Celine herpesvirus circular RNA vaccine and application thereof

Abstract

The invention discloses a cat herpesvirus circular RNA vaccine and application thereof. According to the circular RNA vaccine, four glycoproteins gB, gD, gE and gI of feline herpes virus type 1 (FHV-1) are subjected to systematic antigen optimization, and a fusion protein is constructed on the basis, so that synergistic immunization of multiple antigens is realized; furthermore, the circular RNA vaccine is prepared on the basis of the optimized antigens and fusion proteins. Compared with the prior art, the application realizes the development and production of the cat virus vaccine which is safer, more stable and more lasting in immune effect, the process is stable and reliable, and the large-scale production of high-quality vaccine products can be realized.

Description

Technical Field

[0001] This application specifically relates to a feline herpesvirus circular RNA vaccine and its application, belonging to the field of animal immunotherapy technology. Background Technology

[0002] Feline herpesvirus type 1 (FHV-1) belongs to the alphaherpesvirus subfamily of the herpesviridae family and is a double-stranded DNA virus. Its genome is relatively large, approximately 135 kb in length, and consists of a unique long region (UL) and a unique short region (US). It has been confirmed to encode at least 74 proteins, including 23 viral particle-associated proteins and 13 envelope proteins. Among the various FHV-1 proteins, the envelope glycoproteins gB and gD are key antigens that induce the host to produce neutralizing antibodies, while genes such as gI and gE are important virulence factors involved in the viral pathogenesis process. Based on their central role in the immune response and their high degree of conservation, gB and gD proteins have become ideal targets for developing FHV-1 subunit vaccines, effectively stimulating the body to produce high levels of neutralizing antibodies, thus possessing significant value in vaccine development. FHV-1 is the main pathogen causing feline infectious rhinotracheitis and is one of the most important pathogenic factors in feline upper respiratory tract diseases. Statistics show that over 50% of upper respiratory tract infections in cats are caused by this virus. Infected cats often exhibit high fever, keratitis, and chronic sinusitis; in severe cases, it can develop lobular pneumonia and even periorbital skin ulcers, posing a systemic threat to their health. The disease is particularly dangerous for kittens, with a near 100% morbidity rate and a mortality rate of approximately 50% in kittens aged 2–4 months. The mortality rate in adult cats remains between 20% and 30%. It is noteworthy that recovered cats often carry the virus for life, forming a latent infection that can recur under stress or when immunity is weakened, greatly increasing the difficulty of prevention and control.

[0003] Currently, there is no specific treatment for feline herpesvirus type 1 (FHV-1) infection, and prevention mainly relies on vaccination. Existing FHV-1 vaccines are mostly feline triple inactivated vaccines, requiring annual booster immunizations, and suffer from short-lasting protection and limited efficacy. To overcome the shortcomings of existing inactivated vaccines, the development of new, highly effective vaccines is imperative.

[0004] Circular RNA (circRNA), as a core technology carrier for next-generation nucleic acid vaccines, stands out due to its naturally unique covalently closed circular structure. Its structure lacks the 5' and 3' free ends of linear nucleic acid molecules, allowing it to circumvent exonuclease recognition and degradation at the molecular level. Compared to traditional nucleic acid vaccine carriers such as linear RNA and DNA, it exhibits superior in vivo stability and a longer biological half-life. Crucially, circRNA can achieve persistent and efficient antigen expression in host cells without relying on complex nucleotide modifications, thereby fully activating the body's immune system. It can induce high levels of specific neutralizing antibodies and stimulate strong and durable humoral and cellular immune responses, providing a novel technological path to overcome the bottlenecks of traditional vaccines in terms of immune persistence and protective efficacy. It has become a highly promising innovative direction in the field of nucleic acid vaccines.

[0005] However, in the development of feline herpesvirus type 1 (FHV-1) vaccines, the application of circular RNA technology faces key technical obstacles: the full-length coding sequences of key wild-type FHV-1 envelope glycoproteins (gB / gD / gE / gI) are not well-suited to the circular RNA expression framework. Direct introduction into the circular RNA system often leads to low translation efficiency of the antigen protein or even ineffective expression, which limits the full realization of its immunogenicity. Summary of the Invention

[0006] The main objective of this application is to provide a feline herpesvirus circular RNA vaccine and its application, in order to overcome the shortcomings of the prior art.

[0007] To achieve the aforementioned objectives, this application employs the following technical solution.

[0008] The first aspect of this application provides a circular RNA comprising a nucleotide sequence encoding a feline herpesvirus fusion protein. The nucleotide sequence encoding the fusion protein may be the sequence shown in SEQ ID NO:16 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:16. More preferably, the nucleotide sequence encoding the fusion protein is as shown in SEQ ID NO:16.

[0009] A second aspect of this application provides a circular RNA comprising one or more nucleotide sequences of gB, gD, gE, and gI proteins encoding FHV-1.

[0010] Furthermore, the nucleotide sequence encoding the gB protein can be the sequence shown in SEQ ID No. 17 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO: 17. More preferably, the nucleotide sequence encoding the gB protein is as shown in SEQ ID NO: 17.

[0011] Furthermore, the nucleotide sequence encoding the gD protein can be the sequence shown in SEQ ID No. 18 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO: 18. More preferably, the nucleotide sequence encoding the gD protein is as shown in SEQ ID NO: 18.

[0012] Furthermore, the nucleotide sequence encoding the gE protein can be the sequence shown in SEQ ID No. 19 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO: 19. More preferably, the nucleotide sequence encoding the gE protein is as shown in SEQ ID NO: 19.

[0013] Furthermore, the nucleotide sequence encoding the gI protein can be the sequence shown in SEQ ID No. 20 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO: 20. More preferably, the nucleotide sequence encoding the gI protein is as shown in SEQ ID NO: 20.

[0014] A third aspect of this application provides a circular RNA vector comprising the following structural elements; Promoter, 5' intron, IRES, target sequence, 3' intron; The target sequence can be any one of SEQ ID NO:16 to SEQ ID NO:20, or an extended or truncated sequence of any one of them, for example, a sequence that is more than 95% identical to the full-length sequence of any one of them. Preferably, the target sequence is as shown in SEQ ID NO:16.

[0015] In some embodiments, the carrier includes, but is not limited to, a pCVB carrier.

[0016] A fourth aspect of this application provides a method for preparing circular RNA, comprising: The circular RNA vector was transformed into E. coli cells, the E. coli cells were amplified, and the circular RNA expression plasmid was extracted. The circular RNA expression plasmid was linearized, then transcribed and circularized in vitro to obtain circular RNA.

[0017] A fifth aspect of this application provides a feline herpesvirus fusion protein, wherein the nucleotide sequence encoding the fusion protein can be the sequence shown in SEQ ID NO:16 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:16. More preferably, the nucleotide sequence encoding the fusion protein is as shown in SEQ ID NO:16.

[0018] The sixth aspect of this application provides a recombinant protein, including one or more of recombinant gB, gD, gE, and gI proteins.

[0019] The nucleotide sequence encoding the recombinant gB protein can be the sequence shown in SEQ ID No. 17 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO: 17. Preferably, the nucleotide sequence encoding the recombinant gB protein is as shown in SEQ ID NO: 17.

[0020] The nucleotide sequence encoding the recombinant gD protein can be the sequence shown in SEQ ID No. 18 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO: 18. More preferably, the nucleotide sequence encoding the recombinant gD protein is as shown in SEQ ID NO: 18.

[0021] The nucleotide sequence encoding the recombinant gE protein can be the sequence shown in SEQ ID No. 19 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO: 19. Preferably, the nucleotide sequence encoding the recombinant gE protein is as shown in SEQ ID NO: 19.

[0022] The nucleotide sequence encoding the recombinant gI protein can be the sequence shown in SEQ ID No. 20 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO: 20. Preferably, the nucleotide sequence encoding the recombinant gI protein is as shown in SEQ ID NO: 20.

[0023] The seventh aspect of this application provides a biomaterial for preventing or treating feline herpesvirus infection in animals, the biomaterial comprising the circular RNA and lipid nanoparticles (LNPs) for encapsulating the circular RNA.

[0024] Furthermore, the animals include felines, such as cats.

[0025] The eighth aspect of this application provides the use of the circular RNA, the circular RNA vector, the feline herpesvirus fusion protein, or the biological material in the preparation of a pharmaceutical agent for inducing an immune response against feline herpesvirus infection in test animals.

[0026] Furthermore, the animals include felines, such as cats.

[0027] The ninth aspect of this application provides the use of the circular RNA, the circular RNA vector, the feline herpesvirus fusion protein, or the biological material in the preparation of a medicament for preventing animals from being infected with feline herpesvirus.

[0028] Furthermore, the animals include felines, such as cats.

[0029] The tenth aspect of this application provides a feline herpesvirus circular RNA vaccine comprising the circular RNA.

[0030] Furthermore, the feline herpesvirus circular RNA vaccine may also include a pharmaceutically acceptable carrier.

[0031] Compared with the prior art, the technical solution provided in this application has at least the following advantages: Firstly, based on optimized antigen and circular RNA technology, this application establishes for the first time a complete vaccine development system for feline herpesvirus, creating an integrated and innovative solution from antigen screening and optimization to circular RNA preparation, validation, and animal evaluation. This effectively addresses the potential safety issues of traditional vaccines such as attenuated live vaccines, inactivated vaccines, and subunit vaccines, including the risk of virulence reversion and weak immunogenicity, as well as the challenges of insufficient stability and immunogenicity in linear mRNA vaccines. This application provides a novel pathway for developing safer, more stable, and longer-lasting feline viral vaccines.

[0032] Secondly, this application systematically optimized the antigens of four FHV-1 glycoproteins (gB, gD, gE, and gI). By truncation of the sequences, expression fragments of the antigens were identified, and fusion proteins were constructed to achieve synergistic immunization with multiple antigens, which is expected to elicit a more comprehensive and stronger immune response. The Flag tag was modified from the N-terminus to the C-terminus, optimizing protein detection and characterization, demonstrating the correct folding and functionality of the protein, and providing a key tool for vaccine quality control.

[0033] Third, this application encapsulates circular RNA with lipid nanoparticles and completes preclinical animal immunization efficacy evaluation. Animal experiments have confirmed that the circular RNA vaccine can elicit a strong humoral immune response, providing direct experimental evidence for the vaccine's effectiveness and laying a solid foundation for subsequent development. Attached Figure Description

[0034] Figure 1 This is a gel electrophoresis image of the preliminary verification of the expression of the synthesized plasmid in Example 1 of this application; Figure 2 This is a schematic diagram showing the truncated four proteins of FHV-1 in Example 1 of this application; Figure 3 This is a gel electrophoresis image of the expression of four truncated proteins of FHV-1 verified in Example 1 of this application; Figure 4 This is a gel electrophoresis image of the FHV-1 fusion protein expression verified in Example 1 of this application; Figure 5 This is a gel electrophoresis image of the expression of four C-terminal Flag proteins verified in Example 1 of this application; Figure 6 This is Example 2 of this application, which verifies the circularity of the circular RNA encoding the four C-terminal Flag tag proteins of FHV-1 gB, gD, gE, and gI. Figure 7 This is Example 2 of this application, which verifies the circularity of the circular RNA encoding the FHV-1 fusion protein; Figure 8 This is an ELISA antibody level detection graph of immunized mice in Example 3 of this application; Figure 9 This is a graph showing the detection of neutralizing antibody levels in the serum of immunized mice in Example 3 of this application. Detailed Implementation

[0035] To address the problems existing in the development of feline herpesvirus type 1 (FHV-1) vaccines using circular RNA technology in the prior art, this application proposes a circular RNA adaptability optimization strategy targeting the FHV-1 antigen. This strategy involves the systematic reconstruction of the antigen structure and expression units to achieve efficient and stable expression on the circular RNA platform, thereby developing an FHV-1 circular RNA vaccine with significantly enhanced immunogenicity. This approach can overcome the limitations of existing vaccines in terms of immune protection efficacy and duration, and is of great significance for controlling the prevalence of feline rhinotracheitis and other diseases caused by upper respiratory tract pathogens.

[0036] In one embodiment of this application, a method for preparing an FHV-1 circular RNA vaccine includes the following steps: S1. Synthesize pCDNA3.1-FHV-gB, pCDNA3.1-FHV-gD, pCDNA3.1-FHV-gE, and pCDNA3.1-FHV-gI plasmids. Each plasmid consists of a T7 promoter, a flag tag, a target sequence, and a terminator. The flag tag sequence is: gattacaaggatgacgacgataag. These four plasmids contain four FHV-1 genes (gB, gD, gE, and gI), and their nucleotide sequences are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively.

[0037] These nucleotide sequences can come from the same genes of different circulating strains or genotypes of FHV virus, as long as they encode homologous proteins with immunogenicity.

[0038] In addition, these nucleotide sequences can be replaced with sequences optimized using different codon strategies to adapt to different expression systems.

[0039] In some cases, the pCDNA3.1 vector in the aforementioned plasmid can also be replaced with a prokaryotic-eukaryotic shuttle vector suitable for eukaryotic cell expression.

[0040] S2. The four synthesized plasmids were transfected into F81 cells using Lipofectamine™ 3000 (Thermo Fisher). Verification showed that the gene sequences encoding the full-length glycoproteins FHV-1 gB, gD, gE, and gI had low expression efficiency or were undetectable in F81 cells. Therefore, the pCDNA3.1-FHV-gB, pCDNA3.1-FHV-gD, pCDNA3.1-FHV-gE, and pCDNA3.1-FHV-gI sequences were optimized in this application. The optimization method is as follows: (1) Construction and expression screening of truncated plasmids: To determine the optimal antigenic fragments of FHV-1 gB, gD, gE, and gI glycoproteins, a series of truncated plasmids were designed and constructed. Based on amino acid positions, six fragments of gB, the extracellular domain fragments of gD and gI, and three fragments of gE were amplified, with nucleotide sequences shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15, respectively, and cloned into the pCDNA3.1 vector.

[0041] (2) Construction and validation of fusion protein expression units: To obtain a multi-antigen fusion protein with synergistic immune potential, the previously screened effective truncated sequences were combined. Specifically, gene fragments from the following plasmids were linked using sequences encoding flexible peptide linkers (GS: GGGGS) to construct fusion expression units: The fusion expression unit was constructed by linking gB (481~700), gD (60~260), gE (20~170), and gI (20~165), and its nucleotide sequence is shown in SEQ ID No. 16. The plasmid was labeled pCDNA3.1-gB'+gD'+gE'+gI'. The construct was transformed into E. coli for amplification, and the recombinant plasmid was obtained.

[0042] (3) Modification of protein tag sites: To optimize protein detection and characterization, the tag sites of plasmids pCDNA3.1-gB, pCDNA3.1-gD, pCDNA3.1-gE, and pCDNA3.1-gI constructed in step S1 were modified. The Flag tag, originally located at the N-terminus of the plasmid, was moved to the C-terminus, and its nucleotide sequences are shown in SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, and SEQ ID No. 20, respectively.

[0043] S3. Construction of circular RNA vector: The circular RNA vector pCVB includes (a) the T7 promoter; (b) a 5' intron fragment; (c) IRES; (d) the target sequence; and (e) a 3' intron fragment.

[0044] In some cases, the pCVB vector can also be replaced with other circular RNA expression vectors known in the art.

[0045] The circular RNA vector pCVB was linearized using BsmBI restriction enzyme; the target sequence was amplified, and BsmBI restriction sites were introduced at both ends of the sequence. Each antigen fragment was ligated to the linearized pCVB vector using T4 DNA ligase (ABclonal), and the recombinant products were transformed into competent E. coli cells for amplification. Finally, a series of circular RNA expression plasmids were successfully obtained, which can be named pCVB-FHV-gB, pCVB-FHV-gD, pCVB-FHV-gE, pCVB-FHV-gI, and pCVB-FHV-gB'+gD'+gE'+gI', respectively.

[0046] S4. In vitro transcription and circularization of circular RNA: The circular RNA plasmids obtained in step S3 were digested with Xba I restriction endonuclease to achieve complete linearization. Using the linearized plasmids as DNA templates, in vitro transcription and circularization reactions were performed according to the instructions of an in vitro transcription kit (e.g., the Promega in vitro transcription kit) to form circular RNA molecules. After the reaction, the circular RNA products were initially purified using methods such as lithium chloride precipitation.

[0047] S5. Further purify the RNA product obtained after preliminary purification. For example, if HPLC purification is used, different RNAs will have different elution times. Generally speaking, the RNA precursor will elute first, followed by circular RNA, and finally introns. Collect fractions from different times for detection. Detection methods include, but are not limited to, gel electrophoresis.

[0048] S6. The collected circular RNA was transfected into F81 cells, and the functional activity and immunogenicity of the encoded protein were verified by in vitro expression experiments.

[0049] Furthermore, the circular RNAs of pCVB-FHV-gB, pCVB-FHV-gD, pCVB-FHV-gE, pCVB-FHV-gI, and pCVB-FHV-gB'+gD'+gE'+gI' can be encapsulated using lipid nanoparticles (LNPs). The encapsulated circular RNA can then be used to immunize test animals, such as 6-8 week old BALB / c mice, and the immunization efficacy can be evaluated by performing ELISA and neutralization assays on serum samples at different time points.

[0050] The above-described embodiments of this application establish a mature circular RNA production process, encompassing DNA template linearization, in vitro transcription, circularization, and purification. This process is stable and reliable, enabling large-scale production of high-quality circular RNA, providing technical support for vaccine industrialization. The F81 cell line is utilized as a highly efficient platform for antigen expression and functional validation. This ensures that all optimized antigens and the final circular RNA can be correctly expressed and processed in mammalian cells, validating their functional activity in vivo in advance and reducing subsequent development risks.

[0051] The present application is further described below with reference to embodiments and accompanying drawings. The reagents and raw materials used in the following embodiments are all commercially available, and the experimental methods without specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. Furthermore, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this application all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature.

[0052] Example 1 Antigen Screening 1.1 Plasmid Synthesis and Expression Validation The desired sequences pCDNA3.1-FHV-gB, pCDNA3.1-FHV-gD, pCDNA3.1-FHV-gE, and pCDNA3.1-FHV-gI were synthesized by Genewiz, with nucleotide sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively. The synthesized sequences were transfected into F81 cells. After 48 hours, the cells were collected, lysed with RIPA, and used for subsequent Western blotting experiments. Cells transfected with the plasmids were digested and collected in 1.5 mL centrifuge tubes. The supernatant was discarded after centrifugation, and the cells were washed two to three times with PBS. Appropriate amounts of RIPA cell lysis buffer and protein loading buffer were added. The protein solution was denatured in a 100°C metal bath for 10 min. The comb was removed from the electrophoresis gel, and 20 μL of protein sample was added to each well. The gel was incubated at 140V for 55 min. For membrane transfer, methanol, PVDF membrane, transfer buffer, and transfer plate were prepared. Transfer buffer: 3.0 g Tris base, 14.4 g glycine, 200 mL methanol. First, activate the PVDF membrane in methanol. Place the membrane on the transfer plate, remove the gel from electrophoresis, place it on the membrane, add a sponge, and clamp the transfer plate. Place the plate in the electrophoresis tank, incubate at 200 mA for 120 min. Results are as follows. Figure 1 As shown, none of the four proteins were expressed. Figure 1 pCDNA3.1 is the blank vector, and Flag-GFP is the bifunctional fusion protein tag, abbreviated as F-GFP in the figure.

[0053] 1.2. Sequence Optimization and Expression Screening 1.2.1 Optimization and construction of FHV-1 viral gB, gD, gE, and gI proteins None of the four FHV-1 proteins were expressed after transfection into F81 cells, so the sequences of the four proteins were modified. The FHV-1 gB, gD, gE, and gI genes were predicted using AlphaFold. Based on amino acid positions, gB was divided into 6 truncated genes, gD and gI retained their extracellular sequences, and gE was divided into 3 truncated genes. The sequences of each gene are shown in SEQ ID No. 5 (pCDNA3.1-FHV-gB(150-480)), SEQ ID No. 6 (pCDNA3.1-FHV-gB(150-700)), SEQ ID No. 7 (pCDNA3.1-FHV-gB(150-940)), SEQ ID No. 8 (pCDNA3.1-FHV-gB(481-700)), SEQ ID No. 9 (pCDNA3.1-FHV-gB(481-940)), and SEQ ID No. 10 (pCDNA3.1-FHV-gB(701-940)). ID No.11 (pCDNA3.1-FHV-gD(60-260)), SEQ ID No.12 (pCDNA3.1-FHV-gE(20-170)), SEQ ID No.13 (pCDNA3.1-FHV-gE(20-380)), SEQ ID No. 14 (pCDNA3.1-FHV-gE(185-380)), shown in SEQ ID No. 15 (pCDNA3.1-FHV-gI(20-165)). like Figure 2 As shown in Table 1, different truncated sequences were amplified, and the amplification systems and primers are shown in Table 2.

[0054] Table 1 Amplification System

[0055] Table 2 Primer Sequences

[0056] The amplification program was as follows: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s; 55℃ annealing for 10 s; 72℃ extension for 1 min, 30 cycles; 72℃ extension for 5 min; storage at 4℃.

[0057] The amplified target fragment and linearized vector were mixed according to the system shown in Table 3 below and reacted at 50℃ for 30 min. The obtained ligation product was transformed into E. coli competent cells DH5α. After successful verification by PCR and sequencing, recombinant plasmids pCDNA3.1-FHV-gB(150-480), pCDNA3.1-FHV-gB(150-700), pCDNA3.1-FHV-gB(150-940), pCDNA3.1-FHV-gB(481-700), pCDNA3.1-FHV-gB(481-940), and pCDNA3 were obtained. The following plasmids were used to verify the expression of each truncated plasmid: .1-FHV-gB (701-940), pCDNA3.1-FHV-gD (60-260), pCDNA3.1-FHV-gE (20-170), pCDNA3.1-FHV-gE (20-380), pCDNA3.1-FHV-gE (185-380), and pCDNA3.1-FHV-gI (20-165). To verify the expression of each truncated plasmid, we transfected them into cells and analyzed the results using Western blotting. The results are as follows: Figure 3 As shown, the protein bands corresponding to plasmids pCDNA3.1-FHV-gB (481-700), pCDNA3.1-FHV-gD (60-260), pCDNA3.1-FHV-gE (20-170), and pCDNA3.1-FHV-gI (20-165) have the highest signal intensity. Figure 3 F-GFP is short for Flag-GFP.

[0058] Table 3 Connection System

[0059] 1.2.2 Construction of FHV-1 fusion protein Based on the expression screening results, the four truncated fragments with the highest expression levels—gB (481-700), gD (60-260), gE (20-170), and gI (20-165)—were tandemly linked using overlap extension PCR with GS flexible adapters. Primers were designed so that each fragment's end carried a sequence complementary to the adjacent fragment. The four fragments were ligated through multiple rounds of PCR, using primers shown in Table 4. The amplification program was the same as described above. Finally, the ligated four fragments were ligated into the pCDNA3.1 vector according to the system in Table 3. The construct was transformed into *E. coli* competent cells DH5α. After successful PCR and sequencing verification, the recombinant plasmid pCDNA3.1-FHV-gB'-gD'-gE'-gI' was obtained, with its nucleotide sequence shown in SEQ ID No. 16. Subsequently, expression verification was performed by transfecting eukaryotic F81 cells using the method described above. Figure 4 As shown, the tandemly expressed protein was successfully expressed with a high signal intensity.

[0060] Table 4 Primer Sequences

[0061] 1.2.3 Site Modification of Protein Tags Specifically, the original N-terminal Flag tag coding sequence in the FHV viral plasmid synthesized by Genewiz was first deleted. Specific primers were then synthesized, and their sequences are shown in Table 5. Following the amplification procedure and method described above, the sequences encoding the Flag tag and the GS flexible adapter were precisely inserted into the 3' end of each target gene using homologous recombination. The recombinant products were transformed into E. coli competent cells, successfully constructing recombinant plasmids with C-terminal Flag tags: pCDNA3.1-FHV-gB-GS-Flag, pCDNA3.1-FHV-gD-GS-Flag, pCDNA3.1-FHV-gE-GS-Flag, and pCDNA3.1-FHV-gI-GS-Flag. These were then transformed into F81 cells for verification. Verification showed that all plasmids with the Flag tag moved to the C-terminus were expressed. Figure 5 . Figure 5 , Figure 6 , Figure 8 and Figure 9 In Chinese, Flag is abbreviated as F.

[0062] Table 5 Primer Sequences

[0063] The C-terminal flag-tagged pCDNA3.1-FHV-gB-GS-Flag, pCDNA3.1-FHV-gD-GS-Flag, pCDNA3.1-FHV-gE-GS-Flag, and pCDNA3.1-FHV-gI-GS-Flag from FHV-1 are shown in SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, and SEQ ID No. 20, respectively. The C-terminal flag-tagged pCDNA3.1-FHV-gB'+gD'+gE'+gI' protein is used for subsequent construction of circular RNA, the nucleotide sequence of which is shown in SEQ ID No. 16.

[0064] Example 2: Preparation of Circular RNA 2.1. Construction of circular RNA vectors The circular RNA vector pCVB was linearized using BsmB I restriction enzyme. The reaction system was prepared according to Table 6 and incubated in a 37°C water bath for half an hour. The target sequence (nucleotide sequences shown in SEQ ID No. 16~SEQ ID No. 20) was amplified by introducing BsmB I restriction sites homologous to the linearized pCVB vector at both ends of the sequence using PCR primers. The primer sequences are shown in Table 6, and the amplification system and conditions are as described above. The circular RNA vector pCVB and the target sequence were digested with BsmB I restriction enzyme, and the digestion system is shown in Table 7.

[0065] Table 6 Primer Sequences

[0066] Table 7 Enzyme digestion system

[0067] The target fragments and linearized plasmid pCVB were recovered separately by gel electrophoresis. Each antigen fragment was ligated to the linearized pCVB vector using homologous recombinase. The recombinant products were then transformed into competent E. coli cells for amplification, resulting in the successful acquisition of multiple circular RNA expression plasmids, defined as pCVB-FHV-B'+gD'+gE'+gI', pCVB-FHV-gB-GS-Flag, pCVB-FHV-gD-GS-Flag, pCVB-FHV-gE-GS-Flag, and pCVB-FHV-gI-GS-Flag.

[0068] 2.2 Preparation of circular RNA The above circular RNA plasmids were linearized by single-enzyme digestion with Xba I restriction endonuclease. The system was prepared according to Table 6. The reaction was carried out in a water bath at 37°C for 30 min. The template DNA obtained by enzyme digestion was extracted with phenol-chloroform at a volume ratio of 1:1 and then precipitated with 2.5 volumes of anhydrous ethanol for purification.

[0069] The purified template DNA was transcribed in vitro using T7 RNA polymerase (Promega). The transcription reaction was carried out under the conditions recommended in the manufacturer's instructions, and the transcription system is shown in Table 8.

[0070] Table 8 In vitro transcription system

[0071] The reaction system can be scaled up or down proportionally according to template requirements. 1 ml of reaction can usually synthesize 2-5 mg of RNA in 2-4 hours. Gently pipette to mix the reaction solution and incubate at 37°C for 2-4 hours.

[0072] After transcription and circularization, RQ1 RNase-Free DNase (Promega) was added to a final concentration of 1u / µg template DNA, and the mixture was incubated at 37°C for 15 minutes. The product was then initially purified using lithium chloride solution, followed by washing with 75% ethanol to remove impurities. Finally, after drying, a mixture of circular RNA, circular RNA precursor, and other RNAs was obtained. Low-melting-point gel electrophoresis was used to verify the bands. The circular RNA precursor moved slowly during electrophoresis, while the circular RNA moved faster, resulting in two RNAs of different sizes. The unspliced ​​RNA was larger and appeared at the top of the gel electrophoresis pattern; the spliced ​​RNA was smaller and appeared at the bottom of the gel electrophoresis pattern. Figures 6-7 As shown. This step yields preliminarily purified circular RNAs: CircRNA-FHV-gB-GS-Flag, CircRNA-FHV-gD-GS-Flag, CircRNA-FHV-gE-GS-Flag, CircRNA-FHV-gI-GS-Flag, and CircRNA-FHV-gB'+gD'+gE'+gI'.

[0073] 2.3 Purification and Expression of Circular RNA The preliminarily purified circular RNA was further purified using an SEC1000 HPLC column (7.8*300mm 5μm 1000A) and an Agilent 1260 HPLC system. The conditions were: Waters B EH450A, column temperature: 40℃, flow rate: 1 min / ml, elution conditions: 0–30 min, 100% buffer A (prepared with 10 mM Tris, 0.5 mM EDTA, and DEPC in water). Based on the peak characteristics in the chromatogram, the eluted fractions corresponding to the retention times of the target circular RNA were collected. RNA concentration was determined using NanoDrop and stored at -80℃. The collected circular RNA was transfected into F81 cells for expression validation. Finally, the cells were sent to Hanxin Biotechnology (Suzhou) Co., Ltd. for LNP encapsulation. The resulting products were named CircRNA-FHV-gB-GS-Flag / LNP, CircRNA-FHV-gD-GS-Flag / LNP, CircRNA-FHV-gE-GS-Flag / LNP, CircRNA-FHV-gI-GS-Flag / LNP, and CircRNA-FHV-gB'+gD'+gE'+gI' / LNP. A mixture of LNPs and PBS was used as a blank control under the same conditions, named LNP-PBS.

[0074] The encapsulated product was diluted with PBS, with a target dilution of 50 μL of PBS containing 5 μg of circular RNA. The diluted product was then used for injection in animal experiments.

[0075] Example 3 Animal Experiment (1) Thirty female BALB / c mice (weighing 18.0±2.0g) aged 6-8 weeks were randomly divided into 6 groups and vaccinated by intramuscular injection. The mice were immunized twice, once every 2 weeks. The mice were immunized according to the immunization schedule shown in Table 9.

[0076] Table 9 Immunization Schedule

[0077] Blood was collected from mice via the posterior orbital venous plexus at different times after the second immunization. The collected blood was placed in a 2 mL centrifuge tube, incubated at 37°C for 1 hour, then incubated at 4°C overnight, and centrifuged at 3000g for 10 minutes at 4°C. The serum was collected into another new centrifuge tube, aliquoted into 20 μL per tube, and stored at -80°C for later use.

[0078] (2) The level of anti-FHV IgG antibody in mouse serum was detected by indirect ELISA.

[0079] The FHV-1 recombinant antigen was diluted to 5 μg / mL with 0.05 mol / L carbonate buffer (pH=9.6) and added to 100 μL per well of a 96-well high-adsorption flat-bottom polystyrene microplate. The plate was fixed overnight at 4°C. The plate was washed three times with 200 μL of PBST (pH=7.4) containing 0.05% Tween 20 each time. 100 μL of blocking buffer (3% BSA-PBS) was added to each well, and the plate was blocked at 37°C for 2 h, followed by three washes with PBST. Mouse serum was diluted 1:100 using blocking buffer (100 μL per well), and incubated at 37°C for 2 h, followed by three washes with PBST. Horseradish peroxidase-labeled rabbit anti-mouse IgG was diluted 1:2000 using blocking buffer, and incubated at 37°C for 2 h, followed by three washes with PBST. 100 μL of TMB substrate chromogenic solution was added to each well, and the plate was incubated at room temperature in the dark for 5-10 min. 100 μL of 2 mol / L... The reaction was terminated with H2SO4; the absorbance of each well was measured at 450 nm using a microplate reader. Antibody levels were expressed as absorbance values.

[0080] Experimental results are as follows Figure 8As shown, the ELISA results indicate that the FHV-1 vaccines constructed based on the circular RNA platform in this application all elicited strong specific antibody responses. At 28 days post-secondary immunization, antibody levels (mean OD values: 3.33-6.18) in all experimental groups were several times higher than those in the negative control. The fusion protein vaccine CircRNA-FHV-gB'+gD'+gE'+gI', composed of tandemly selected antigen fragments, demonstrated the most outstanding immunogenicity, with a mean OD value as high as 6.18. More importantly, this immune response exhibited excellent durability; even at 91 days post-secondary immunization, the antibody level in the fusion protein vaccine group remained at a high level of 5.04, significantly higher than other groups.

[0081] (3) Detect the titer of neutralizing antibodies in mouse serum.

[0082] Seed cells into 96-well plates, adding 100 μL of F81 cell suspension to each well, at a cell density of 1–2 × 10⁶ cells / well. 4 Cells / well; Incubate 96-well plates at 37°C, 5% CO2 for approximately 24 hours until a cell monolayer forms; Serially dilute the serum to be tested with maintenance medium, preparing 6 replicates for each dilution; Dilute known titers of FHV-1 virus to 100 TCID50 / 0.1 mL, mix 60 μL of diluted serum with 60 μL of virus solution, and incubate at 37°C for 1 hour to allow antibody-virus binding; Aspirate the culture medium from the 96-well plate, gently wash the cells once with PBS, and add 100 μL of virus-serum mixture to each well. In the virus control group, add only virus, no serum; in the cell control group, add only maintenance medium, no virus or serum; in the serological control group, add only serum, no virus. Incubate the 96-well plate at 37°C, 5% CO2 for 1–2 hours to allow virus adsorption; then aspirate the virus-serum mixture and add 100 μL of fresh maintenance medium to each well. Cytopathic effect (CPE) was observed daily, and the number of wells without CPE in each dilution was recorded. Neutralizing antibody titers were calculated using the Reed-Muench method, and the final results were expressed in Log 10 form.

[0083] Experimental results are as follows Figure 9As shown, mouse serum was collected on days 28 and 91 after the second immunization for virus neutralization experiments. Results showed that all vaccine groups successfully induced high levels of neutralizing antibodies with a durable response. On day 28 after the second immunization, the neutralizing antibody GMTs of each monomeric antigen vaccine (gB, gD, gE, gI) ranged from 416.2 to 544.0, all significantly higher than the negative control. The fusion protein vaccine (CircRNA-FHV-gB'+gD'+gE'+gI') induced the strongest immune response, with a GMT as high as 693.8, significantly superior to the monomeric groups, demonstrating that selecting highly expressed sequence tandem has a significant effect on vaccine-induced neutralizing antibody activity. By day 91 after the second immunization, the neutralizing antibody levels in all vaccine groups remained high, with the fusion protein vaccine maintaining a GMT of 603.6, demonstrating excellent immune durability.

[0084] In summary, this application successfully overcomes the core technical bottleneck of low expression efficiency of FHV-1 antigen in circular RNA systems by employing the core strategies of "antigen truncated fragment screening" and "multi-antigen fusion protein construction," combined with optimized protein characterization design. Experimental data fully validate that this strategy not only significantly enhances immunogenicity but also induces durable immune memory, providing a powerful solution to the problems of short protection period and limited efficacy of existing FHV-1 vaccines.

[0085] It should be understood that the above embodiments are preferred embodiments of this application, but the implementation of this application is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this application shall be considered equivalent substitutions and shall be included within the protection scope of this application.

Claims

1. A circular RNA, characterized in that, The nucleotide sequence encoding the feline herpesvirus fusion protein comprises SEQ ID NO:

16.

2. A circular RNA vector, characterized in that, The structural elements comprise: a promoter, a 5' intron, an IRES, a sequence of interest, a 3' intron; The sequence of interest is as shown in SEQ ID NO:

16.

3. The circular RNA vector of claim 2, wherein: The vector comprises a pCVB vector.

4. A method for preparing a circular RNA, characterized by, The vector comprises: The circular RNA vector of any one of claims 2-3 is transformed into E. coli cells, the E. coli cells are amplified, and the circular RNA expression plasmid is extracted; The circular RNA expression plasmid is linearized, followed by in vitro transcription and circularization to obtain the circular RNA.

5. A feline herpesvirus fusion protein, characterized in that: The nucleotide sequence encoding the fusion protein is as shown in SEQ ID NO:

16.

6. A biomaterial for preventing or treating an animal from being infected with a feline herpesvirus, characterized by: The biomaterial comprises the circular RNA of claim 1 and a lipid nanoparticle for encapsulating the circular RNA.

7. Use of the circular RNA of claim 1, the circular RNA vector of any one of claims 2-3, the feline herpesvirus fusion protein of claim 5, or the biomaterial of claim 6 in the preparation of a medicament for inducing an immune response against feline herpesvirus infection in a subject animal; the animal comprising a feline.

8. Use of the circular RNA of claim 1, the circular RNA vector of any one of claims 2-3, the feline herpesvirus fusion protein of claim 5, or the biomaterial of claim 6 in the preparation of a medicament for preventing an animal from being infected with feline herpesvirus; the animal comprising a feline.

9. A feline herpesvirus circular RNA vaccine, characterized in that, The circular RNA of claim 1.

10. The feline herpesvirus circular RNA vaccine of claim 9, characterized in that, The pharmaceutically acceptable carrier is also included.

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