific site, an immediate or prolonged release, so as to provide a suitable composition. Preferably, according to the present invention, the composition is a vaccine composition.
According to a preferred embodiment, the composition of the invention comprises at least one immunologically active element against Trypanosoma cruzi and a pharmaceutically acceptable excipient. This element is preferably a recombinant chimeric protein comprising a peptide sequence with at least 80% identity with SEQ ID No 1. Even more preferably, the protein comprises the peptide sequence of SEQ ID No 1.
For the preparation of the compositions according to the present invention, any methods known to those skilled in the art may be used.
As used herein, the term “immunologically active”, or any reference to the immunological activity of an element, such as the protein of the invention, refers to the element's ability to stop, prevent or treat an infection by Trypanosoma cruzi.
As used herein, the term “biologically acceptable” refers to a vehicle or carrier that can be safely administered to an animal, particularly mammals and humans, without excessively negative or toxic side effects.
According to one embodiment of the invention, the composition can be formulated in the form of a pharmaceutical composition.
The present invention also relates to the use of a chimeric protein comprising a peptide sequence with at least 80% identity with SEQ ID No 1, for the manufacture of a composition for the prevention and/or treatment of Trypanosoma cruzi infection. Preferably, the protein comprises the peptide sequence of SEQ ID No 1. Although the compositions of the present invention are preferably administered parenterally, they can also be administered by any other suitable route. In a preferred embodiment, the pharmaceutical compositions may be administered intravenously, intramuscularly, or subcutaneously.
Examples of a preparation suitable for parenteral administration include injection, intravenous fluid, solution or suspension for infusion, lyophilized powder for suspension or solution, inhalation powder, and the like.
The formulation according to the present invention may also be in a form for oral administration. Examples of a preparation suitable for oral administration include tablet, capsule, powder, fine granule, pellet, solution, suspension, syrup, and the like.
However, the form of the preparation should not be limited to these alone.
As a pharmaceutically acceptable excipient, for example, an adjuvant, disintegrant or disintegrant auxiliary, binder, coating agent, colorant, diluent, base, solubilizer or solubilizer auxiliary, isotonicity agent, pH regulator, stabilizer, propellant, adhesive and the like can be used.
A preparation suitable for oral administration may contain, as an excipient, for example, glucose, lactose, lactose monohydrate, D-mannitol, starch, crystalline cellulose and the like; disintegrant or disintegrant auxiliary, such as carboxymethylcellulose, starch, calcium carboxymethylcellulose, silicon dioxide and the like; binder, such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gelatine and the like; lubricant, such as magnesium stearate, talc and the like; base, such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, gelatine, kaolin, glycerol, purified water, hard fat, and the like.
A preparation suitable for injection or intravenous fluid may contain pharmaceutical excipients for preparation, such as solubilizer or solubilizing auxiliary, capable of constituting an aqueous injection or an injection to be dissolved when in use, such as in distilled water for injection, saline solution, propylene glycol, and the like; isotonicity agent, such as glucose, sodium chloride, D-mannitol, glycerol, and the like; pH regulator such as an inorganic acid, organic acid, inorganic or organic base or the like. According to the present invention, the adjuvants are preferably selected from the group comprising CpG and aluminum hydroxide.
In general, the appropriate dose can be administered in one to several portions or can be administered every few days according to a defined initial-boost dose protocol. In a preferred embodiment of the invention the composition comprises 1 to 500 μg of protein, 10 to 500 μg/mL of CpG, preferably 180 g/mL, 20 to 40% v/v of aluminum hydroxide, preferably 30% v/v. In another preferred embodiment of the invention the composition comprises 1 to 500 μg of protein and 100 to 1000 μg/mL of Poly-IC, preferably 500 μg/mL.
According to the present invention, the disease caused by Trypanosoma cruzi infection is Chagas disease.
The present invention is further described by the following non-limiting examples, which are merely illustrative. Several modifications and variations of the embodiments are evident to the person skilled in the art, without departing from the spirit and scope of the invention.
Several variations to the scope of protection of the present application are allowed. This reinforces the fact that the present invention is not limited to the particular configurations/embodiments described above.
Using the programs SYFPEITHI, Bimas and The Immune Epitope Database and Analysis Resource (IEDB), epitopes of the TS and ASP-2 proteins were predicted. From the epitopes found, the five best ranked for each HLA-ABC allele were selected, i.e., those with the highest probability of binding to MHC class I. Subsequently, the frequency with which the same peptide appeared among the top five was evaluated for all the HLA alleles analyzed, in search of immunodominant epitopes. The peptides were highlighted by colors in the amino acid sequence of the proteins, according to their frequency. After identifying these regions, the chimeric protein and sequence (SEQ ID No 1) was defined by joining the immunogenic region of the TS protein with that of the ASP-2 protein. In addition, three SAPA repeats were added, present in the TS protein, which, as described in previous studies, induce an intense antibody production. Therefore, the sequence of the chimeric protein includes the TS portion from amino acids 1 to 234, followed by SAPA (aa 235 to 270), a spacer region of three glycines (aa 271 to 273) and finally the ASP-2 region (aa 274 to 573).
The trials were carried out on C57BL/6 mice (6 animals/group). Vaccination consists of an initial dose and two booster doses, all with the same constructs, with an interval of 21 days from one to the other. One group receives the TS protein, another ASP-2 (only the regions contained in Chimera) and another the Chimera protein (SEQ ID No 1) in individual doses of 100 μg/mL of protein+180 μg/mL of CpG+30% v/v of aluminum hydroxide, in a final volume of 100 μL per mouse. CpG and aluminum hydroxide are vaccine adjuvants. The injection is made subcutaneously in the dorsal region. One control group receives only the adjuvants and the other only PBS. Thirty days after the last booster, part of the animals is euthanized to analyze the immune response and part is challenged with 104 trypomastigote forms of T. cruzi strain Y to evaluate parasitemia.
From the animals that were euthanized 30 days after the booster, in order to assess the efficacy of the vaccination, the spleen was removed to obtain splenocytes. The cells obtained were incubated with erythrocyte lysis solution (Ammonium-Chloride-Potassium—ACK), washed with RPMI 1640 medium containing 1% of antibiotic and counted. The splenocytes were then plated in 96-well plates (1×106 cells per well) and stimulated for 48 hours with the proteins TS, ASP-2 and Chimera, individually, at a concentration of 10 μg/mL. After the incubation period, the plates were centrifuged, and the culture supernatant was stored at −20° C. Subsequently, the levels of IFN-γ and IL-10 in the culture supernatant were assessed using the enzyme-linked immunosorbent assay (ELISA) technique, following the manufacturer's instructions (Mouse IFN-γ DuoSet ELISA, R&D Systems). The samples were assessed in duplicate and the colorimetric reaction was read in a microplate reader at 450 nm.
The results are shown in
To assess the specific humoral response before the challenge, blood samples (taken through the orbital plexus) from the immunized and challenged animals were collected 30 days after the last boost. The blood was centrifuged, and the plasma obtained was stored at −20° C. Subsequently, antibody production was assessed using the ELISA technique. Initially, a 96-well plate was sensitized with 5 μg of TS or ASP-2, and incubated overnight at 4° C. Subsequently, the animals' plasma was added (in duplicate) at a dilution of 1:800. This was followed by incubation with secondary antibodies for total IgG, IgG1 and IgG2a conjugated to streptavidin-HRP (dilution 1:5,000). The colorimetric reaction was read at 450 nm. As shown in
To evaluate the specific humoral response after challenge, blood samples (taken through the orbital plexus) from immunized and challenged animals were collected 30 days after challenge with T. cruzi. The blood was centrifuged, and the plasma obtained was stored at −20° C. Subsequently, the production of antibodies was assessed using the ELISA technique. Initially, a 96-well plate was sensitized with 5 μg of TS or ASP-2, and incubated overnight at 4° C. Subsequently, the animals' plasma was added (in duplicate) at a dilution of 1:800. This was followed by incubation with secondary antibodies for total IgG, IgG1 and IgG2a conjugated to streptavidin-HRP (dilution 1:5,000). The colorimetric reaction was read at 450 nm. The result is shown in
Once again, the production of total IgG and IgGI anti-TS was observed in animals vaccinated with TS and Chimera. In addition, in these groups there was also an increase in the production of anti-TS IgG2, and of total IgG and anti-ASP-21gG1.
Parasitemia was assessed 15 days post-challenge by viewing the parasites under a microscope. According to the Pizzi-Brener method (1962), 5 μL of blood collected from the animal's tail was placed between a slide and a coverslip (20 mm×20 mm) and 50 random fields were evaluated. After counting, the total number of parasites per mL is calculated using the following formula:
It was observed that the animals vaccinated with Chimera were more resistant to infection, showing parasitemia 96.7% lower than the PBS group on the seventh day post-infection (DPI). On the other hand, the animals vaccinated with TS and ASP-2 had partial protection.
During the course of the infection, the animals' weight was measured. To make it easier to see the differences between the groups, the percentage change in weight was calculated, with the initial weight taken as 100%.
In addition, the deaths that occurred during the experiment were recorded. As shown in
Subsequently, a comparison of vaccine formulations was carried out using Chimera supplemented with two different adjuvants. Because it showed excellent results in human clinical trials for cancer and HIV vaccines (AMMI et al., 2015; SAXENA et al., 2019), polyinosinic:polycytidylic acid (Poly (I:C)) was the adjuvant chosen to be compared with CpG and Alumen.
The formulation of Chimera+CpG+Alumen was maintained at the concentrations described above. For Chimera+Poly (I:C), 100 μg/mL of protein+500 μg/mL of Poly (I:C) were used, in a final volume of 100 μL per dose. The vaccine protocol was the same as previously used, consisting of three vaccine doses with an interval of twenty-one days between administrations.
The cellular immune response was induced in different experimental groups by detecting the cytokines IFN-γ and IL-10 in the splenocyte culture supernatant. In this experimental trial, in addition to the stimuli with rASP-2 and rTS, Chimera was added.
As can be seen in
The production of IL-10 (
The humoral immune response was assessed by the detection of total IgG, IgG1 and IgG2c antibodies specific to the rASP-2 and ITS proteins. It can be seen that both immunization with CpG+Alumen+Chimera and with Poly (I:C)+Chimera stimulated the production of anti-rASP-2 (
In order to compare the ability of the two vaccine formulations to induce an anti-parasitic response, immunized mice were challenged with 104 trypomastigotes of the Y strain from T. cruzi. Parasitemia was assessed every 2 days for 15 days after the challenge, starting on the third day after infection. As can be seen in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2 021010 469 4 | May 2021 | BR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/BR2022/050150 | 5/3/2022 | WO |
