Immunogenic mRNA Compositions Against Leishmaniasis and Uses

Description

1 / 17 “IMMUNOGENIC mRNA COMPOSITIONS AGAINST LEISHMANIASIS AND USES”

[01] The present technology deals with immunogenic compositions comprising the mRNA sequence defined by SEQ ID No. 1, lipid nanocarriers and pharmaceutically acceptable excipients. It also deals with the use of the mRNA sequence and the immunogenic compositions for the production of vaccines against leishmaniasis. The mRNA sequence encodes a recombinant protein associated with the kinetoplast of L. infantum. The immunogenic compositions containing the mRNA have demonstrated the ability to induce a humoral immune response and reduce parasite load in infection against L. amazonensis infection. Total IgG production was higher with the use of mRNA compared to the use of the recombinant protein.

[02] Leishmaniasis is a zoonotic disease caused by protozoa of the genus Leishmania, transmitted to humans by the bite of sandflies. It manifests in three main forms, each associated with different species of the parasite. Cutaneous leishmaniasis, the most common form, is characterized by skin ulcers and is mainly caused by L. major, L. tropica, and L. mexicana. Mucocutaneous leishmaniasis, which affects the mucous membranes of the nose, mouth, and throat, can lead to severe deformities and is associated with L. braziliensis. The most severe form of the disease, visceral leishmaniasis or kala-azar, affects internal organs such as the liver and spleen and is caused by L. donovani and L. infantum. With high mortality rates, mainly in East Africa and South Asia, visceral leishmaniasis is also the most lethal form of the disease. According to the World Health Organization (WHO), leishmaniasis results in approximately 3.3 million disabilities and 50,000 deaths annually.Considered the second deadliest parasitic disease in the world, leishmaniasis represents a serious public health problem. Petition 870250071082, dated 12 / 08 / 2025, page 5 / 25 2 / 17 exacerbated by the lack of effective treatments and vaccines. Although some medications and chemotherapy regimens exist, there is still no vaccine or specific treatment that offers robust protection against the disease.

[03] Among the neglected tropical diseases recognized by the WHO, leishmaniasis stands out for its high mortality and morbidity. With the increasing global burden of disease and the growing threats of climate change, urbanization, and drug resistance, the development of vaccines against leishmaniasis remains an urgent priority.

[04] mRNA vaccines have shown promise in combating infectious diseases, stimulating adaptive immunity through humoral and cellular responses. Although the therapeutic use of mRNA has been recognized for almost 30 years, the first RNA interference (siRNA)-based therapy, patisiran, was only approved in 2017. However, it was during the COVID-19 pandemic in 2020 that mRNA vaccines gained global prominence, leading to the approval of the Pfizer-BioNTech and Moderna vaccines.

[05] These vaccines offer several advantages over other vaccine technologies, such as recombinant protein-based vaccines, DNA vaccines, and live attenuated virus-based vaccines. They are safe because they pose no risk of infection or integration into the genome, eliminating concerns about insertional mutagenesis. mRNA can be synthesized rapidly and on a large scale, which facilitates efficient and low-cost production. In addition, mRNA vaccines only require the delivery of genetic material to the cell cytoplasm, unlike DNA vaccines, which rely on viral vectors to reach the nucleus. These characteristics make the Petition 870250071082, dated 12 / 08 / 2025, page 6 / 25 3 / 17 mRNA vaccines: a promising technology for the future of immunizations.

[06] mRNA vaccines have the ability to induce temporary expression of antigens, a process controlled by the lifespan of the coding mRNA, which is regulated by innate degradation pathways. In vitro transcribed mRNA (IVT) was recognized as effective in vivo in 1990 when it was administered to mice, allowing the delivery of antigenic information to antigen-presenting cells. The production of IVT mRNA involves the use of a linear DNA template of the gene of interest, which is transcribed by bacteriophage-derived RNA polymerases such as T7, T4, or SP6. The resulting mRNA, with a structure similar to natural mRNA, contains a 5' cap, 5' and 3' untranslated regions (UTRs), and a poly(A) tail. Modification of the UTRs is critical as it can increase the stability and efficiency of mRNA translation.Frequently, endogenously found protein UTRs with long half-lives that do not actively synthesize mRNA are selected for this purpose (e.g., α and β-globins, which are functional proteins found abundantly in erythrocytes). To protect mRNA against degradation by exonucleases and endonucleases, it is essential to have a poly(A) tail and a 5' cap. A common practice is to add a poly(A) tail containing between 100 and 150 nucleotides to the nucleotide sequence. Furthermore, the immunogenicity of the mRNA can be adjusted by incorporating modified nucleosides, such as (1-methyl)-pseudouridine, which decrease the activation of innate immune sensors and interferon alpha (IFN-α) signaling, thus minimizing the degradation of the invading mRNA.

[07] Despite the advantages of mRNA as a vaccine candidate, in vivo delivery of these molecules remains a challenge. mRNA is a negatively charged molecule, which generates repulsion with the Petition 870250071082, dated 12 / 08 / 2025, page 7 / 25 4 / 17 cell membrane, hindering its spontaneous entry into cells. Furthermore, it is highly unstable, being easily degraded by alkaline hydrolysis or the action of RNases. Therefore, the development of suitable systems for the cytoplasmic delivery of these molecules is essential to ensure the efficacy of mRNA-based vaccines.

[08] Lipid nanoparticles (LNPs) are an effective platform for delivering RNA and therapeutic agents, stably encapsulating genetic material and acting as a protective envelope that facilitates cell transfection. Due to the lipid composition of cell membranes, encapsulating RNA in lipid vesicles allows interaction with the membrane and release of RNA into the cytosol. LNPs utilize positively charged lipids, which interact electrostatically with negatively charged RNA, ensuring efficient encapsulation. In addition to the ionizable cationic lipid, LNPs are composed of auxiliary lipids including DSPC and cholesterol, and a polyethylene glycol (PEG)-lipid conjugate.Each lipid has a specific function, such as mRNA encapsulation, endosomal membrane disruption, cellular delivery, lipid bilayer formation and stabilization, particle size homogeneity and dispersion, particle stability during preparation and storage, circulation half-life, and biodistribution.

[09] LNPs can be prepared in several ways, with microfluidic methods being preferred due to their good reproducibility. LNPs can be produced by injecting organic solutions containing lipids and aqueous solutions into a microfluidic device, where the rapid mixing of the two miscible phases induces an increase in the polarity of the environment, lipid supersaturation, and consequent formation of LNPs. mRNA encapsulation occurs by association Petition 870250071082, dated 12 / 08 / 2025, page 8 / 25 5 / 17 electrostatic interaction between negatively charged nucleic acids and positively charged lipids.

[10] Several studies are underway to develop vaccines for the prevention of leishmaniasis. However, to date, no specific study on mRNA-based vaccines for this disease has been found.

[11] Patent document BR1020230095925, filed on 05 / 18 / 2023, entitled “Immunogenic compositions against visceral leishmaniasis, chimeric recombinant protein and uses”, describes the recombinant protein encoded by the mRNA used in the present technology and its use in immunogenic compositions for the production of vaccines against leishmaniasis. The patent document demonstrates that the recombinant protein induced the production of total IgG, IgG1 and Ig2 / 3, in addition to promoting partial protection against infection with L. infantum. In immunization with the mRNA encapsulated in LNPs, the production of total IgG was higher, indicating a greater humoral response compared to immunization with the recombinant protein. Furthermore, this approach resulted in partial protection against infection by L. amazonensis.

[12] US patent document 20130236484, whose priority date is 03 / 08 / 2013, entitled “Leishmaniasis antigen detection assays and vaccines”, describes a polypeptide comprising an immunogenic portion of an L. infantum or L. donovani antigen for use in diagnostics or vaccines. Although it claims the amino acid sequence and indicates the mRNA sequence that encodes it, it does not address the development of immunogenic compositions or vaccines that use mRNA sequences.

[13] The article by Gomes and colleagues, published in March 2022 in the journal Microbes Infect, entitled “Intranasal immunization with chitosan microparticles enhances LACK-DNA vaccine protection and Petition 870250071082, dated 12 / 08 / 2025, p. 9 / 25 The study, titled “6 / 17 induces specific long-lasting immunity against visceral leishmaniasis,” reports the development of a plasmid-based vaccine encoding the p36 / LACK antigen. In this study, chitosan microparticles associated with the plasmid were used to increase long-lasting immunity against late-onset L. infantum challenge. Infection at 7 days, 3, or 6 months after immunization resulted in significantly lower parasite loads compared to unvaccinated controls. The protection achieved was correlated with increased responsiveness of spleen cells to parasite antigens, marked by increased proliferation and IFN-γ, as well as decreased production of IL-10 and TNF-α.

[14] The article by He and colleagues, entitled “DNA prime-protein boost vaccine encoding HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2 against visceral leishmaniasis” and published in January 2019 in the journal Immunology, also presents protein- and DNA-based vaccines, this time encoding HLA-A2, HLA-A24 and HLA-DR1 against visceral leishmaniasis. BALB / c mice were immunized with a prime-boost regimen and subsequently challenged with a Leishmania strain recently isolated from a patient with visceral leishmaniasis. The DNA-based vaccine induced a strong immune response, with high titers of IgG antibodies and a robust cellular immunity response, showing long-term immunogenicity. Evaluation of the parasite load in the spleen of the different groups indicated that the vaccine conferred partial protection against infection, with a 76% reduction in the number of amastigotes.

[15] In the state of the art, no technology was found that presented the mRNA sequence defined by SEQ ID No. 1 for use in the production of vaccines against leishmaniasis, nor immunogenic compositions that included lipid nanocarriers for immunization. Petition 870250071082, dated 12 / 08 / 2025, page 10 / 25 7 / 17 human, in which immunization in an animal model was capable of inducing robust cellular and humoral responses, as well as prophylactic potential against L. amazonensis infection, validated by parasite load analysis in challenged animals. BRIEF DESCRIPTION OF THE FIGURES

[16] Figure 1 shows, in (A), the agarose gel for confirmation of in vitro mRNA synthesis defined by SEQ ID No. 1. The pcDNA3.1 plasmid modified with the Leishmania infantum gene encoding the AYU79903.1 protein (GenBank) was digested with Nhel and Xhol enzymes to confirm its identity. Channel 1 (PM) contains the molecular weight standard, with weights indicated in base pairs (bp). Channel 2 (n digested) indicates the pcDNA3.1 plasmid with the undigested protein sequence. Channel 3 (digested) indicates the pcDNA3.1 plasmid with the protein sequence, digested with Nhel and Xhol enzymes. In (B), the plasmid was linearized with the Xhol enzyme, purified, and analyzed on a 0.7% agarose gel stained with SyberSafe. Channel 1 (PM) contains the molecular weight pattern, with weights indicated in base pairs (bp). Channel 2 (n digested) indicates the undigested pcDNA3.1 plasmid. Channel 3 (Xhol digested) indicates the pcDNA3 plasmid.1 with the protein sequence digested with the enzyme Xhol. In (C), after the in vitro transcription reaction, the mRNA defined by SEQ lD No. 1 was purified and analyzed on a 1% agarose gel under denaturing conditions, stained with ethidium bromide. Channel 1 (PM) contains the molecular weight standard, with weights indicated in base number (bases). Channel 2 (mRNA SEQ lD No. 1) indicates the mRNA encoding the LinKAP protein transcribed from the plasmid. In (D) is represented the final structure of the mRNA, defined by SEQ lD No. 1, after in vitro transcription, with a Cap5' sequence, a 5' sequence and a 3' sequence. Petition 870250071082, dated 12 / 08 / 2025, page 11 / 25 8 / 17 UTR, the protein-coding sequence in the center, and a tail of 110 adenines.

[17] Figure 2 represents, in A, the dynamic light scattering (DLS) size distribution curve and, in B, the cryo-TEM image of the LNPs encapsulating the mRNA defined by SEQ ID No. 1 (LNP-mRNA) by immersion freezing technique.

[18] Figure 3 shows, in A, the agarose gel representative of the mRNA defined by SEQ ID No. 1 synthesized in vitro. Lane 1 contains the molecular weight standard, with weights indicated in number of bases. Lane 2 (mRNA SEQ ID No. 1) indicates the mRNA transcribed in vitro from the plasmid and, in B, the Western blot performed with total protein extract from HEK 293T cells 48 hours after transfection with LNP-mRNA, demonstrating the encoded protein. Lane 1 contains the extract from untransfected cells. Lane 2 contains the extract from cells incubated with mRNA without any transfection reagent. Lane 3 contains the extract from cells incubated with the LinKAP mRNA formulation encapsulated in lipid nanoparticles (LNPs). Lane 4 contains the LinKAP protein produced in bacteria and purified. Below, the same membrane is incubated with anti-β-actin antibody, as a control for the amounts of cell extract that were used in each channel.

[19] Figure 4 shows the in vivo bioluminescence assays of luciferase encapsulated in LNPs (LNP-Luc) in mice. Female BALB / c mice were inoculated with 10 µg of LNP-Luc via IM and subjected to IVIS spectrum imaging at the times indicated after administration, as shown in A. B shows the quantification of luminescence expressed in photons per second (p / s) after IM inoculation, and C shows the tissue distribution of LNP-Luc in Petition 870250071082, dated 12 / 08 / 2025, page 12 / 25 9 / 17 mice and, in D, the quantification of the tissue distribution of LNPLuc in mice.

[20] Figure 5 represents cytokine production in response to LNPs with or without mRNA defined by SEQ ID No. 1 and SM-102. Mouse splenocyte cultures were stimulated with LNPs and cytokine levels were measured 16 hours after incubation for TNF, IFN-γ, IL-6, IL-10, IL-2, IL-4 and IL-17.

[21] Figure 6 represents the evaluation of the immune response and protection in a homologous immunization protocol, in which mice were immunized in a prime-boost-boost regimen (dose, booster and 2nd booster) with LNPs, with blank LNP (without mRNA), with LNP-mRNA and only with the protein already encoded as a comparator. Serum was collected on days 20 and 40 for analysis of specific total IgG (A) and subclasses (B). In (C) there is the evaluation of IFN-γ production in splenocyte culture, in RPMI medium, with protein stimulation and with Concanavalin A (ConA), in (D) the parasite load by limiting dilution assay 60 days after challenge with L. amazonensis (5*105 parasites). DETAILED DESCRIPTION OF THE TECHNOLOGY

[22] The present technology relates to immunogenic compositions comprising the mRNA sequence defined by SEQ ID No. 1, lipid nanocarriers and pharmaceutically acceptable excipients. It also relates to the use of the mRNA sequence and the immunogenic compositions for the production of vaccines against leishmaniasis. The mRNA sequence encodes a recombinant protein associated with the kinetoplast of L. infantum. The immunogenic compositions containing the mRNA have demonstrated the ability to induce a humoral immune response and reduce parasite load in the infection against L. Petition 870250071082, dated 12 / 08 / 2025, page 13 / 25 10 / 17 amazonensis. Total IgG production was higher with the use of mRNA compared to the use of recombinant protein.

[23] More specifically, the mRNA sequence defined by SEQ ID No. 1 encodes the recombinant protein corresponding to amino acid sequence No. 1 to 200 of protein AYU79903.1 (GenBank). The sequence must be carried in lipid nanoparticles, composed of 1,2-dioleyioxy-3-dimethylaminopropane (DODMA), dimyristoylglycerol associated with polyethylene glycol-2000 (DMG-PEG2000), cholesterol and 1,2-diestearoylsn-glycero-3-phosphocholine (DSPC), in the ratio 50:10:39:1 to 40:20:35:5. The compositions can be liquid or lyophilized.

[24] The mRNA sequence and immunogenic compositions of the present technology can be used to prepare a vaccine against leishmaniasis. The vaccine can be administered, preferably, intramuscularly or subcutaneously.

[25] The present invention can be better understood through the following examples, which are not limiting. EXAMPLE 1 - MESSENGER RNA SYNTHESIS

[26] The pcDNA3.1 vector was modified with the gene encoding the AYU79903.1 protein (GenBank), cloned between the cleavage sites of the Nhel and Xhol restriction enzymes, the T7 RNA polymerase promoter sequence and a synthetic 5' UTR sequence (doi: 10.1038 / s41591021-01574-5) above the protein coding sequence, a 3' UTR sequence of the human alpha globin gene immediately below it, and a sequence of 110 adenines adjacent to the 3' UTR sequence. It also includes an ampicillin resistance gene. The use of this vector is one, but not a limiting, embodiment of the technology. Other vectors can be used at this stage, such as pUC19, or any vector containing an origin of replication, a T7 RNA polymerase promoter sequence, and an antibiotic resistance gene. Petition 870250071082, dated 12 / 08 / 2025, page 14 / 25 11 / 17

[27] To obtain larger quantities of plasmid, it was transformed into E. coli XL-1 Blue bacteria and purified on silica columns. After purification, the plasmid was digested with NheI and XhoI enzymes to confirm its identity. Agarose gel analysis of the digestion product showed plasmid DNA fragments with the expected sizes of 5.3 kb (kilobases) and 891 bp (base pairs) (Figure 1A). To be used as a template in the in vitro transcription reaction, the plasmid was linearized with the XhoI enzyme and purified on a silica column (Figure 1B).

[28] For the in vitro transcription reaction, the plasmid was incubated with an appropriate buffer, with the Cap analog for the addition of the 5' Cap, and with the T7 RNA polymerase enzyme. In addition, ribonucleotides rATP, rGTP, rCTP, and the rUTP analog (N1-Methylpseudouridine-5'-triphosphate) were added. The reaction was incubated at 37°C for 3 hours, and at the end, DNase was added to degrade the plasmid used as a template. Subsequently, the transcribed mRNA was purified with phenol and chloroform and analyzed on an agarose gel under denaturing conditions. As can be seen in Figure 1C, an intact band corresponding to the expected mRNA size defined by SEQ ID No. 1, of 909 bases, was detected. Figure 1D shows the final mRNA structure defined by SEQ ID No. 1 after in vitro transcription and the addition of Cap5'. EXAMPLE 2 - PREPARATION OF LIPID NANOPARTICLES

[29] The LNP formulations were produced by the nanoprecipitation method. The organic phase consists of a commercially obtained ionizable cationic lipid (1,2-dioleyloxy-3-dimethylaminopropane - DODMA), 1,2-diestearoylsn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and cholesterol, solubilized in ethanol (in the molar ratio of Petition 870250071082, dated 12 / 08 / 2025, page 15 / 25 12 / 17 The organic phase consisted of mRNA in 20 mM citrate buffer, pH 4.0, at concentrations between 30 and 175 µg / mL. The organic and aqueous phases were combined in a microfluidic device (ANP, Particle Works, UK) at a flow ratio of 1:3 (ethanol:aqueous phase), with a total flow rate between 9 and 12 mL / min. The LNP containing the mRNA defined by SEQ ID No. 1 was ultrafiltered through a 30 kDa Amicon column by centrifugation at 10,000 x g for 30 minutes to remove ethanol and citrate buffer and concentrate the formulation. It was then resuspended in 0.1 M PBS buffer, pH 7.4, to a final volume of 1.0 mL.

[30] The particle size, polydispersity index (PDI), and zeta potential of mRNA-containing LNPs defined by SEQ ID No. 1 (LNPmRNA) were determined by dynamic light scattering (DLS) at a fixed angle of 90° and 25°C using the Zetasizer Nano ZS90 (Malvern Instruments, UK). Nanoparticle tracking analysis (NTA) was performed using the NanoSight NS300 instrument and NTA 3.1 software (Malvern Instruments, UK). To assess encapsulation efficiency and mRNA concentration in LNPmRNA, the Quant-iT™ RiboGreen™ RNA reagent and kit (Invitrogen) were used. Fluorescence was measured in freshly prepared formulations, both in the presence and absence of Triton X100. The morphology of LNP-mRNAs was examined by cryo-TEM transmission electron microscopy, using the immersion freeze technique, in which the samples were distributed in a thin film on a microscope grid and immersed in liquid ethane.It was possible to encapsulate both luciferase mRNA and mRNA defined by SEQ ID No. 1. DLS analysis showed that the average particle size of LNPs in PBS was close to 100 nm, with a low PDI. Petition 870250071082, dated 12 / 08 / 2025, page 16 / 25 13 / 17 (< 0.2), indicating a high degree of homogeneity between the particles, and a slightly negative zeta potential (Figure 2 A, Table 1).

[31] High encapsulation efficiency (> 85%) was achieved for both formulations, which is crucial for achieving the in vivo dose required for immunizations. The concentration of LNPmRNA particles and size distribution (i.e., the average diameter at which 10, 50, and 90% of the particles are smaller) were evaluated by NTA (Table 1). Consistent with DLS analysis, particle sizes ranged from 84 to 207 nm, with 90% measuring less than 200 nm, for both mRNAs. For all formulations, the particle concentration was close to 10⁸ particles / ml. Cryo-TEM images (Figure 2B) revealed that the mRNA-loaded LNP particles have rounded, smooth-surfaced, nanometer-sized particles, consistent with the DLS and NTA results. A thin layer surrounding the shiny inner portion, indicating the presence of a semi-structured lipid bilayer, was also observed.The presence of an electron-dense core and 'bleb' cavities in the structure indicates the location of mRNA within the particles. Table 1 - Average diameter, polydispersity index (PDI), zeta potential, encapsulation efficiency (%EE), size distribution (D10, D50, D90) and particle concentration for LNP-mRNA and LNP-Luc. Average size (nm) Zeta Potential PDI (mV) %EE LNP-Luc 106.20 ± 9.26 0.17 ± 0.07 -4.80 ± 1.64 92.29 ± 1.89 LNP-mRNA 106.05 ± 6.15 0.21 ± 0.04 -4.37 ± 2.31 86.83 ± 3.90 Particles / ml Distribution (nm) D10 D50 D90 LNP-Luc 4.48x10⁸ 84.6 ± 1.2 113.3 ± 3.7 119.4 ± 15.0 LNP-mRNA 1.07x10⁹ 95.2 ± 0.9 124.8 ± 1.9 207.1 ± 4.4 Petition 870250071082, dated 12 / 08 / 2025, page 17 / 25 14 / 17 EXAMPLE 3 - PRODUCTION OF FUNCTIONAL LNPs, WITH IN VITRO DELIVERY CAPABILITY

[32] The integrity of the newly transcribed mRNA was confirmed by denaturing agarose gel electrophoresis, in which a 909 nt band corresponding to the mRNA defined by SEQ ID No. 1 was observed (Figure 3A). To evaluate in vitro cellular delivery and translation efficiency of the encapsulated mRNA, HEK 293T cells were transfected with LNP-mRNA. 48 h after transfection, Western blot assays were performed using extracts from the transfected cells and anti-leishmania antibodies. The results indicated that transfection with LNP-mRNA resulted in the production of a protein with approximately 50 kDa (Figure 3B). Although the predicted molecular weight for the protein is 25 kDa, corresponding to a 235 amino acid protein, the anomalous mobility observed in SDS-PAGE, probably due to the presence of a large repetitive domain, has been previously reported.The effectiveness of mRNA vaccines depends on the expression of the protein antigen, followed by an antigen-specific immune response. Delivering sufficient quantities of mRNA remains a crucial aspect of vaccine development. The results demonstrated that the developed formulation efficiently delivers mRNA to cells, resulting in detectable levels of protein expression. In contrast to naked mRNA, which exhibits a low transfection rate in immortalized cell lines unless complexed with cationic polymers or lipid reagents (such as TransIT or Lipofectin), LNPs act as transfection agents, facilitating mRNA entry into the cell cytoplasm via endocytic pathways. Petition 870250071082, dated 12 / 08 / 2025, page 18 / 25 15 / 17 EXAMPLE 4 - PRODUCTION OF FUNCTIONAL LNPs, WITH IN VIVO DELIVERY CAPACITY

[33] To evaluate the tissue distribution of the formulation, LNP containing mRNA encoding the luciferase enzyme (LNP-Luc) was administered into the thigh muscle of BALB / c mice. Bioluminescence imaging (BLI) analyses were performed at 6, 24, 48, and 72-hour intervals. Intramuscular injection generated robust luciferase expression, which remained predominantly at the injection site throughout the observation period (Figure 4A). Prolonged retention of LNP-Luc was confirmed, with bioluminescence signal detected up to 3 days after application (Figure 4B). Ex vivo analyses revealed elevated levels of bioluminescence in the liver and spleen, while the heart, kidneys, and lungs did not show significant luciferase activity (Figures 4C and D). EXAMPLE 5 - EVALUATION OF THE ADJUVANT CHARACTERISTICS OF LNPs

[34] Several studies have shown that LNPs can increase the effectiveness of mRNA vaccines by stimulating innate immunity. The ability of LNPs, with or without mRNA, to activate the innate immune response was evaluated by incubating the nanoparticles with mouse splenocytes and measuring the levels of different cytokines. For comparative control, LNPs formulated with the ionizable lipid SM-102, known for its ability to stimulate innate immunity, were used. The results showed that both LNPs prepared with DODMA and with SM-102 induced a significant production of TNF, indicating a strong activation of the innate immune response (Figure 5). In addition, a significant increase in the production of IFN-γ and IL-6 was observed in response to both LNPs. No statistically significant differences were identified between the LNPs without Petition 870250071082, dated 12 / 08 / 2025, page 19 / 25 16 / 17 mRNA and LNPs with mRNA, highlighting the overall effectiveness of LNPs. In contrast, free RNA did not induce the production of any of the cytokines evaluated, emphasizing the crucial role of lipids as essential components in mRNA vaccines for effective activation of innate immunity. The absence of IL-10 production, an anti-inflammatory cytokine, suggests that LNPs do not trigger an anti-inflammatory response that could compromise the desired immunological efficacy. These findings indicate that the new LNP formulation used is effective in inducing initial inflammatory responses, characterized by the production of pro-inflammatory cytokines such as TNF, IFN-γ, and IL-6, essential elements for the adjuvant activity of the delivery system and for the efficacy of mRNA vaccines. EXAMPLE 6 - HUMORAL AND CELLULAR RESPONSE INDUCED BY LNP-mRNA IMMUNIZATION AND PROTECTION AGAINST LEISHMANIA CHALLENGE

[35] To evaluate the immunogenic and protective capacity of the vaccine, female C57BL / 6 mice were immunized intramuscularly with: 10 pg of LNP-mRNA, and controls, following a homologous primeboost-boost regimen with 21-day intervals between doses. Sera were collected on days 20, 40, and 71 for analysis of specific total IgG and subclasses. Thirty days after the last immunization, spleens from some mice were collected and used in splenocyte cultures, appropriately stimulated to evaluate IFN-γ production. The mice were challenged with L. amazonensis (5*10⁵ parasites) by subcutaneous inoculation in the paw. Sixty days after the challenge, the parasite load in the lesion of the animals was evaluated using a limiting dilution assay. The results of the ELISA, performed with the recombinant protein and sera from immunized animals, demonstrated high levels of antibodies generated in mice that received the Petition 870250071082, dated 12 / 08 / 2025, page 20 / 25 17 / 17 LNP-mRNA formulation (Figure 6A-C), with a predominance of the IgG2c subclass. Furthermore, IFN-γ production was observed (Figure 6D). Notably, immunization with LNP-mRNA induced the production of higher antibody titers and similar ex vivo IFN-γ levels compared to immunization with the corresponding adjuvanted recombinant protein. Evaluation of parasite load revealed that LNP-mRNA significantly reduced the number of parasites, comparable to the recombinant protein, thus confirming the protective potential of the formulation. Petition 870250071082, dated 12 / 08 / 2025, page 21 / 25

Claims

1 / 1 CLAIMS 1. IMMUNOGENIC COMPOSITIONS AGAINST LEISHMANIASIS, characterized by comprising the mRNA sequence defined by SEQ ID No. 1, lipid nanocarriers and pharmaceutically acceptable excipients.

2. COMPOSITIONS, according to claim 1, characterized in that the lipid nanocarriers comprise 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), distearolphosphatidylcholine (DSPC), cholesterol and polyethylene glycol (PEG), in the ratio of 50:10:39:1 to 40:20:35:

5.

3. COMPOSITIONS, according to claim 1, characterized by being presented in liquid or lyophilized form.

4. USE OF THE mRNA SEQUENCE defined by SEQ ID No. 1, characterized as being for the preparation of a vaccine against leishmaniasis.

5. USE OF THE IMMUNOGENIC COMPOSITIONS defined in any of claims 1 to 2, characterized by being for the preparation of a vaccine against leishmaniasis. Petition 870250014418, dated 21 / 02 / 2025, pp. 31 / 37

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