Vaccine composition for preventing malaria comprising EF-2 recombinant protein derived from plasmodium falciparum as active ingredient

Abstract

The present invention relates to a vaccine composition for preventing malaria, comprising, as an active ingredient, an elongation factor-2 (EF-2) recombinant protein derived from Plasmodium falciparum and, more specifically, to a vaccine composition for preventing malaria, comprising, as an active ingredient, EF-2 recombinant peptides derived from Plasmodium falciparum consisting of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and use thereof.

Description

A vaccine composition for preventing malaria comprising EF-2 recombinant protein derived from Plasmodium falciparum as an active ingredient

[0001] The present invention relates to a vaccine composition for preventing malaria comprising an EF-2 (elongation factor-2) recombinant protein derived from Plasmodium falciparum as an active ingredient, and a method for preventing malaria infection using the vaccine composition.

[0002]

[0003] This work was supported by the Future Growth High Value-Added Vaccine Development Project of the Korea Health Industry Development Institute, the Core Technology Development Project of the National Research Foundation of Korea, and the Science and Engineering Research Infrastructure Establishment Project (Project Numbers: HV22C0192000023, 2024M3A9H5043148, 2015R1A6A1A03032236).

[0004] Malaria is an infectious disease caused by the parasite Plasmodium, which enters the bodies of humans, monkeys, rats, and birds through mosquito bites. Malaria causes malaria when the parasite lives in the bloodstream, and it is the single most common cause of death worldwide (2.4 million people annually). Approximately 3 to 5 million people are infected with malaria each year, with relatively high morbidity and mortality rates. The World Health Organization estimates that 2 to 3 million children die from malaria each year in Africa alone. Furthermore, the widespread occurrence and increasing prevalence of malaria caused by drug-resistant parasites (Plasmodium malariae) and insecticide-resistant vectors (Anopheles) have highlighted the need for developing novel malaria control methods in many countries.

[0005] Existing malaria vaccines are limited to surface proteins, but the parasite's high mutation rate makes preclinical testing difficult. To address this issue, the present invention selected an intracellular antigen with high homology, enabling preclinical testing, as the vaccine material and evaluated its efficacy through preclinical testing.

[0006] Meanwhile, Korean Patent Publication No. 2020-0104066 discloses a 'malaria prevention vaccine composition comprising an EF-1α recombinant protein derived from Plasmodium falciparum as an active ingredient', and Korean Patent Publication No. 2023-0060818 discloses a 'virus-like particle combination vaccine comprising MSP-8, MSP-9, and RAP1 of malaria parasites', but there is no description of the 'malaria prevention vaccine composition comprising an EF-2 recombinant protein derived from Plasmodium falciparum as an active ingredient' of the present invention.

[0007] The present invention was derived from the above-mentioned needs, and the inventors of the present invention selected an EF-2 protein derived from Plasmodium falciparum, which has a homology of 98% or more among the intracellular antigens of Plasmodium falciparum and P. berghei, and then recombined it into two segments [fragment 1 (1F), fragment 2 (2F)], and then immunized a mouse model with the recombinant peptides of 1F and 2F, and confirmed the infection protection effect against rodent malaria. As a result, it was confirmed that the combination of the 1F and 2F recombinant peptides of the EF-2 protein has an antimalarial effect, thereby completing the present invention.

[0008] To solve the above problem, the present invention provides a vaccine composition for preventing malaria, which comprises as active ingredients an EF-2 (elongation factor-2) recombinant peptide derived from Plasmodium falciparum having an amino acid sequence of sequence number 1 and an EF-2 recombinant peptide derived from Plasmodium falciparum having an amino acid sequence of sequence number 2.

[0009] In addition, the present invention provides a method for preventing malaria infection, comprising a step of administering the vaccine composition to an individual expected to be infected with Plasmodium.

[0010] In addition, the present invention provides a kit for diagnosing malaria infection, comprising an EF-2 recombinant peptide derived from Plasmodium falciparum, comprising an amino acid sequence of sequence number 1 or sequence number 2.

[0011] The vaccine composition of the present invention contains an EF-2 recombinant peptide exhibiting an antimalarial effect as an active ingredient, and thus can be used to prevent malaria.

[0012] Figure 1 is an SDS-PAGE gel photograph confirming the first segment (1F) and second segment (2F) recombinant peptides of EF-2 derived from Plasmodium falciparum after expression and purification.

[0013] Figure 2 shows the results of analyzing the survival rate of experimental animals after infection with rodent malaria parasite (P. berghei). Mock: Uninfected normal control group, Pb: Rodent malaria parasite-infected group, BSA (Alum): Rodent malaria parasite-infected group after bovine serum albumin immunization, 1F+2F (Alum): Rodent malaria parasite-infected group after immunization with 1F and 2F recombinant peptides of EF-2.

[0014] Figures 3 and 4 show analyses of parasitemia in experimental animals after infection with rodent malaria parasite (P. berghei). Figure 3 shows the results for the rodent malaria parasite infection group after immunization with bovine serum albumin (BSA), and Figure 4 shows the results for the rodent malaria parasite infection group after immunization with 1F and 2F recombinant peptides of EF-2.

[0015] Figure 5 shows the blood analysis results of the vaccine antigen (1F and 2F recombinant peptides of EF-2) immunization experimental group that survived 30 days after infection with rodent malaria parasite (P. berghei). WBC: white blood cell, RBC: red blood cell, Hgb: hemoglobin, Hct: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RDW: red blood cell size distribution width, PLT: platelet, MPV: mean platelet volume, PCT: platelet volume percentage, PDW: platelet distribution width.

[0016] Figure 6 shows the results of analyzing the changes in white blood cells in the blood of the vaccine antigen (1F and 2F recombinant peptides of EF-2) immunized experimental group that survived 30 days after infection with rodent malaria parasite (P. berghei). LYM%: lymphocyte percentage, MON%: monocyte percentage, GRA%: granulocyte percentage, LYM#: lymphocyte count, MON#: monocyte count, GRA#: granulocyte count.

[0017] Figure 7 shows the results of analyzing parasitemia in experimental animals after infection with rodent malaria parasite (P. berghei) in the experimental groups immunized with 1F and 2F recombinant peptides of EF-2 derived from P. falciparum and the experimental group immunized with the whole recombinant protein of the same EF-2. (a) shows the parasitemia of each experimental group for 23 days from rodent malaria parasite infection, and (b) and (c) show the parasitemia results on the 21st and 23rd days, respectively. *: p < 0.05.

[0018] Figure 8 shows the results of ELISA analysis of the detection limit using 4-fold serial dilutions to determine whether 1F and 2F recombinant antigen-specific IgG antibodies were produced in serum isolated 14 days after the final immunization of the experimental group immunized with 1F and 2F recombinant antigens of EF-2 derived from P. falciparum. Non-immunized mouse serum served as the control group. *: p < 0.05.

[0019] Figure 9 shows the results of an ELISA experiment to evaluate antigen-specific IgG subclasses using sera isolated 14 days after the final immunization of the 1F and 2F recombinant antigen immunization experimental group of EF-2 derived from P. falciparum. Serum from non-immunized mice served as the control group. *: p < 0.05.

[0020] In order to achieve the purpose of the present invention, the present invention provides a vaccine composition for preventing malaria, which comprises as active ingredients an EF-2 (elongation factor-2) recombinant peptide derived from Plasmodium falciparum having an amino acid sequence of SEQ ID NO: 1 and an EF-2 recombinant peptide derived from Plasmodium falciparum having an amino acid sequence of SEQ ID NO: 2.

[0021] In the vaccine composition of the present invention, the malaria refers to an acute febrile infectious disease caused by infection with the malaria parasite (Plasmodium).

[0022] The scope of the EF-2 recombinant peptide according to the present invention includes peptides having amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 and functional equivalents of the peptides. The term "functional equivalent" refers to a peptide having at least 70%, preferably 80%, more preferably 90%, and even more preferably 95% sequence homology with the amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 as a result of addition, substitution, or deletion of amino acids, and exhibiting substantially the same physiological activity as the peptides represented by SEQ ID NO: 1 and SEQ ID NO: 2.

[0023] The vaccine composition of the present invention preferably has immunogenicity, but is not limited thereto.

[0024] In addition, the vaccine of the present invention is preferably a subunit vaccine, but is not limited thereto. The term "subunit vaccine" refers to a vaccine containing an infection-protective antigen isolated from a microorganism, and refers to a protein subunit vaccine that uses only a portion of a microorganism's protein through protein purification or genetic recombination technology.

[0025] In the present invention, the term “prevention” means any act of suppressing or delaying the onset of malaria parasite infection by administering a vaccine composition according to the present invention.

[0026] The vaccine composition of the present invention further comprises a pharmaceutically acceptable carrier or diluent. Suitable carriers for vaccines are well known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. The carriers may be aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate.

[0027] Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous carriers include electrolyte replenishers, liquids, and nutritional supplements, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may be additionally present. Preferred preservatives include formalin, thimerosal, neomycin, polymyxin B, and amphotericin B.

[0028] In addition, the vaccine composition may further comprise an adjuvant. The adjuvant refers to a compound or mixture that enhances the immune response and / or promotes the absorption rate after vaccination, and includes any absorption-promoting agent. Acceptable adjuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, ketolimpet hemocyanin, dinitrophenol, and the like.

[0029] The vaccine composition of the present invention can be administered through any one route of administration selected from oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or nasal, and can be administered as a bolus or slowly injected, but is preferably administered as an injection.

[0030] The vaccine composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to exhibit a vaccine effect, but not to cause side effects or serious or excessive immune responses. The level of the effective dose may vary depending on various factors, including the disorder to be treated, the severity of the disorder, the activity of the specific compound, the route of administration, the clearance rate of the recombinant EF-2 peptide, the duration of treatment, drugs used in combination or concurrently with the recombinant EF-2 peptide, the age, weight, sex, eating habits, general health of the subject, and other factors known in the medical field. Various general matters to be considered when determining a "therapeutically effective amount" are well known to those skilled in the art.

[0031] The present invention also provides a method for preventing malaria infection, comprising administering the vaccine composition to an individual expected to be infected with Plasmodium.

[0032] In the preventive method of the present invention, the malaria parasite may be, but is not limited to, Plasmodium falciparum or Plasmodium berghei.

[0033] Additionally, in the preventive method according to the present invention, the subject means a host subject that can be infected by the malaria parasite (Plasmodium), and may be a mammal other than a human, but is not limited thereto.

[0034] The present invention also provides a kit for diagnosing malaria infection, comprising a recombinant peptide of EF-2 (elongation factor-2) derived from Plasmodium falciparum, comprising an amino acid sequence of sequence number 1 or sequence number 2.

[0035] The kit of the present invention may include an EF-2 recombinant peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 according to the present invention and a reagent capable of detecting an antibody specifically binding to the EF-2 recombinant peptide. In the kit of the present invention, the EF-2 recombinant peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 functions as an antigen, and the reagent for detecting the peptide (antigen)-antibody complex may include, but is not limited to, a reagent for radial immunoassay, ELISA (Enzyme linked immunosorbent assay), or immunofluorescence analysis.

[0036] A method for diagnosing malaria infection using a kit according to the present invention may be performed by reacting a sample (e.g., immune serum) isolated from a subject suspected of being infected with malaria with an EF-2 recombinant peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention under conditions in which an antigen / antibody complex can be formed, and then detecting the formation of an antigen / antibody complex.

[0037]

[0038] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0039]

[0040] Materials and Methods

[0041] 1. Culturing of malaria parasites

[0042] The blood stage of Plasmodium falciparum 3D7 was cultured in vitro. Cultures were performed in RPMI 1640 medium containing human O-type red blood cells at a hematocrit of 5%, supplemented with 24 mM sodium bicarbonate, 25 mM HEPES, 0.8% hypoxanthine, 0.9% albumin, and 25 μg / mL gentamicin. Cultures were performed at 37°C in a CO2-O2-N2 incubator (atmosphere composition: 5% CO2, 5% O2, and 90% N2), and the medium was replaced daily. Cultures were monitored by periodic smears and Giemsa staining.

[0043]

[0044] 2. Antigen expression

[0045] The first segment of the PfEF-2 (GenBank accession number: XM_001348624.1) gene was amplified using the first primer set [forward (5'-CTCTCTAAGCTTATGGTGAACTTTACGGTA-3', SEQ ID NO: 5) and reverse (5'-CTCTCTCGAGGGATCCTTATACTATATATTGATC-3', SEQ ID NO: 6)] using genomic DNA extracted from Plasmodium falciparum 3D7 as a template, and the second segment of the PfEF-2 gene was amplified using the second primer set [forward (5'-CTCTGGATCCGATGAAATCAGGTACTATT-3', SEQ ID NO: 7) and reverse (5'-CTCTCTCGAGGGATCCTTAAAGTTTATCTAAATA-3', SEQ ID NO: 8)]. PCR was performed using a T100 Thermal Cycler (Bio-Rad, USA), and the reaction conditions were as follows: 95°C, 5 min → [95°C, 30 sec → 56°C, 45 sec → 72°C, 1 min] (30 cycles) → 72°C, 5 min. The PCR products were each cloned into the pET21b+ vector (Novagen, USA), and the recombinant vector was transformed into Escherichia coli BL21 (DE3). For the expression of the recombinant antigen of PfEF-2, the transformed E. coli was inoculated into 200 ml of LB liquid medium containing 100 μg / ml of ampicillin, and the culture medium was incubated at 37°C for OD 600The cells were cultured until the value reached 0.4. After that, 0.5 mM IPTG (isopropyl β-d-1-thiogalactopyranoside) was added and cultured at 37°C for 4 hours to induce the expression of the antigen protein. The cell pellet was collected by centrifugation at 8,000 rpm for 10 minutes, washed with PBS, resuspended in lysis buffer, and then sonicated. The sonicated lysate was centrifuged at 12,000 rpm for 20 minutes to obtain the pellet and inclusion bodies (IB). The IB was then solubilized using 6 M Gu-HCl (guanidine hydrochloride), and the solubilized supernatant was purified using a His-tag column to purify the recombinant protein. The purified recombinant protein was incubated with a refolding buffer for 36–48 hours to induce protein refolding, and then dialyzed to remove the refolding buffer. The purified recombinant protein was confirmed by loading it onto an SDS-PAGE gel.

[0046]

[0047] 3. Animal testing to verify vaccine efficacy

[0048] Six-week-old C57BL / 6 female mice (Orient Bio Co., Korea) were purchased and acclimated for one week. All experimental animals were maintained under restricted conditions with limited food and water. Animal experiments were conducted in accordance with experimental procedures approved by the Seoul National University Institutional Animal Care and Use Committee.

[0049] For immunization, 50 μg each of the first segment of rPfEF-2 (1F, SEQ ID NO: 1) and the second segment of rPfEF-2 (2F, SEQ ID NO: 2) and the adjuvant alum were mixed in a 1:1 ratio, and 100 μl (1F 0.5 μg / μl, 2F 0.5 μg / μl per mouse) was administered once a week at 3-week intervals for a total of 3 times. The first dose was administered subcutaneously, and the second and third doses were administered intraperitoneally. Two weeks after the final immunization, 1 × 10 rodent malaria parasite (P. berghei) NK65 obtained from MR4 (Malaria Research and Reference Reagent Resource Center) was administered. 3 Each mouse was infected by injection of a sera sample into the tail vein. Bovine serum albumin (BSA) was mixed with alum in the same amount as rPfEF-2 1F and rPfEF-2 2F and used as a control for immunization. Six mice were used for each experimental group. After infection with P. berghei, blood samples were collected from the tail vein every two days to analyze parasitemia, and the survival rate of the mice was analyzed daily for 30 days.

[0050]

[0051] 4. Analysis of antigen-specific immune effects

[0052] The serum of the immunized mice above was obtained by collecting only the serum from buccal blood (facial vein blood collection) or orbital blood collection 14 days after the final immunization. 100 ng of the expressed protein antigen was added to a 96-well plate and coated by incubation overnight at 4°C. After three washes, the plate was blocked with 5% skim milk for 2 hours at 37°C. After three washes, 100 μl of mouse serum diluted in blocking buffer was added for 1 hour and reacted for 1 hour at 37°C. After three washes, 100 μl of anti-mouse IgG-HRP was diluted 1:10,000 in blocking buffer as a secondary antibody and added for 1 hour at 37°C. After five washes, 100 μl of TMB solution was added, and 2 minutes later, stop solution was added to prevent nonspecific reactions and stop the reaction. Absorbance was measured at 450 nm.

[0053]

[0054] Example 1. Expression of EF-2 (elongation factor-2) recombinant protein

[0055] The first fragment (1F) of recombinant rPfEF-2 expressed in E. coli cells and the second fragment (2F) of rPfEF-2 were identified in the cell pellet after IPTG expression induction, and the finally purified recombinant rPfEF-2 1F and rPfEF-2 2F were identified to have sizes of 55 kDa and 44 kDa, respectively (Fig. 1).

[0056]

[0057] Example 2. Evaluation of vaccine efficacy of rPfEF-2

[0058] To evaluate the efficacy of rPfEF-2 1F and rPfEF-2 2F as vaccine antigens, animal experiments were conducted. As a result of checking the survival rate after P. berghei infection, all animals in the non-immunized P. berghei-infected group (Pb) died on the 21st day, and only 2 animals in the BSA comparison control group survived on the 30th day, showing a survival rate of 33.3%. In the experimental group simultaneously immunized with rPfEF-2 1F and rPfEF-2 2F, 5 animals survived for 30 days, showing a survival rate of 83% (Fig. 2).

[0059] In addition, the non-immunized P. berghei-infected group (Pb) died after maintaining a blood parasite count of approximately 20% on the 21st day, and two out of six surviving BSA control groups showed blood parasite counts of less than 1% (Fig. 3), and among five surviving experimental groups simultaneously immunized with rPfEF-2 1F and rPfEF-2 2F, only one showed a blood parasite count of 17% for 30 days, and the remaining four were confirmed to maintain blood parasite counts of less than 1% (Fig. 4).

[0060] In addition, as a result of examining the changes in blood cells of experimental mice co-immunized with rPfEF-2 1F and rPfEF-2 2F that survived 30 days after P. berghei infection, it was confirmed that the levels of blood cell indicators of the experimental mice did not change compared to uninfected normal mice (Mock) (Figs. 5 and 6). The above results show that the combination of rPfEF-2 1F and rPfEF-2 2F of the present invention has an antimalarial effect, and that it can be used as an antigen for a malaria vaccine.

[0061]

[0062] Example 3. Comparison of vaccine efficacy of rPfEF-2 whole and 1F and 2F combination treatments.

[0063] The present inventors recombined the whole protein of EF-2 derived from P. falciparum (GenBank accession number: XP_001348660.1) and compared and analyzed the vaccine efficacy between the whole protein and 1F and 2F combination treatments.

[0064] Six-week-old C57BL / 6 female mice were used, and 50 μg of rPfEF-2 whole and alum, an adjuvant, were mixed in a 1:1 ratio and administered intramuscularly. 25 μg each of rPfEF-2 1F and rPfEF-2 2F (total 50 μg) and alum, an adjuvant, were mixed in a 1:1 ratio and administered intramuscularly. Two weeks after the final immunization, 1 × 10 rodent malaria parasite (P. berghei) NK65 3 Each mouse was infected by intravenous injection of P. berghei. Mice were immunized with bovine serum albumin (BSA) mixed with alum in an equal amount to the recombinant protein and used as a control. After infection with P. berghei, blood samples were collected from the tail vein every two days and analyzed for parasitemia.

[0065] As a result of the analysis, the rPfEF-2 whole immunization experimental group showed a low level of blood parasite count until the 13th day after P. berghei infection, but the blood parasite count began to increase from the 15th day after P. berghei infection, and on the 23rd day after P. berghei infection, the blood parasite count was similar to that of the P. berghei infection group, whereas the rPfEF-2 1F and 2F simultaneous immunization experimental group showed an increase in blood parasite count from the 13th day after P. berghei infection, but showed a tendency of decreasing blood parasite count after the 19th day after P. berghei infection. P. On the 23rd day after infection with S. berghei, the parasitemia in the rPfEF-2 1F and 2F simultaneous immunization experimental group was 54.2%, while that in the rPfEF-2 whole immunization experimental group was 27.1%. The rPfEF-2 1F and 2F simultaneous immunization showed a blood parasitemia count that was twice as low as that in the rPfEF-2 whole immunization, which was statistically significant (Fig. 7). These results indicate that the combination of EF-2 1F and 2F antigens effectively suppresses the proliferation of parasites in the blood compared to the whole protein antigen of EF-2, demonstrating an excellent protective effect against malaria infection.

[0066]

[0067] Example 4. Antigen-specific antibody production and immune response analysis of 1F and 2F combination treatments

[0068] To analyze the antigen-specific antibody production and antigen-specific immunological serum response in mice immunized with rPfEF-2 1F and 2F combinations, ELISA was performed with mouse sera obtained through animal experiments. The control group was set as sera from non-immunized mice, and the limit of detection was determined by ELISA reaction on plates coated with 1F and 2F. While the sera from non-immunized mice did not exceed 0.2 in absorbance at 450 nm (OD), the OD value of the sera from immunized mice was higher than that of the sera from non-immunized mice up to a 204,800-fold dilution, which could be determined as the limit of detection (Fig. 8).

[0069] To predict antigen-specific immunological responses, an IgG subclass ELISA was performed. Serum from non-immunized mice served as a control, and the levels of IgG1 and IgG2c were assessed by ELISA on plates coated with 1F and 2F. Mouse sera were diluted 1:10,000, and the levels of IgG1 were significantly higher than those of IgG2c, indicating that the antigen induces a Th2 immune mechanism (Fig. 9).

Claims

1. A vaccine composition for preventing malaria comprising, as active ingredients, an EF-2 (elongation factor-2) recombinant peptide derived from Plasmodium falciparum having an amino acid sequence of sequence number 1 and an EF-2 recombinant peptide derived from Plasmodium falciparum having an amino acid sequence of sequence number 2.

2. A vaccine composition according to claim 1, characterized in that the vaccine is a subunit vaccine.

3. A vaccine composition according to claim 1, characterized in that the composition further comprises at least one selected from the group consisting of a pharmaceutically acceptable carrier, a diluent, and an adjuvant.

4. A vaccine composition according to claim 1, characterized in that the composition is administered through any one route of administration selected from oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and nasal routes.

5. A method for preventing malaria infection, comprising administering a vaccine composition according to any one of claims 1 to 4 to an individual expected to be infected with Plasmodium.

6. A preventive method according to claim 5, characterized in that the malaria parasite is Plasmodium falciparum or Plasmodium berghei.

7. A kit for diagnosing malaria infection, comprising a recombinant peptide of EF-2 (elongation factor-2) derived from Plasmodium falciparum, consisting of an amino acid sequence of sequence number 1 or sequence number 2.

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