NL2039834A - Messenger ribonucleic acid vaccine targeting envelope protein of chikungunya virus and preparation method therefor - Google Patents

Abstract

The present invention belongs to the technical field of messenger ribonucleic acid (mRNA) vaccine preparation, and specifically relates to an mRNA vaccine targeting an envelope protein of Chikungunya Virus (CHIKV) and a preparation method therefor. Specifically, the present invention provides an application of a nucleotide sequence as shown in SEQ ID NO: 1 in the preparation of an mRNA vaccine against CHIKV. In the present invention, a vaccine with cross—protective capabilities against different lineages of CHIKV is designed, reducing production costs and immunization cycles, and being capable of effectively responding to the outbreak of CHIKV. Fig. l

Description

MESSENGER RIBONUCLEIC ACID VACCINE TARGETING ENVELOPE

PROTEIN OF CHIKUNGUNYA VIRUS AND PREPARATION METHOD

THEREFOR

Technology Field

The present invention belongs to the technical field of messenger ribonucleic acid (mRNA) vaccine preparation, and specifically relates to an mRNA vaccine targeting an envelope protein of Chikungunya Virus (CHIK V) and a preparation method therefor.

Background

CHIKV, a virus transmitted through mosquitoes, originating in Africa and belonging to the alphavirus genus. It has broad cell and tissue tropism, capable of replicating in most cell types except B and T cells. It spreads throughout the body through severe viremia, primarily triggering joint inflammation. Although CHIK V fever is a self-limiting disease, it can cause severe sequelae and is more harmful to the elderly and children.

At first, CHIKV breaks out in Eastern Africa, subsequently spreading across various

African countries, followed by erupting in Southeast Asia, and later in the Indian subcontinent. CHIKV includes four major lineages: (1) West African; (2)

East/Central/South African (ECSA); (3) Asian; and (4) Indian Ocean. Initially, CHIKV 1s transmitted only by Aedes aegypti, and accordingly, it only breaks out in local parts of tropical regions. However, with the emergence of the Indian Ocean lineage, CHIKV can be transmitted by Aedes albopictus distributed in temperate regions, gradually spreading to the temperature regions, showing a global outbreak trend. Currently, there are almost no safe and effective vaccines available on the market. In prior art, there are preparation methods for vaccines as follows. (1) Attenuated live vaccine, which are prepared by modifying the virus to reduce its virulence. However, there is a tendency for reversion once the virus enters the body, posing safety risks and having potential side effects. (2) Inactivated vaccines, which are prepared by inactivating CHIKV. However, it involves live virus preparation and requires repeated inactivation verification, resulting in slow updates and generations. These vaccines lack good T-cell immune responses and have short-lived neutralizing antibodies. (3) Recombinant protein vaccines, which utilize expression systems to directly express viral proteins and can even be designed as multivalent or multimeric vaccines, capable of preventing multiple strains simultaneously. However, multimeric proteins may fail to fully show the antigen profile after folding, and have relatively poor immunogenicity.

In addition, suitable adjuvants are required and the immunization cycle is longer. mRNA vaccines are a form of vaccine, with the basic principle of introducing mRNA expressing antigen targets into the body through specific delivery systems. The proteins are expressed in vivo and the body is stimulated to produce specific immunological responses, enabling immune protection to the body. mRNA-based drugs, especially mRNA vaccines, have been widely proven as a promising immunotherapy strategy. The mRNA vaccines have unique advantages including high efficiency, relatively low side effects, and low cost.

Currently, there are no reports on mRNA vaccines targeting CHIKYV.

Given that, a conserved amino acid sequence targeting the envelope protein has been designed in the present invention using a method of identifying conserved sequences, providing a safer and more effective vaccine for the prevention and treatment of CHIKV.

Summary

A main objective of the present invention is to provide an mRNA vaccine targeting an envelope protein of CHIKV and a preparation method therefor, providing a new method for preventing and treating CHIKV. Specifically, the present invention provides the following technical solutions.

The present invention provides an application of a nucleotide sequence as shown in

SEQ ID NO: 1 in the preparation of an mRNA vaccine against CHIKV. 23 In a further solution, the present invention provides an application of a protein as shown in SEQ ID NO: 2 in the preparation of an mRNA vaccine against CHIKYV, the protein as shown in SEQ ID NO: 2 being obtained through transcription and translation of the sequence as shown in SEQ ID NO: 1.

In an embodiment, the present invention provides a recombinant plasmid, having a structure of PUC57-T7-5°UTR-E3-E2-6k-E1-3°UTR-PolyA. A nucleotide sequence of a coding region E3-E2-6k-E!1 is as shown in SEQ ID NO: 1, a nucleotide sequence of S"UTR is as shown in SEQ ID NO: 3, and a nucleotide sequence of 3’UTR is as shown in SEQ ID

NO: 4. 3 In a further solution, the present invention provides an mRNA vaccine against CHIKV.

The vaccine includes a vaccine body and a vaccine carrier. The vaccine body targets an envelope protein of CHIKYV, and a sequence of the envelope protein is as shown in SEQ ID

NO: 2.

In a further solution, the vaccine body is obtained by enzymatic linearization of the recombinant plasmid in vitro, followed by transcription.

In a further solution, a sequence of the vaccine body is as shown in SEQ ID NO: 5.

In a further solution, the vaccine carrier is a lipid nanoparticle (LNP).

In a preferred solution, the LNP is obtained by: mixing SM102, DMG-PEG2000, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol in molar percentages of 50%, 1.5%, 10%, and 38.5%, respectively, and dissolving them in an anhydrous ethanol solution.

In an embodiment, the present invention provides a kit. The kit contains the recombinant plasmid or the vaccine described above.

In an embodiment, a preparation method for an mRNA vaccine against CHIKV includes the following steps:

Sl: constructing a recombinant plasmid containing a nucleotide sequence as shown in

SEQ ID NO: 1;

S2: performing enzymatic linearizing of the recombinant plasmid, and performing in vitro transcription on a linearized template, to obtain an mRNA vaccine body;

S3: preparing a vaccine carrier: an LNP solution; and

S4: dissolving the mRNA vaccine body in a citrate buffer solution, followed by mixing with the LNP solution, and performing ultrafiltration to obtain the mRNA vaccine.

The present invention has the following technical effects:

In the present invention, a method of identifying conserved sequences is employed to design the vaccine, and a conserved amino acid sequence targeting the envelope protein is designed. Given the outstanding performance of mRNA vaccine during the SARS-CoV-2 outbreak, it is designed into an mRNA vaccine, mRNA-CHIKV-E, which is prepared into

LNP using microfluidic methods and administered to Balb/c mice. Neutralizing antibody levels in mice immunized with this mRNA vaccine are assessed through authentic virus neutralization tests. Cellular immunity is evaluated using inactivated viruses from the Asian lineage and the E2 protein (Sino Biological 40440-VO8B) from the Indian Ocean lineage in an Elispot experiment. It is found that mice develop neutralizing antibodies against the

Malaysian strain after two immunizations and show high-level T-cell immune responses against both the Asian and Indian Ocean lineages of the virus. Furthermore, the immunized mice completely control the occurrence of viremia, indicating that the designed vaccine has certain immunogenicity, can elicit better T-cell immune responses, and possesses good protective efficacy.

Brief Description of the Drawings

FIG. 1 is a schematic diagram of mRNA-CHIKV-E.

FIG. 2 shows the detection results of mRNA-CHIKV-E preparation process, where: a) shows electrophoresis of in vitro transcribed RNA, lanes 2 and 3: mRNA-CHIKV-E; b) shows encapsulation verification, lane 1: mMRNA-CHIKV-E-LNP, and lane 2: mRNA-CHIKV-E-LNP after full release of mRNA with tritonx-100 treatment; and c) shows particle size distribution of prepared mRNA-CHIKV-E-LNP.

FIG. 3 shows western blotting (WB) experimental results of protein expression after mRNA-CHIKV-E transfection into cells; where a) shows mRNA-CHIKV-E-LNP {abbreviated as "CV-E-LNP" in the figure) transfected into 2931/17 cells compared with commercial kit Mirus (MIR2225) transfected with bare RNA expression; and b) shows mRNA-CHIKV-E-LNP (abbreviated as "CV-E-LNP" in the figure) transfected into Vero cells compared with Mirus expression. (-) represents negative control, Mirus represents transfection using a commercial kit, CV-E-LNP represents transfection using the prepared vaccine, and f-actin is the internal reference protein.

FIG. 4 shows immunofluorescence images of Vero cells.

FIG. 5 shows body weight changes in Balb/c mice within five days after immunization with different doses.

FIG. 6 shows IgG binding antibody titers against E2 protein (Sino Biological 40440-VO08&B) in sera collected from mice immunized with mRNA-CHIKV-E on days 7, 14, 5 21, and 28.

FIG. 7 shows representative results of Elispot experiments using E2 protein from the

Indian Ocean lineage and inactivated virus from the Asian lineage as stimulants; where: a)

IFN-~y; and b) IL-2.

FIG. 8 shows analysis of Elispot experimental results; where a) shows spot count statistics after detection of IFN-y with E2 protein from the Indian Ocean lineage as stimulant; b) shows spot count statistics after detection of IFN-y with inactivated virus from the Asian lineage as stimulant; c) shows spot count statistics after detection of IL-2 with E2 protein from the Indian Ocean lineage as stimulant; and d) shows spot count statistics after detection of IL-2 with inactivated virus from the Asian lineage as stimulant.

FIG. 9 shows neutralizing antibody titers against Malaysian strain (a) and its mutant strain (b) in Balb/c mice on days 7, 14, 21, and 28 after immunization.

FIG. 10 shows joint swelling in four limbs of mice within five days after challenge; where: a) left forelimb; b) right forelimb; c) left hindlimb; and d) right hindlimb.

FIG. 11 shows virus load detection in blood of mice from different dose groups after challenge.

FIG. 12 shows virus load detection results in systemic tissues and organs seven days after challenge.

FIG. 13 shows pathological injury analysis results of lung tissue.

FIG. 14 shows pathological injury analysis results of liver tissue.

FIG. 15 shows pathological injury analysis results of hind leg muscle tissue.

FIG. 16 shows pathological analysis results of brain tissue.

FIG. 17 shows pathological analysis results of spleen tissue.

FIG. 18 shows pathological analysis results of kidney tissue.

FIG. 19 shows pathological analysis results of heart tissue.

FIG. 20 shows statistical analysis of pathological scores of lung, liver, hind leg muscle, brain, spleen, kidney, and heart tissues.

Detailed Description

To make objectives, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below with reference to the examples. It is to be understood that the specific examples described herein are merely illustrative of the present invention and are not be deemed as limiting the scope of the present invention. The interpretations of some of the materials used in the present invention are shown in Table 1 below. All materials used in the present invention are available on the market.

Uridine triphosphate | 63-39-8 (UTP)

SM102 (amino lipid) 2089251-47-6

DSPC 816-94-4

The sequence information in the present invention is shown in Table 2 below

Name Sequence number mRNA-CHIKV-E coding | SEQ ID NO: 1 region, ie, the nucleotide sequence of E3-E2-6k-E 1 mRNA-CHIKV-E coding | SEQ ID NO: 2 region protein mRNA-CV-E vaccine body | SEQ ID NO: 5 sequence

Example 1. Vaccine preparation

Amino acid sequences of 788 full-length structural proteins from CHIKV strains of different lineages were retrieved from the NCBI Virus database, which were subjected to amino acid sequence alignment, resulting in a total of 1248 amino acids. The most frequent amino acid at each position was selected, and these 1248 conserved amino acids were reassembled in order, to form a new conserved amino acid sequence, which was then subjected to codon optimization according to human codon preferences, to obtain a DNA sequence. An envelope protein E3-E2-6k-E1 sequence was selected and constructed into a plasmid PUCS7 containing a T7 promoter, SUTR, 3UTR, and 100 PolyA (PUC57-T7-5°UTR-E3-E2-6k-E1-3’UTR-PolyA) (FIG. 1). A nucleotide sequence of

E3-E2-6k-E1 is shown in SEQ ID NO: 1, and a protein sequence of E3-E2-6k-E1 is shown in SEQ ID NO: 2. A nucleotide sequence of 5’UTR is shown in SEQ ID NO: 3, and a nucleotide sequence of 3’UTR is shown in SEQ ID NO: 4.

Plasmid amplification was performed using Topl0 bacteria, and enzyme digestion linearization was performed with Bsal after plasmid was extracted. A linearized template was precipitated with 70% isopropanol, followed by washing with 70% ethanol. The linearized template was subjected to in vitro transcription using a commercial kit (NEB).

Before the transcription, the transcription material UTP was completely replaced by

Nl-methyl pseudouridine triphosphate (N1-Me-pUTP). The detection results of the transcription process are shown in FIG. 2. mRNA-CHIKV-E (abbreviated as "mRNA-CV-E") was obtained, which was the vaccine body, with a nucleotide sequence shown in SEQ ID NO: 5. 2. Encapsulation of vaccine carrier, LNP

The mRNA was dissolved in a citric acid buffer at pH of 4 and a concentration of 50 mM to achieve a final concentration of 108 ng/uL. An LNP mixed solution was prepared by mixing SM102, DMG-PEG2000, DSPC, and cholesterol in a molar ratio of 50%, 1.5%, 10%, and 38.5% in an anhydrous ethanol solution. Both the lipid-ethanol solution and the mRNA-citric acid solution were filtered through 0.22 pm microporous membranes, and were then mixed using a microfluidic device at a flow rate ratio of buffer phase to ethanol phase of 15 mL/min:5 mL/min, with the molar ratio of phosphorus in mRNA-CHIKV-E to nitrogen in SM102 being 1:8. The obtained mRNA-CHIKV-E-LNP was immediately diluted with 15 mL of the above citric acid buffer and then ultrafiltered using a 100 kDa ultrafiltration tube at a centrifugal force of 3000 g until the volume was reduced to one-fourth of the original, followed by the addition of 50 mM Tris-HCl buffer (pH=7.5) to 15 mL. This process was repeated twice, and the concentration and encapsulation efficiency were measured, to ensure the encapsulation efficiency of 95% or more (FIG. 2), and the encapsulated CHIKV mRNA vaccine, mRNA-CHIK V-E-LNP was obtained.

After preparation of mRNA-CHIKV-E-LNP, it was transfected into 293T/17 cells and

Vero cells, with the corresponding mRNA serving as a control. Total cell protein was collected 16 hours after transfection, which were analyzed by WB using CHIKV El (GeneTex GTX135187) and E2 (Alpha Diagnostic international #CHIKE21-A) antibodies to confirm the expression of target antigens. High expression of El and E2 proteins was ensured in the mRNA-CHIKV-E-LNP group (FIG. 3). To determine the cellular localization of vaccine antigens on cells, Vero cells transfected with mRNA-CHIKV-E-LNP were immunostained with an E1 antibody (GeneTex GTX135187), confirming antigen localization to the cell membrane for subsequent antigen secretion and presentation (FIG. 4).

After verifying expression, the mRNA-CHIKV-E-LNP vaccine targeting the E protein was administered at high (15 ug/animal), medium (8 pg/animal), and low (4 pg/animal) doses, with LNP serving as a control. Immunizations were given on days 0 and 14. Body weight changes after vaccination were monitored, to ensure basic safety of the vaccine (FIG. 5). The immunogenicity of the vaccine was assessed by detecting specific IgG binding antibodies against the E2 protein in serum, to ensure good immunogenicity (FIG. 6).

To detect whether memory cellular immune effect is generated after immunization with the vaccine, the ElisPot method was employed to detect positive cells of IL-2 and

INF-y in spleen immune cells stimulated by antigen, thus assessing cellular immunity levels (FIGS. 7-8).

To determine serum neutralizing antibody levels and cross-protection effect, the CPE method was used to detect neutralizing antibody levels at different time points after immunization using two different CHIKV strains on Vero cells (FIG. 9).

Finally, the effectiveness of the vaccine was demonstrated through challenge protection experiments. Since CHIKV often causes joint swelling and deformation, the site of challenge was the hind leg muscle of mice, and changes in limb joints were monitored. A vernier caliper was employed to monitor vertical and horizontal values of joints (FIG. 10).

Blood was collected at different time points after challenge to detect viral loads in the blood using qPCR, and the dynamic changes of viremia was monitored (FIG. 11). Seven days after challenge, mice from each group were dissected, and viral loads in various tissues and organs were detected using qPCR, to determine if the vaccine effectively inhibited viral replication (FIG. 12). Simultaneously, HE staining method was used to detect pathological damage in various tissues and organs, and corresponding pathological scores are used for quantitative statistical analysis of the degree of damage (FIGS. 13-19).

Vaccine validation effect is as follows. 1. Electrophoresis of in vitro transcribed RNA and verification of successful preparation of mRNA-CHIKV-E-LNP

As shown in FIG. 2, the transcribed RNA shows no significant degradation or impurity band, and mRNA-CHIKV-E-LNP is successfully prepared with a particle size of less than 100 nm. 2. Cellular expression verification of mRNA-CHIK V-E-LNP

It can be seen from FIG. 3 that mRNA-CHIKV-E is expressed in both 293T/17 cells and Vero cells after transfection, with no target bands observed in the untransfected negative control (-).

Immunofluorescence experiments in FIG. 4 indicate that mRNA-CHIKV-E is successfully expressed in Vero cells, mainly expressed within the cells and on the cell membrane, with no target protein observed in the negative control group. 3. Body weight changes in BALB/c mice after vaccination

It can be seen from FIG. 5 that, the body weights of immunized mice and those injected with empty LNPs slightly decrease but then continue to increase overall, with normal body temperatures, indicating no significant toxicity of the vaccine. 4. Significantly elevated serum binding antibody titers against E2 protein antigen in

BALB/c mice after immunization with mRNA-CHIKV-E: two immunizations at day 0 and day 14

It can be seen from FIG. 6 that, serum binding antibodies can be produced 7 days after the first immunization, and the binding antibodies one week after the second immunization are significantly increased, reaching a titer of 105. 5. mRNA-CHIKV-E induces strong T-Cell immunity m BALB/c mice, and is effective against both Asian and Indian Ocean lineages

As shown in FIGS. 7 and 8, Elispot results indicate that compared to the empty LNP group, all dose groups produce strong T-cell immune responses and are effective against both Asian and Indian Ocean lineages. 6. Neutralizing antibodies against Malaysian strain and its mutant strain after mRNA-CHIKV-E immunization

FIG. 9 shows a significant increase in neutralizing antibodies 21 days after the first immunization, with neutralizing antibodies produced against the two strains. 7. Challenge experiment in immunized BALB/c mice

As seen in FIG. 10, after challenge, the right forelimb swelling in the control and empty LNP groups is significantly greater than that in the vaccine group. 8. mRNA-CHIKV-E completely controls the emergence of viremia in

CHIK V-infected mice

FIG. 11 shows that high, medium, and low dose groups effectively control the emergence of viremia, while the severe viremia are observed in the empty LNP and control groups on the first day post-infection. 9. Given that CHIKV virus infection can cause systemic tissue and organ infiltration and damage, viral loads in systemic tissues and organs are measured; and the results show that mRNA-CHIKV-E can effectively eliminate or significantly reduce viral loads in systemic tissues and organs

As shown in FIG. 12, mice immunized with mRNA-CHIKV-E after CHIKV challenge

U can clear viral loads in most tissues and organs, and the virus load in the spleen and hind foot muscle after in-situ injection of the vaccine 1s significantly decreased, indicating that the vaccine can effectively clear or significantly reduce the virus load in systemic tissues and organs. 10. mRNA-CHIKV-E immunization can significantly reduce the degree of pathological damage to systemic tissues and organs caused by CHIKV challenge

It effectively alleviates pathological features in lung tissue such as hemorrhage or congestion, inflammatory cell infiltration, integrity of alveolar structure, and bronchial obstruction, with significant differences observed at medium and low doses (P<0.05), as shown in FIG. 13 and the analysis results in FIG. 20.

It effectively alleviates pathological features in liver tissue, including hemorrhage, intravascular thrombosis, inflammatory cell infiltration, and hepatocyte swelling and fusion, with significant differences observed at high and medium doses (P<0.05, P<0.01), as shown in FIG. 14 and the analysis results in FIG. 20.

The pathological features such as inflammatory cell infiltration, haemorrhage and myofibrosis in the hind leg muscles at the site of intramuscular injection for challenge are effectively attenuated, with significant differences in the three dose groups (P<0.001), as shown in FIG. 15 and analysis results in FIG. 20.

In brain tissue, it effectively alleviates pathological features such as microglial proliferation, mild hemorrhage, and partial neuronal death, with significant differences observed in three dose groups (P<0.0001, P<0.01), as shown in FIG. 16 and the analysis results in FIG. 20.

In spleen tissue, it effectively alleviates pathological features such as hemorrhage, increased inflammatory cells, and active or absent germinal centers, with significant differences observed in three dose groups (P<0.0001), as shown in FIG. 17 and the analysis results in FIG. 20.

In kidney tissue, it effectively alleviates pathological features such as hemorrhage or congestion, inflammatory cell infiltration, and exudate, with significant differences observed in the high and medium dose groups (P<0.01, P<0.001), as shown in FIG. 18 and the analysis results in FIG. 20.

In heart tissue, it effectively alleviates pathological features such as inflammatory cell infiltration, muscular fibrosis, hemorrhage, or congestion, with significant differences observed in all three dose groups (P<0.0001), as shown in FIG. 19 and the analysis results s in FIG. 20.

In summary, the present invention has designed a vaccine with cross-protective efficacy against different lineages of CHIKV, which reduces production costs and immunization cycles, and can effectively respond to the outbreak of CHIKV. The above shows and describes the basic principles, main characteristics and advantages of the present invention. It 1s to be understood by those skilled in the art that the above examples do not limit the present invention in any way, and that any technical solution obtained by means of equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims (10)

CONCLUSIONS 1. Use of a nucleotide sequence as shown in SEQ ID NO: 1 in the preparation of a messenger ribonucleic acid (mRNA) vaccine against Chikungunya virus (CHIKV). 2. Use of a protein as shown in SEQ ID NO: 2 in the preparation of an mRNA vaccine against CHIKV, wherein the protein as shown in SEQ ID NO: 2 is obtained by transcription and translation of the sequence as shown in SEQ ID NO: 1. 3. Recombinant plasmid, having a structure of PUCHT7-TT7~5"UTR-E3-E2~6k-E1-3'UTR-PolyA, wherein a nucleotide sequence of a coding region E3-E2-6k-E1 as shown in SEQ ID NO: 1, a nucleotide sequence of S'UTR as shown in SEQ ID NO: 3, and a nucleotide sequence of 3'UTR as shown in SEQ ID NO: 4. 4, mRNA vaccine against CHIKV, consisting of a vaccine body and a vaccine carrier, wherein the vaccine body is directed against an envelope protein of CHIKV, and a sequence of the envelope protein as shown in SEQ ID NO: 2. 5. A vaccine according to claim 4, wherein the vaccine body is obtained by enzymatic linearization of the recombinant plasmid according to claim 3 in vitro, followed by transcription, 6. The vaccine of claim 5, wherein a vaccine body sequence is as shown in SEQ ID NO: 5. 7. A vaccine according to claim 4, wherein the vaccine carrier is a Lipid Nanoparticle (LNP). 8. The vaccine according to claim 7, wherein the LNP is obtained by mixing SM102, DMG-PEG2000, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol in molar percentages of 50%, 1.5%, 10% and 38.5% respectively, and dissolving in an anhydrous ethanol solution. ©. Kit containing the recombinant plasmid according to claim 3 or the vaccine according to any one of claims 4 to 8. 10. A method of preparing an mRNA vaccine against CHIKV, comprising the steps of: S1: constructing a recombinant plasmid containing a nucleotide sequence as shown in SEQ ID NO: 1; S2: enzymatically linearizing the recombinant plasmid and performing in vitro transcription on a linearized template to obtain an mRNA vaccine body; S3: preparing a vaccine carrier: an LNP solution; and S4: dissolving the mRNA vaccine body in a citrate buffer solution, followed by mixing with the LNP solution and ultrafiltration to obtain the mRNA vaccine.